Abstracts

To overcome the shortage of endocrine beta cells needed for improving and implementing beta cell therapy in type-1 diabetes patients, additional sources of beta cells must be found. Reprogramming non-endocrine cell types from different pancreatic compartments presents an attractive approach given their relative abundance. The majority of cells that can be obtained from cadaveric organ donors are exocrine cells. These are discarded during the procedure of islet cell isolation. We previously demonstrated the growth factor induced conversion of rat acinar cells into functional beta cells. This cellular reprogramming is triggered by a combination of EGF and LIF, and requires the re-activation of the pro-endocrine transcription factor Neurogenin-3 (Ngn3). Our hypothesis is that sufficiently high STAT3 and MAPK signalling is capable to reprogram human exocrine cells to endocrine beta-like cells, in concordance to what has been demonstrated in the rodent in vitro. The effect of ectopic expression of activated MAPK and STAT3 by human exocrine cells obtained as remnant fraction after islet isolation from cadaveric donors is investigated. Human exocrine cells were transduced directly after isolation with lentiviruses expressing activated MAPK and STAT3, and placed directly in 3D matrix culture or first as free-floating suspension culture followed by 3D matrix culture. We found that simultaneous introduction of activated STAT3 and MAPK provokes a re-expression of the pro-endocrine transcription factor Neurogenin-3 (Ngn3) in the human exocrine cells. These cell preparations are a mixture of acinar and duct cells, and in vitro, they rapidly adopt a duct-like phenotype. The stable re-expression of Ngn3 is seen in the nucleus of 20% of the keratin-19 positive human exocrine cells only in conditions exposed to activated STAT3 and MAPK. This phenomenon occurs in exocrine cells in 3D culture without or with a preculture period in suspension. However, it is only in the condition where cells are first kept in suspension, and after 7 days transferred to 3D culture that we observe a modest, but significant increase in the number of insulin positive cells. Reprogramming exocrine cells as a source of potentially transplantable beta cells is a novel approach to improve cell therapy in type-1 diabetes. Given the large number of exocrine cells in the (donor) pancreas, this could represent an important boost for beta cell therapy.

Nanobodies (Nbs) derived from camelids are a unique form of monoclonal antibodies characterized by a single antigen binding domain. The small size, the almost limitless repertoire diversity, the high affinity and high specificity towards target molecules, high stability and high expression yields in various heterologous expression systems are among the features which make Nbs an interestingly useful class of molecules as diagnostics and therapeutics, as intrabodies, as crystallization aid and as proteomics tools (for expression profiling, physical mapping of proteins, protein-protein interaction studies, functional analysis and (in)activation of genes, etc.). Moreover, Nbs are excellent affinity reagents for ELISA, western blot, immunohistochemistry and affinity capture. We have developed a robust technology to identify antigen-specific Nbs. Access to antigen-specific customized Nbs is possible for all researchers via VIB Nanobody Service Facility (VIB/NSF). Moreover, support is provided by VIB/NSF to use or to further engineer Nbs according to the customers' needs.

In recent years huge interest has been developed in understanding the molecular mechanisms underlying neurodevelopmental disorders. Though, there is tremendous effort geared towards this, the inability to study the human brain of patients (e.g. patient-derived neuronal material/brain biopsy) limits our knowledge of human neurological disorders and hampers the investigations towards a potential therapy. To circumvent these limitations, we make use of a humanized model for neurodevelopmental diseases, applying the induced pluripotent stem cell (iPSC) technology. iPSC technology was first described in 2006 through the introduction of four basic transcription factors (Oct4, Sox2, Klf4 en cMyc) in fully differentiated somatic cells, which brings them in a de-differentiated state. The resulting cell line then acquires the characteristics of embryonal stem cells that can then be differentiated into one of the three germline lineages. These iPSCs can be differentiated to neuronal progenitors and subsequently to different types of neurons and constitute a very valuable in vitro model for the study of neurological disorders. Apart from this, iPS models are of immense value to evaluate candidate drugs and in later course as a potential therapeutic element. In our study, we aim to investigate the molecular and cellular abnormalities underlying the MECP2 duplication syndrome. This syndrome is characterized by severe to profound neurodevelopmental delay, hypotonia, progressive spasticity, seizures and recurrent infections in male patients. It is caused by genomic duplication of the MECP2 gene with consequently higher MeCP2 protein dosages. The iPSC clones were derived by transducing MECP2 duplication patient and control fibroblasts with trancription factors Oct4, Sox2, Klf4 and c-Myc as described by the Yamanaka group. Control and MECP2dup iPSC lines demonstrated pluripotent gene expression and transgene silencing. The pluripotency of each iPSC line was further assessed by teratoma formation and the presence of the three embryonic germ layers. Further, the presence of the MECP2 duplication in the generated iPSC clones was reconfirmed by quantitative RT-PCR, Western blot analysis and array CGH. In a next step we have generated cortical neurons with fore brain identity starting from these iPSC lines. We are currently performing analyses of maturation, synapse development and morphology. These results will be discussed at the meeting.

The use of scale down models is common practice in biotech industry to support commercial processes. The general purpose of scale down models is to examine numerous process parameters allowing fast process development and optimization. A magnitude of possible tools and systems to support cell culture model development is commercially available and as such, they can serve as a backbone for model design. Bioprocess development is becoming exceedingly important to screen and optimize process parameters of the large scale manufacturing process. Performing these studies at the manufacturing scale is practically unfeasible due to the costs of operation and limited availability of large scale reactors. As a result the development of scale-down models that correspond to the performance of the commercial process is crucial to achieve reliable process characterization. These models can range from several liters (small scale bioreactors) to the micro-liter range (microtiter plates) and should all show a certain amount of equivalence to the key performance parameters of their large scale counterpart. A common strategy is to follow a proportional scale-down for the volume-dependent parameters (e.g., working volume, feed volume, agitation and aeration), while sustaining the set-points of volume-independent parameters (e.g., pH, dissolved oxygen, temperature). In the scope of a viral risk mitigation program within Genzyme, the use of gamma-irradiation is a possible strategy to reduce the risk of viral contamination through the raw materials. However, introduction of gamma-irradiated raw materials into the cell culture process could have an impact on cell culture performance and productivity. Therefore to understand this risk, an impact of a single gamma-irradiated media component was evaluated using different small scale tools: a) microtiter plate assays; b) real-time cell monitoring and c) flask model as well as a qualified scale-down bioreactor model. This study indicates that small scale tools can be used as a first line screening mechanism prior to introducing experiments at more labor intensive, time consuming and costly qualified scale down models and further, at commercial scale. However, it is a prerequisite to understand the limitations of each model in order to proceed with a correct decision on which small scale model to be used as a support for a specific study and will be further discussed.

Despite increased awareness and rigor controls around virus, microbial and endotoxin contamination and associated safety implications, there is still a probability that contaminant can breach the manufacturing process and can lead to loss of product due to regulatory concerns towards potential health risks. These events can also result in manufacturing shutdowns for extended periods of time. The most recent virus contaminations in mammalian cell culture manufacturing facilities are today being associated with the raw materials and therefore it is becoming increasingly more important to address the potential entry of different contaminants into the process and to identify remediation plans in order to reduce manufacturing downtime, thereby reducing the risk to patient supply as well as the overall production costs of a contamination. This presentation will discuss impact and possible remediation approaches to address manufacturing facility contamination related to raw materials.

The current therapy for myocardial infarction (MI) is not adequate enough since mortality after MI remains very high. Nowadays, the pharmacological and surgical treatments only delay the progression towards heart failure but do not significantly improve the cardiac function. The administration of stem cells is a promising therapy to repair the function of the heart after a MI. Recently, our research group reported the existence of a new cardiac stem cell (CSC) population, the cardiac atrial appendage stem cells (CASCs). These CASCs do possess a greater myocardial differentiation potential compared to the c-kit+ CSCs or the cardiosphere-derived stem cells. After co-culture with neonatal rat cardiomyocytes, CASCs demonstrate sarcomeric organised troponin T and I expression associated with ion currents that are characteristic for adult cardiomyocytes. Based on these promising in vitro results we decided to study the functional characteristics of these CASCs in the Göttingen minipig infarct model. In this pilot study, 6 animals (3 controls, 3 transplant) were included. MI was induced by an occlusion of the left anterior descending (LAD) artery for 2 hours. Prior to transplantation, CASCs were labelled with green fluorescent protein (GFP) and ex vivo expanded to clinical relevant numbers without losing their differentiation capacities. To enhance cell retention, an average of 63±28x106 CASCs were dissolved in a fibrin matrix and administered on the infarct- and peri-infarct zone immediately after reperfusion of the LAD. Two months after transplantation, compared to the control group, no significant decrease in left ventricular ejection fraction (LVEF) could be detected in the transplant group. The LVEF decreased from 64,9±11,5% to 54,9±7,2% and from 65,7±2,0% to 58,4±1,7% in controls and transplants respectively. On the other hand a significant change in infarct size was measured. In the controls, only a decrease of 9% was detected while this was 43,7% in the transplant group. Histological analysis by an H&E staining on tissue sections confirmed these MRI data. However, no CASCs could be found in the transplanted tissue. Probably, CASCs were not able to migrate out of this fibrin matrix to integrate in the cardiac tissue so only paracrine effects were measured. In the near future, the cell administration protocol has to be optimized in order to obtain more efficient transplantation and cell retention in the heart. In this way we want to develop a good transplantation model that allows us to study the clinical potential of CASCs after a MI.

Activation of cAMP-responsive element binding protein (CREB) dependent gene expression is a crucial step in the molecular cascade that mediates synaptic plasticity and memory processes. The aim of this study was to set up a method to investigate brain activity related to the CREB pathway. Benzyl Quinolone CarboxylicAcid (BQCA), a highly selective allosteric potentiator of the M1 muscarinic acetylcholine receptor (M1mAChR) is well characterizedin vitro and in vivo and was therefore used as tool compound (1). To avoid the use of laborious and high time-consuming western blot analysis we optimized the Alphascreen® SureFire® technology in brain homogenates. We demonstrated that focused microwave irradiation is necessary to preserve the protein phosphorylation state of CREB and show increased pCREB in hippocampus and cortex of C57BL/6JCrl mice after administration of BQCA. We confirmed that BQCA increases CREB phosphorylation. Maximal levels of pCREB were reached 15 min after subcutaneous administration of 15 mpk BQCA. We also demonstrated that Xanomeline, a non selective M1/M4 agonist, and in addition AC- 260584, an allosteric M1 agonist, increased pCREB levels. These results add to the evidence that M1 activation via the orthosteric as well as the allosteric site activates the PKA/CREB signaling pathway in the hippocampus known to play an important role in spatial memory formation. We can conclude that this ex vivo measurement can be used as a screening system to investigate the central effect of M1 receptor activation. This Alphascreen® technique combined with microwave irradiation can in future be applied to the measurement of other phosphoproteins.

Data standards automate the specification and validation process. The upfront implementation of the data standards in a sponsor’s data management and statistical environment enables sponsors to automate the exchange of specifications and instructions to their internal and external partners. A central data standards library and a metadata repository are not only used to generate study specifications, but will also enable the verification of the study data and metadata against the specifications. The Workflow In the sponsor’s environment a global Data Standards Library is built. This library includes: •The (e)CRF Library containing all sponsor standard (e)CRF templates, annotated with CDASH and/or SDTM variables. Each individual standard (e)CRF template is linked to a cluster of metadata. •The SDTM Library containing the metadata of all domains defined in SDTM v1.2 and SDTMIG v3.1.2 •The Therapeutic Area Library containing all metadata of the sponsor specific domains. All elements in the data standards library are stored on the 5 levels of metadata: table level, variable level, value level metadata, controlled terminology and computational algorithms. The Study Specification process is performed by the sponsor’s data manager and specifies the visit schedule and the needed (e)CRF templates. The selected metadata from the data standards library, including the clusters of metadata attached to the (e)CRF templates are used to auto-populate the Study Metadata Repository. This repository contains the selected metadata of all studies. This enables the sponsor to compare the metadata of all studies for consistency. The Study Metadata Specifications (define.xml, visit schedule, selected (e)CRF templates, …) extracted from the study metadata repository for one specific study are provided to the partner that is responsible for the operational data management activities of that study. After the study build process and during the data management cycle, the partner provides the study data and metadata back to the sponsor. The uploaded data and metadata is compared electronically against the original specifications in the study metadata repository by the sponsor. In addition, the SDTM Validation Checks are executed to verify whether the uploaded study is compliant with the data standards library. Reports with the output of both electronic checks are automatically generated.

BAMMBO will provide innovative solutions to overcome existing bottle-necks associated with culturing marine organisms in order to sustainably produce high yields of value-added products for the pharmaceutical, cosmetic and industrial sectors. BAMMBO will screen and identify target marine organisms (e.g. bacteria, fungi, sponges, microalgae, macroalgae and yeasts) from diverse global locations for potential as sustainable producers of high–added value molecules (HVAB’s). Our project will apply analytical methods for the extraction, purification and enrichment of targeted bioactive compounds. A detailed life cycle analysis of the production pathways developed in the project will be undertaken to fully evaluate the sustainability of production of biologically active products from marine organisms. BAMMBO will exploit knowledge and technologies developed during the project and effectively manage their transfer to relevant stakeholders in industry and the research community, as well as to policy-makers. Innovative technologies developed in the project will be demonstrated with the involvement of industry partners, and the results will be of interest not only to companies directly involved in the marine sector, but to other large scale industry players such as pharmaceutical companies with interest in added-value bioactive compounds.

Obesity is a chronic and complex disorder and has become a major contributor to global burden of disease and morbidity. It leads to the development of hyperglycemia and diabetes, dyslipidemia and hypertension (all components of the metabolic syndrome), cardiovascular disease, and may be associated with osteoarthritis, and breast and prostate cancer. Treatment of obesity includes lifestyle modifications, pharmacological therapy, and surgical interventions. Although it can be argued that the entire population should be targeted with a goal of prevention of weight gain and obesity, there are currently so many ‘at risk’ individuals that there is need for simple, rapid and cheap strategies to identify individuals at highest risk of developing co-morbidities in order to identify those who might benefit the most from aggressive weight management, application of dietary regimens, and pharmacotherapy. To improve selection strategy, we propose to develop multiplex point-of-care tests. Our target cells to discover sensitive and specific RNA markers that can be included in these tests are monocytes because they are readily accessible (blood) and are activated during obesity. Monocyte activation is associated with increased infiltration of adipose, aortic and cardiac tissues where they actively induce insulin resistance, atherosclerosis, and heart failure. First, we modelled RNA interactions in pathways related to oxidative stress and inflammation, which have synergic effects in the development of obesity-associated diseases (1). These interactions provide information on how to treat or prevent obesity and associated co-morbidities (2). As an example, we found that the combination of low interleukin-1 receptor associated kinase-3 (IRAK3) and high mitochondrial superoxide dismutase (SOD2) was associated with a high prevalence of metabolic syndrome (odds ratio: 9.3; sensitivity: 91%; specificity: 77%). By comparison, the odds ratio of high-sensitivity C-reactive protein, the most widely used risk marker, was 4.3 (3). Second, we identified novel microRNAs which modulate these interacting RNA molecules and are associated with resistance to adiponectin and insulin, metabolic syndrome and cardiovascular disease (4, 5) (International PCT Patent application filed (with diagnostic and medical use claims)). We used our systems biology platform to establish an active role of the identified markers in the disease process, rendering them excellent candidates for companion diagnostic development related to monocyte-specific interventions in collaboration with commercial partners. This work is performed in collaboration with Divisions of Cardiology and Endocrinology of the University Hospital Leuven. (1) Hulsmans M, Holvoet P. J Cell Mol Med. 2010;14(1-2):70-8 (2) Holvoet P, et al. Gene signatures. European Patent 2 262 301, granted on 30-11-2011 (3) Hulsmans M, et al. PLoS ONE. 2012;7(1):e30414 (4) Hulsmans M, De Keyzer D, Holvoet P. FASEB J. 2011;25(8):2515-27 (5) Hulsmans M, et al. PLoS ONE. 2012; DOI: 10.1371/journal.pone.0032794

A novel method will be presented to determine the pharmacokinetic properties of protein drugs in an early phase of drug development. The technology enables first-in-human drug clearance studies in therapeutic protein development. Benefits of the method are a faster time-to-market, a reduction of costs, a reduction of the number of animal tests and a safer testing of biopharmaceutical drugs. We will present results that show that our technology is a feasible technology to investigate the pharmacokinetics of protein drugs at very low (microdose) levels. Glycosylated EPO will be used as a show case. Though some technical hurdles still have to be taken, the results up to now are promising. Our innovative approach meets the need in the biopharmaceutical industry for a strategy to find the most effective therapeutic protein production process early in drug development by measuring the pharmacokinetic profile of protein drugs in humans in a safe manner. It can greatly speed up the drug discovery process and the development of biosimilars and biobetters. Other applications include new protein drugs ranging from small synthetic peptides to large conjugated antibody molecules, nanoscale drugs and delivery systems and gene therapy.

Industrial process and product optimization is impossible without meaningful models and insights on significant features controlling process or product performance. Real-world modeling and feature selection problems have many issues - high-dimensional, non-linear, with unbalanced measurements, correlated features, missing experiments, etc., which makes it difficult for most people to know what the right approach is in any given situation. We present a function discovery technology based on symbolic regression that routinely converts these problems into meaningful and insightful models with robust driver features identification. Without requiring a Ph.D. in Computer Science or Statistics, it is now possible to easily develop robust nonlinear models (complete with trust measures), identify data outliers and interactively explore the model dynamics and response sensitivities. Our presentation will illustrate the ease and power of automatic conversion of a spreadsheet of data into an interactive data story report using examples drawn from infectious disease modeling, industrial process monitoring, and economics.

Complex recombinant glycoproteins typically consist of a diverse set of isoforms which slightly differ in their glycosylation, phosphorylation and/or other post-translation attributes. As most of these modifications directly impact the protein´s pharmacokinetic or dynamic properties, it is clear that recombinant processes should consist of the appropriate mechanisms to control and target the composition of the complex final protein pool to meet the required target product profile. While perfusion processes are characterized by different trends throughout the harvest phase (which in some cases extends to numerous weeks) the harvested protein can also show differing properties, relating to cell culture attributes such as media use, cell densities, protein titers and bioreactor controls. To ensure consistent performance and protein quality, a well-designed and robust downstream process is essential. Even more so, variability in the upstream process should at least be partially mitigated by the mechanisms applied in the downstream process. In terms of glycosylation and other post-translational attributes, which characteristically contribute to the chemical properties of the protein such as overall charge (pI) and surface hydrophobicity, the downstream process can selectively isolate the desired protein isoforms and remove any undesired species. Careful selection of chromatography media with ion exchange, hydrophobic interaction or mixed mode properties in combination with the correct load, wash and elution conditions (pH, ionic strength,…) can assure the required amount of flexibility to overcome any upstream variability. For this, a thorough characterization of protein isoform behavior throughout the upstream and downstream processes is the foundation of a well-controlled and consistent recombinant glycoprotein process.

Introduction The daily use of two languages may lead to conflict-specific brain adaptations in bilinguals. To evaluate the non-verbal conflicts in bilingual children we compared simultaneous and sequential bilingual to monolingual 8-11 year old children in an fMRI study using a colour-Simon (stimulus-response) and a numerical Stroop (stimulus-stimulus conflict) paradigm. Methodology A 3T MR system (Philips Achieva Release 2.5) with an 8 channel SENSE head coil was used. Groups consisting of 12 monolinguals, 15 sequential bilinguals and 15 simultaneous bilinguals,respectively, were scanned. Simultaneous bilinguals acquired both languages from birth at home while sequential bilinguals acquired the second later at school. A SE-EPI with 130 dynamics was used. (FOV: 212x230 mm2, matrix:104x105, 22-4mm slices, TR:3) In the Simon task red or green squares were shown in a jittered event-related design in two categories: congruent (position and colour match) and incongruent (position and colour mismatch). The participants had to press the right button for a red and the left for a green figure. In the Stroop task two digits were shown. The numerically larger digit had to be selected. There were three categories (Figure1): congruent (physical and numerical comparisons match), incongruent (physical and numerical comparison mismatch) and neutral (only numerical size differed). Analysis The analysis was done with SPM5 software (Wellcome Department of Cognitive Neurology, London, UK). All functional volumes were realigned, normalized, smoothed (8-mm FWHM) and filtered (1/128 Hz cutoff). For each task, the BOLD signal was analyzed by HRF+ temporal+ dispersion derivatives, including six motion parameters. For each scan of each subject, t-maps were calculated. Second-level analysis (repeated measures ANOVA) was performed for the groups. Results and Discussion Figure2 shows larger conflict effect in bilinguals compared to monolinguals in both tasks. fMRI data indicates clear bilingualism-related changes in the caudate nucleus, superior temporal gyrus and cingulate gyrus during the execution of non-verbal conflict tasks. The largest effect was found in simultaneous bilinguals, followed by sequential bilinguals and then monolinguals. This study provides a direct comparison of the performance on S-S and S-R conflict tasks. It is the first to report fMRI and concurrent reaction time measurements in a population of monolingual and bilingual children. It is also the first study to make a distinction between simultaneous and sequential bilingualism.

Introduction Previous studies have shown a relationship between corpus callosum (CC) morphology and the lateralization of language processing and the correlation between the size of the CC and developmental language disorders. Various parts of the CC are associated with language. We used DT-MRI based tractography to investigate the morphology of the CC. We compare five bundles arising for the sub-regions of the mid-sagittal CC in simultaneous bilinguals and bilingual interpreters; 1) arising from anterior third of the CC connecting to the orbital lobe (OL), 2) form the anterior mid-body of the CC to the primary motor cortex (PMC), 3) from the mid-posterior part of the CC,4 ) from the posterior part of the CC and 5) connection to the temporal lobe. Aim The hypothesis of our study was that the sub-regions of the CC associated with language exhibit structural differences between bilinguals and interpreters and that MRI DTI can be used to assess these differences. The idea was that among multiple language users the microstructure of the brain of trained professional language switchers might be different from the normal bilinguals. Materials and Methods A 3T MR system (Philips Achieva Release 2.5) with an 8 channel SENSE head coil was used. 12 bilinguals and 12 interpreters (right-handed, healthy males and females, 21-27 year old), were scanned. Scan parameters: SS-SEEPI, 15 non-collinear diffusion gradient directions,b= 700 s/mm2,TR/TE=6484ms/60ms, FOV=224x224x120 mm3, 60 oblique axial 2mm slices, scan duration=454s. Figure 1 shows 5 bundles: orbital, motor cortex, mid-posterior, posterior and temporal lobes. The mean fractional anisotropy (FA) in the bundles was calculated for each subject using DTIstudio (Johns Hopkins University, Baltimore, USA). An independent-samples t-test was undertaken to compare the mean FA values between groups. A P-value of less than 0.05 was considered significant. Results and Discussion The FA values are summarized in Table1. The results show that the mean FA value is significantly lower in interpreters compared to bilinguals in four of the bundles (PMC, mid-posterior, posterior and temporal). No significant difference between the two groups could be found for the FA values of fibres going from the CC to the orbital lobe. These results support the hypothesis that the structure of the CC can be changed by training to switch between two languages.

Arctix is a bioinformatics service unit within the VIB Department of Plant Systems Biology, Division of Bioinformatics and Systems Biology. Established upon more than a decade of scientific and computational experience within the fields of systems biology and genomics, Arctix is offering a variety of bioinformatics solutions and services. With the recent technological advances, huge amounts of transcriptome, proteome, and metabolome data are being produced on daily basis, while whole genome sequences are being determined at an ever-increasing pace. It is therefore essential to integrate these various kinds of data to describe and understand more complex biological systems. Genes and proteins are no longer studied as isolated entities but as part of regulatory and interacting biological networks and novel systems biology approaches enable us to better comprehend the entirety of processes that happen in a biological system on multiple scales of organisation, in time and space, from genomes to cells and from organs to organisms. The analysis, integration and visualisation of these high-throughput data asks for specialised and state-of-the-art software solutions. One of the simplest ways to reach this goal is to simply let Arctix help you in finding appropriate solutions for your bioinformatics problems. We offer flexible, automated and user-friendly gateways in challenging and fast-evolving scientific fields such as systems biology, genomics and next-generation sequencing. The Arctix team relies on years of experience in bioinformatics, systems biology and genomics, proven by a track record of many proof-of-concept publications in high impact factor journals and services provided for third parties such as industrial partners and wet-lab research groups. The Arctix team can offer top-down bioinformatics, basic to sophisticated data analyses, custom software development and expert consultancy. If you are interested in or want to learn more about our services, do not hesitate to contact us. More information and details can be found on our website http://www.arctix.be.

Introduction: Inspired by the discovery of dendritic cells (DCs) as specialized antigen-presenting cells (APCs), antitumor immunotherapy by genetic manipulation of DCs to present tumor antigens has been explored. Lentiviral vectors (LVs) have been shown to be highly efficacious in DC transduction thereby providing a powerful tool to deliver tumor antigens to DCs. Moreover, direct administration of tumor antigen encoding LVs has highlighted their potential as an off-the-shelf vaccine. Aim: The translation of LV-vaccines to the clinic has been hampered by concerns about their safety. Most of these can be tackled by transductional targeting of LVs to DCs. Therefore we developed the Nanobody (Nb) display technology. Methods: The Nb display technology is an innovative approach which exploits the budding mechanism of LVs to incorporate a bindingdefective but fusogenic form of VSV.G (VSV.GS) together with an APC-specific Nb. The Nbs under investigation are Nb R3_13, DC1.8 and DC2.1, respectively specific for human DCs, mouse DCs and APCs in general. Nb BCII10, specific for β-lactamase, was used as a control throughout the study. Broad tropism LVs were applied for comparison. Results: We generated producer cells that stably express a membrane bound form of the aforementioned Nbs. As shown by the reverse transcriptase assay this enabled us to produce Nb displaying LVs at similar titers as broad tropism LVs. Incorporation of Nbs and VSV.GS on the viral surface was demonstrated using an in-house developed ELISA and by western blot. Selective in situ transduction of DCs (Nb DC1.8) or DCs and macrophages (Nb DC2.1) was demonstrated by flow cytometry in which Thy1.1+ cells, obtained from lymph nodes injected with Thy1.1 encoding LVs, were characterized in detail. In vivo bioluminescence imaging on mice of which the inguinal lymph node was injected with Firefly Luciferase (FLuc) encoding LVs, was used to evaluate the biokinetics of Fluc+ cells. We observed long-term (20 days) presence of transduced cells after injection of broad tropism LVs in contrast to short-term (3 days) presence of transduced cells when Nb DC1.8 or Nb DC2.1 displaying LVs were administered. These data were confirmed by PCR and can be explained by the short half-life of transduced APCs. Nb R3_13 and Nb DC2.1 displaying LVs were further used to specifically transduce human lymph node DCs in vitro, highlighting the promise of the technology. Currently we are evaluating the potential of Nb displaying LVs encoding the antigen ovalbumin (OVA) to induce OVA-specific CD4+ and CD8+ T cells. Preliminary data show the induction of qualitatively different immune responses upon immunization with the LVs under investigation. Conclusion: We report on the Nb display technology to target LVs to specific APCs, a strategy that can be exploited for fundamental research exploring the stimulatory capacity of APC types and to facilitate the translation of LV-vaccines from the bench to the clinic.

Collaboration models are highly debatable items in the current challenging environment for drug design and development. Industry gradually starts to realize the potential of combining different ideas, skills, expertise and resources in technologically demanding areas and is tapping more and more into early phase research conducted at universities and small and medium-sized enterprises specialized in biotechnology (Kling, 2011). In addition, sharing knowledge with competitors at the precompetitive and even at a competitive stage becomes debatable 2 (Mullard, 2011). Currently, alternative and complex models of collaboration between stakeholders are being explored (Mullard, 2011; Lenford 2011). There is an increased interest in the formation of ‘multi-partner’ consortia, often based on a public-private partnership (PPP) model 5 (Gallagher, 2009; Croft, 2005). R&D projects conducted in precompetitive partnerships are all about sharing ideas and knowledge development. Taking into account the multifaceted partnering model of a PPP and the heterogeneity of the partners, their different and often conflicting missions, goals, objectives and cultures, it is not surprisingly that intellectual property (IP) rights, so key in pharma R&D, play a pivotal role and that IP and trust issues seem to secure their share in the negotiation process (Mullard, 2011; Nat Rev Drug Discov, 2011, Cappellaro, 2011; Cressey, 2011; Goldman, 2011; Munos, 2010; Napa, 2011; Vargas, 2010). The synergy of roles of the different stakeholders, however, can bring drug development to a higher level through partnering (Schmidt, 2011). It is worthwhile exploring the black and white view on the motives of the different stakeholder groups on participation in precompetitive biopharmaceutical R&D PPPs by answering some fundamental questions. The article explores the current state of knowledge on public-private partnerships (PPPs) in the life sciences. The emerging trend towards precompetitive PPPs is highlighted. Motives for participation in PPPs and expectations of stakeholders are explored. Certain motives, such as the drive to innovate and desire to speed up the drug development process, are common for all stakeholders and widely accepted. There are however barriers to overcome, such as different interests in intellectual property (IP) rights. The role of precompetitive PPPs in creating value of IP for all stakeholders is underestimated. Precompetitive PPPs are important for networking and establishing further collaborations.

Histological images show the effects of disease and treatment on the cellular level, making them an invaluable tool for medical researchers studying these effects. However, evaluating these images by hand is time-consuming and inherently subjective. A fully automatic software tool would be a very valuable asset for this work. Still, no sufficiently accurate solution exists. Available software tools that segment histological images into individual cells generally show mediocre results. Detected cells often lack correct borders, groups of touching cells are identified as individual cells, or single cells are over-segmented into smaller parts. Irrelevant tissue is also often kept as though they were cells. Furthermore, these tools require a set of parameters to be tuned per image, severely limiting support for processing large batches of images, especially if taken under variable lighting conditions. Lastly, possible image artifacts such as spots or tissue creases are not taken into account, leading to incorrect results. In this research we combine various image processing and machine learning techniques to provide an image segmentation algorithm that outperforms the state of the art tools by a large margin. To achieve this, we first create a rough segmentation of the image using an intelligent and image-aware color thresholding, separating the cells from the background and other objects. This provides us with some individual cell segments, but may also contain unfiltered remnants of irrelevant tissue or segments that contain so-called clumps of very proximate cells. In order to identify segments as belonging to one of these three categories, a classification model is trained using a database of labeled example segments of all categories. The model is based on a number of key differentiating features, mostly based on size, shape and color of the segment. We were able to train a successful model, allowing us to accurately label each segment resulting from the initial step as clump, irrelevant, or cell. To attain a complete segmentation of the image, detected cell clumps have to be divided into their constituent individual cells. For this we use an innovative approach which forms an improvement upon existing work by combining it with novel techniques and metrics. This allows us to not only detect the best split locations in a clump, but also to split it using a flexible and probable boundary, instead of a simple line. Our method is applied to images of skeletal muscle cells, but can be extended to other types of histological images. This method outperforms currently available packages in matching cell annotations by experts in an experiment with a large number of muscle cell images. Moreover, it requires no parameter tuning and works for large batches of images, thus saving researchers a large amount of time and work, while simultaneously creating an objective standard for image evaluation.

In tropical regions (South America, Asia and Africa) diseases like malaria and dengue cause many deaths. These diseases are transmitted through mosquitoes bites (Anopheles sp. and Aedes aegypti respectively). The current practice to protect against transmission of these diseases is by use of mosquito repellents. Common mosquito repellents used today are synthetic in nature and are suspected or have been proved to be harmful to the user and environment (e.g. DEET, DDT, dimethylphylphthalate, parathion etc). This research work is part of the FP7 No-Bug project (Novel release system and bio-based utilities for insect repellent textiles). The main interest of the project is personal protective textiles against insects (mosquitoes) for application not only in tropical areas where vector borne diseases are a major threat to the public health but also in European countries where the presence of mosquitoes can be nuisance. To solve the problems associated with the synthetic repellents, novel bio-repellents will be identified and an innovative slow release system established. Our aim is to develop a novel insect repellent personal protective equipment to be used by professional travelers (education, business, research, volunteers, missionary and peace corps) when they travel for duty in mosquito prone areas. The target mosquitoes are Anopheles stephensi which cause malaria and Aedes aegypti that transmit dengue. Keywords: Personal protective equipment (PPE), Bio-repellents, professional travelers, malaria, dengue, mosquito.

n-butanol is an important commodity chemical used as a solvent and as a precursor for chemical synthesis. In addition, n-butanol holds tremendous promise as a second-generation biofuel. It has a considerably higher combustion value than ethanol and its chemical properties allow blending in fuels more readily than ethanol, therefore it is a genuine “drop-in” biofuel. The production of n-butanol with solvent-producing strains of Clostridium, commonly known as ABE (acetone-butanol-ethanol) fermentation, is one of the largest biotechnological processes ever developed. The inability of the fermentation process to compete with the booming petrochemical industry in the 1950s and 1960s lead to its almost complete demise. The economics of the ABE fermentation are hampered by two bottlenecks: the high cost of the substrates (i) and butanol toxicity leading to low space-time-yields and high purification costs (ii). Improvements can be expected from process and metabolic engineering. Our research and development efforts are focused on improving the fermentation by process engineering. Significant economic gains are expected by increasing the efficiency of the recovery step. Integration of the first step of the downstream process with the fermentation by using a suitable in-situ product removal technique is an interesting strategy to overcome this bottleneck. Pervaporation is identified as one of the most promising unit operations for in situ product recovery of n-butanol. Organophilic pervaporation membranes selectively remove organics from dilute aqueous streams. The driving force for selective mass transport is achieved by maintaining a vacuum or applying a sweep gas at the permeate side of the membrane. Results will be presented about butanol recovery in a bench scale integrated unit designed for continuous ABE fermentation with pervaporation as in-situ product removal technique.

Heparanase is strongly implicated in tumor angiogenesis and metastasis. Specific inhibition of heparanase is thus of great clinical and commercial interest. Traditionally, the role of heparanase in tumor progression has been attributed to the degradation of heparan sulfate (HS) in the extracellular matrix, by secreted enzyme (released by tumor cells, by tumor-associated platelets and inflammatory cells). Degradation of HS might allow cancer cells to cross tissue boundaries (i.e. invasion of the tissue around the tumor eventually leading to metastasis) and render HS-bound growth factors available for receptor activation and subsequent signal transduction. Therefore, the development of heparanase inhibitors has so far been focused on blocking the enzymatic activity of the protein. Although showing some efficacy, these inhibitors suffer from a lack of specificity and undesired side effects. However, the biological activity of heparanase is more intricate. Heparanase also specifically activates several signal transduction cascades, ultimately leading to enhanced cell migration, cell proliferation and angiogenesis. All of these processes are directly involved in cancer progression. Interestingly, this signal transduction is independent of the enzymatic activity of heparanase. Although cellular receptors initiating this signaling cascade are unknown and the link between signaling, the cellular effects and tumor progression is poorly understood, several lines of evidence show that the non-enzymatic activity of heparanase might be primarily responsible for enhancing tumor progression, and not (only) the enzymatic activity. Our long-term goal is to develop heparanase inhibitors and diagnostics for clinical use in cancer treatment, while at the same time deciphering the molecular and cellular mechanism that support the role of heparanase in tumor angiogenesis and metastasis. To identify heparanase domains, cellular receptor(s) and signaling pathways involved in heparanase-induced cell migration, we have developed a library of heparanase-specific Nanobodies - single chain antigen binding domains of camelid antibodies - originating from immunizations in llama, dromedary and alpaca, in collaboration with the NSF of VIB. At present, several Nanobodies inhibiting different (non enzymatic) activities of heparanase have been identified and are being tested, for instance in cell migration and invasion assays. These function-blocking Nanobodies should clarify which aspects of heparanase biology are relevant for tumor angiogenesis and metastasis. At the same time, Nanobodies that block important functions or activities of heparanase could serve as lead components for developing specific and effective heparanase inhibitors suited for clinical use. Apart from applications in cancer therapy and diagnostic, heparanase inhibitors might also be of importance in the treatment of several auto-immune diseases and conditions related to chronic inflammation.

Second-harmonic imaging microscopy (SHIM) has become a viable tool in high-resolution microscopy for biomedical applications. It started off as a valuable addition to two-photon excited fluorescence (TPEF) microscopy, where all the available optics and light sources for TPEF can be used, with the addition of a specific filter for the second-harmonic wavelength in a separate detection channel. SHIM also offers the benefits of other nonlinear imaging techniques, like deeper penetration in the sample, lower phototoxicity and higher axial resolution. The additional value of SHIM is the collection of structural information and the opportunity to image highly ordered structures like collagen, myosin, tubulin and starch without the need to label them, making it an even less invasive imaging technique. Fluorescent proteins (FPs) are widely used in biomedical research. Two key elements in the use of FPs are the automatic formation of a chromophore due to a post-translational modification, and the possibility to express these proteins in living systems as fusion proteins, allowing the study of one specific protein in vivo. For the real-time visualisation of multiple proteins simultaneously, as well as for interaction studies based on Förster resonance energy transfer (FRET), an extensive rainbow of FPs with different absorption and emission properties has been discovered and developed. The different optical properties are adapted by engineering longer or shorter pi-conjugated systems as chromophores. In this study, we characterize the 2nd order nonlinear optical properties to find out the applicability of FPs in SHIM. We have demonstrated earlier that the chromophore embedded in the protein matrix of bacteriorhodopsin is non-centrosymmetric and capable of generating second-harmonic scattering. Based on the similarity for FPs (a non-centrosymmetric chromophore in a protective protein barrel) we were able to show the second-order nonlinear optical response for a selection of the FP rainbow: eGFP, eYFP, DsRed, mStrawberry, mCherry and the photoswitchable Dronpa. We have observed a general trend in the relationship between the linear optical properties and the first hyperpolarisability, β, being a measure for the 2nd order nonlinear optical properties of the FPs. Knowing that a bathochromic shift of the maximal absorption and fluorescence is linked to a more extended conjugated system for the chromophore, we demonstrated that for most of the FPs, these trends are also linked to an increasing β. Only for eYFP an extremely low beta was found, not following the generally observed rule. This could be explained by the chemical structure of the eYFP chromophore, containing an inversion center that makes more than half of the chromophore a centrosymmetric structure. In an effort to validate this hypothesis and find a yellow fluorescent protein with an acceptable β, we will look for a different (natural) yellow FP, and make an eYFP mutant lacking the inversion center.

Introduction: Compound Logistics is an essential part of the Drug Discovery process. The Compound Logistics & Formulations department (CL&F) in Beerse is responsible for the storage and distribution of high quality compound samples. Currently, more than 700,000 unique chemicals from J&J’s Corporate Compound Library are stored in Beerse and those samples are frequently ordered by different screening teams globally. Compounds are an important part of the Hit-to-Lead screening cost and a reduction in compound volume needed for screening could significantly increase cost savings. Therefore, CL&F decided to invest in a new liquid handling platform that can dispense nanovolume droplets, called Octopus. Compound logistics: do more with less … The Octopus platform is based on Labcyte Echo liquid handling instruments which revolutionized liquid transfer by using a new “touch less” transfer technology based on focused acoustic energy. Sound waves eject precisely-sized nano-volume droplets from the source liquid into a micro plate, without using tips, pin tools, or nozzles. The cost of consumables and wash fluids and disposing of that wash fluid can now be decreased dramatically. This technology also prevents fast compound depletion due to small volume transfer and because many processes no longer require intermediate plates. To improve efficiency, the Echo Liquid handling instruments were arrayed into integrated and flexible work cell configurations. Mitsubishi industrial robots have been chosen for moving plates from one instrument to another. To protect the source plates as much as possible from external influences, heat sealers and seal remover equipment were also added to the Octopus platform which was build in a clean room environment. The system is able to run day and night, without operator intervention, because a high capacity stacker has been integrated. Moreover, a small plate centrifuge and standard liquid handler are also part of the Octopus platform to ensure the production of high-quality assay-ready compound plates. To control the movements of the robot and to drive the different instruments, PAA’s Overlord 3 scheduler software is being used, as well as a customized user and LIMS interface that runs on top of this scheduler. The operator is optimally protected from the robot and stacker moves, because the Octopus platform is placed in an ergonomic hexagonal enclosure with safety interlocks. Since bringing a new technology into existing work flows is not just a simple substitution of technologies, it has been very important to establish collaborative relationships with the different automation, software, contractor companies and business teams. The Octopus project is an example of an innovative approach to realize our current unambiguous business mission statement: “Do more with less, and do it faster”.

Silica is a well known biomaterial that can be prepared in several different forms: different 3D structures for implants, gels, microsphere powders, films and different kinds of injectable morphologies based on sol and gel structures. It is a natural component of the body, it is non-toxic, and biodegradable. Since it is a synthetic, inorganic material, little or no variation is seen, it does not provoke an immune response and there is no risk for transfer of animal pathogens when brought into patients. We will work with silica hydrogels which are produced by the Turku University of Applied Sciences, Finland. Conditions have to be defined which allow for cells to survive when they come in contact with this material. Specifically for this project, we want to investigate viability of cells which are encapsulated within silica hydrogel. Using WEHI164 and human periosteum derived cells, we will test a set of parameters concerning both cell and biomaterial conditions. In order to do so, we will compare survival assays from the different available techniques and commercial kits. In general, the viability of cells can be monitored by their production of ATP (Cell titer Glo® kit, Promega), by measuring their metabolic activity using tetrazolium salts MTT and MTS (CellTiter 96® kit, Promega), checking the cell-proliferation with reduction of resazurin (Alamar Blue®, Invitrogen), or with propidium iodide test, trypan blue or neutral red. However, for the cell-in-gel system we will have to find a compatible assay which can be applied on encapsulated cells. We will also work out a procedure to visualize the cells growing inside the silica. Finally future directions are suggested for using silica-based hydrogels in 3D cell culture.

IL-6 is a soluble cytokine with established roles in inflammatory, autoimmune and oncology disorders. The therapeutic benefits of blocking the IL-6 signaling are therefore wide-ranging. Monoclonal antibodies are highly specific and have high affinities, and are therefore considered attractive therapeutics. Highly potent human antibodies neutralizing IL-6 were generated with the SIMPLE Antibody™ technology. Active immunization of llamas was done with human IL-6. The generation of a panel of antagonistic antibodies against human IL-6 using the SIMPLE Antibody™ platform generated several promising candidates. Mining naturally occurring affinity variants by chain shuffling resulted in two antibodies showing extremely high affinities and potencies. The process of germlining, during which between 5 and 10 framework residues were changed to bring the sequence closer to the human germline V regions, did not affect the affinity of either antibody. Both antibodies have femtomolar potency in cell based assays and outperformed the benchmark antibodies that are in progressed stages of clinical development. The structural characterization of both SIMPLE antibodies unveiled the molecular mechanism explaining the remarkably high potency. Second to this they also confirm that the SIMPLE antibodies adopt the predicted human canonical folds.

ReGenesys is a biotechnology company focused on the research, development and commercialization of stem cell-based therapies and technologies. MultiStem®, an FDA approved adherent stem cell product developed from bone marrow, is currently being used in Phase II clinical trials for the treatment of ulcerative colitis, ischemic stroke and acute myocardial infarct (AMI). A Phase I study for graft versus host disease (GVHD) prophylaxis in allogeneic bone marrow transplant was recently concluded with good results and will lead to a follow-up Phase II study for chronic GVHD. The company also received permission in Germany to open a Phase I/II trial for support in liver transplantation after alcoholic cirrhosis. In addition, MultiStem® is being evaluated in preclinical studies for the treatment of a range of other indications such as conditions involving ischemic injury, neurological injury or disease and conditions involving inflammation or immune system function. An indispensable part of the development and manufacturing of an advanced cell therapy product is quality control. The legislation concerning the quality requirements of these types of products is still under development and new, more stringent guidelines are expected to be released in the near future. ReGenesys therefore aims to be first in class by designing a high level QC pipeline. A set of standards such as screening for growth, marker expression, immunosuppression, multipotent differentiation and chromosome stability (SNP arrays) is conducted on each newly produced batch of cells. This pipeline is sufficient to guarantee the identity and quality of MultiStem®. However, since we are advanced with the development of xeno-free MultiStem® and are seeking novel production technologies, it is essential to prove that such modifications in the manufacturing process do not lead to altered phenotypes with reduced therapeutic capacity. We are currently extending our QC pipeline with different genome-wide screening methods to comprehensively characterize the molecular phenotype of our product. Next-generation sequencing is employed to analyze DNA methylation of MultiStem® in order to prove epigenetic stability during cell expansion. It is very important to define DNA methylation-specific markers that discriminate MultiStem® from competing products based on e.g. mesenchymal stem cells. Screening for gene expression and miRNA patterns, and subsequent pathway analyses are done to investigate cultured cells and fine-tune their manufacturing. Here, we present our QC pipeline that implements these genomics tools to ensure that no intrinsic changes occur and that the identity of MultiStem® is secured.

New biomarker candidates are urgently required for the screening and early diagnosis of colorectal cancer. Due to the high complexity of human samples, new technologies have to be developed to overcome the problems with the high dynamic range. By using a quantitative Tandem Mass Tag (TMT) method, in which we label the proteins, rather than the tryptic peptides, it is possible to reduce this complexity considerably. TMT-labeled proteins are separated by 1D gel electrophoresis, gel slices are excised all the way down the track of the gel, and after in-gel digestion, the peptides of the different fractions are separated with nano- reverse phase-LC online coupled with high resolution mass spectrometry. By combining two fragmentation methods, Collision Induced Dissociation (CID) for identification of the peptides, and Higher energy Collisional Dissociation (HCD) for quantification of the TMT reporters, high quality peptide spectra are generated in the LTQ-velos orbitrap and confident peptide identifications are obtained. After normalization of the quantitative TMT ratios, potential biomarker candidates can be found.

Purpose: When the vitreous gel is altered either by aging or eye disease, pathologic traction can develop due to vitreomacular adhesion (VMA). When this adhesion becomes symptomatic, it may contribute to the pathophysiology of vision-threatening disorders such as vitreomacular traction and macular hole, as well as other visual disorders. Currently, the treatment of severe symptomatic VMA is surgical removal of the vitreous by vitrectomy. The purpose of the MIVI-TRUST Phase III program was to assess the safety and efficacy of ocriplasmin for the treatment of symptomatic VMA. Methods: The MIVI-TRUST Phase III program consists of two separate studies (MIVI-006 & MIVI-007) which were both randomized, placebo-controlled, double-masked, multicenter trials, investigating a single dose of 125µg(100µl) intravitreal injection of ocriplasmin as compared to a 100µl placebo intravitreal injection. The primary endpoint was resolution of VMA at day 28, determined by Central Reading Centre OCT evaluation. Secondary endpoints included total posterior vitreous detachment(PVD) at day 28, nonsurgical full thickness macular hole (FTMH) closure and visual acuity. Eyes were followed for 6 months. Results: 652 patients (age 18 – 93) were enrolled. 464 patients were randomized to receive a single dose of 125µg ocriplasmin and 188 patients received a placebo injection. Mean baseline BCVA was 64 letters (Range 8-88). Statistical analysis of the combined data demonstrated nonsurgical VMA resolution in 26.5% of microplasmin-injected patients vs 10.1% of placebo-injected patients (P<0.001). Total PVD was more prevalent in microplasmin-injected patients (13.4%) compared with placebo-injected patients (3.7%; P<0.001). Nonsurgical closure of FTMH occurred in 40.6% of microplasmin-injected eyes compared with 10.6% in placebo-injected eyes (P<0.001). Microplasmin-injected patients exhibited a higher incidence of 3-lines improvement in best-corrected visual acuity compared with placebo-injected patients. Conclusions: This is the largest Phase III trial ever conducted to evaluate a pharmacologic intervention for the treatment of symptomatic vitreomacular adhesion. Currently, symptomatic VMA can only be treated surgically by vitrectomy. The single intravitreal dose of 125µg ocriplasmin achieved better outcomes in the primary endpoint (VMA resolution), and secondary outcomes (total PVD occurrence, FTMH closure, visual acuity gains) when compared to placebo. Treatment was generally well tolerated by patients and appears to be a potential minimally invasive pharmacologic option for the treatment of symptomatic vitreomacular adhesion.

PURPOSE. Excessive postoperative wound healing with subsequent inflammation and scarring frequently leads to surgical failure of glaucoma filtration surgery (GFS). We intend to check the hypothesis that placental growth factor (PlGF) plays a role in scar formation after GFS, and that it may be a target for improvement of the outcome of this surgery. METHODS. Aqueous humor and plasma samples of glaucoma and control patients (n=10) were collected and PlGF levels were determined by ELISA. The effect of the anti-murine PlGF-antibody (5D11D4) was investigated in a mouse model of GFS in C75Bl/6 mice. Immediately after surgery 5D11D4 (1µl; 5.2mg/ml) or 1C8, an irrelevant mouse IgG antibody (1µl; 4.8 mg/ml), was injected in the anterior chamber (n=10 eyes for both groups). An anti-murine VEGF-R2 antibody (DC101) was used as a positive control (1µl; 6.2 mg/ml; n=10). Mice were killed on post-operative day 8. Treatment outcome was studied by clinical investigation of intra-ocular pressure (IOP), bleb area and bleb survival every other day. All antibodies were kindly provided by ThromboGenics NV. RESULTS. PlGF levels in aqueous humor were found to be significantly upregulated in glaucoma compared to control patients (17 ± 2 pg/ml versus 12 ± 0.75 pg/ml, p=0.03). No significant differences were found in plasma concentrations of PlGF. In the mouse model of GFS, treatment using the anti-PlGF antibody (5D11D4) significantly improved surgical outcome by increasing bleb survival (p=0.04) and bleb area (p=0.01) with 29% compared to negative control (1C8). A single administration of anti-VEGF-R2 (DC101) also significantly improved bleb area with 7% as compared to 1C8 (p=0.05), but had no effect on bleb survival (p=0.23). A trend towards an increased bleb area after 5D11D4 administration was observed compared to DC101 delivery (p=0.07). IOP was not found to be significantly different in any of the groups (p>0.05). CONCLUSIONS. Local production of PlGF in the eye may indicate an important role for this growth factor in wound healing after GFS. Indeed, targeting PlGF with an inhibitory monoclonal antibody is efficacious in improving GFS outcome, possibly even more effectively than inhibition of VEGF-R2. These results render PlGF an appealing target for ocular wound healing and point to the potential therapeutic benefits of PlGF-inhibition.

Factor VIII (FVIII) is an essential protein in the regulation of the coagulation system. Abnormally high FVIII levels are an important risk factor for thrombosis. Conversely, reduced FVIII levels are responsible for the most frequent hereditary bleeding disorder, haemophilia A. In plasma, circulating FVIII is bound to vWF, which protects it from degradation. Upon activation by thrombin, FVIII dissociates from vWF, binds to negatively charged phospholipids, and participates as a cofactor to factor IXa in the factor X activating (tenase) complex. The presence of a phospholipid containing membrane significantly increases the activity of the tenase complex. The FVIII C2 domain comprises the major region involved in phospholipid binding and is often targeted by FVIII inhibitor antibodies occurring in patients with haemophilia A treated with recombinant FVIII. These antibodies have been shown to block the binding of FVIII to phospholipids. Recent data showed that the C1 domain is also involved in phospholipid binding. The C1 and C2 domains co-operate for optimal binding which is necessary for full activity of the tenase complex. TB-402, a human monoclonal anti-FVIII antibody showing partial inhibition of FVIII, recognizes a conformational epitope in the C1 domain near the C1 and C2 interface. The location of this epitope suggests that TB-402 could reduce the ability of FVIII to interact with a phospholipid surface. Using surface-plasmon resonance and ELISA, we have investigated the effect of TB-402 on the binding of FVIII to phospholipids. In ELISA, a mixture of phosphatidylserine and phosphatidylcholine (70/30) was coated on the plate at high and low density. FVIII was subsequently incubated with increasing amounts of TB-402 and allowed to bind to the coated phospholipids. The bound FVIII was detected using an anti-FVIII A2 domain antibody. We observed that TB-402 blocked the binding of FVIII to the low density phospholipid surface in a dose-dependent manner, although it showed little or no effect on the binding of FVIII to the higher density surface. Similarly, TB-402 also interfered with the binding of the FVIII/FIX complex to phospholipids. Alternatively, the effect of phospholipids on the binding of FVIII to TB-402 was investigated using surface plasmon resonance. For this, FVIII was pre-incubated with increasing amounts of phospholipids after which its binding to a TB-402 coated surface was assessed. Increasing concentrations of phospholipids dose-dependently inhibited the binding of FVIII to TB-402. From this, it can be concluded that TB-402 interferes with the binding of FVIII to phospholipids, thereby resulting in a suboptimal function of the tenase complex. TB-402 is co-developed by ThromboGenics NV (Heverlee, Belgium) and Bio-Invent (Lund, Sweden).

Subcutaneous human tumor xenografts in immunodeficient mice are commonly used to test the preclinical efficacy of new cancer therapies. The golden standard for measuring subcutaneous tumor growth is based on an operator assumption of the most representative tumor length, width and height measured with a caliper. This method requires intensive training and a high level of experience to limit intra-measurement and inter-operator dependent variability. The latter are important for data reproduction and the continuity of tumor xenograft experiments after an operator switch. Peira BVBA and ThromboGenics NV have developed a prototype for an innovative handheld measurement device (named TM900) that visualizes the tumor at high resolution, automatically segments the tumor and calculates the 3D-volume. Two in vivo pilot studies were performed to determine measurement variability and accuracy from the new method. Colo205 and HCT-116 tumor xenografts were implanted in nude mice and the animals were treated intraperitoneally with PBS or the anti-angiogenic antibody Avastin at 20mg/kg twice a week, which is known to inhibit tumor growth. In one experiment, two operators measured in parallel tumor growth with the caliper and the TM900 prototype. Comparison of their measurements demonstrated that the TM900 operator-dependent measurement variability is similar or even tends to be lower than the caliper measurements from experienced operators. TM900 measurements from both operators resulted in an almost equal percentage of tumor growth inhibition (78% and 82%). In another experiment, tumors were measured repetitively to determine the intra-measurement variability. Only small measurement variability was obtained with the TM900 comparable with the caliper measurement variability from a highly experienced operator. At the end of the experiments, tumors were excised to correlate tumor weight with tumor volume as indication for the in vivo measurement accuracy. A strong correlation was observed between TM900 measurements and tumor weights in both studies, indicative for high measurement accuracy. During the pilot in vivo studies, variability was observed in measuring more extreme sized and complex tumors. These issues will be addressed in next versions, where algorithm and hardware will be optimized in order to increase the tumor volume measurement range. Immunodeficient hairy mice are also used as preclinical screening models. Therefore, the TM900 device will be tested to measure tumors in mice with fur. Once the product is optimized and the method characteristics are extensively consolidated in vivo, Peira BVBA will complete product valorization for market introduction. In conclusion, Peira bvba and ThromboGenics NV successfully constructed a prototype of an innovative device for measuring subcutaneous tumors. This device could offer a reliable alternative for the current golden standard measurements since it generates less operator-dependent and traceable data.

Background Biobanks are seen as increasingly important in translating biomedical research into real improvements in health care. Biobanks are defined by the samples and associated (minimal) clinical data. In this regard, information management systems are an indispensable tool in biobank management. In order to make efficient use of the different data sources within the hospital, the tumour biobank@UZA conducted a comparative study between different Laboratory Information Management Systems (LIMS) and conducted a proof-of-concept study. Methods Different LIMS systems (commercial and open-source) were compared based on efficiency, costs, flexibility, user friendliness and connectivity, advantages and disadvantages. Based on these demands, SLims (Genohm, Belgium) was selected and evaluated in the proof-of-concept study over a six month period from April till October 2011. The use cases involved the import of historical oncological data, real-time data integration as well as data export. Other use cases were associated with tissue tumor sample management and processing. In addition, an oncological serum biobank was integrated in the same data management system. Results Based on the use cases, the proof-of-concept study resulted in a custom-made sample management system. The system enables efficient data import and transfer. The use of an integrated biobank system resulted in an overall quality increase of the minimal clinical data associated with the tissue tumour samples through the identification of data inconsistencies. Interconnectivity between data sources and devices enables import of valuable qualitative parameters (i.e. freezer temperature). The system was extensively validated using virtual and real test cases in order to detect errors and optimize the system. Sample management for serum samples was successfully integrated and tested in the existing system. Conclusions Product comparison and the proof-of-concept study resulted in a custom-made sample management system for the tumour biobank@UZA, which can also be used for other biological samples. Further optimization is still required to meet existing and future requirements. Acknowledgements: The tumour biobank at the Department of Pathology(UZA) collaborates with surrounding hospitals for sample collection and as such contributes to the development of a virtual tumour biobank of the Belgian Cancer Registry.

Antibiotics enter the human food chain mainly through therapeutic or preventive dosing of farm animals. Because of possible adverse effects on human health the European Union installed intensive screening programs for antimicrobials and their residues on animals entering the human food chain. One such antibiotic is chloramphenicol (CAM) which is a neutral nitrobenzene derivative. Although it is an effective antimicrobial drug, it has lost favor due to the occurrence of severe adverse effects in humans. Thus, today CAM has been banned from use in the human food chain and its absence from food and feed is thoroughly screened. We developed an electrochemical biosensor to detect CAM. The sensor is made by immobilization of engineered sDNA aptamers, called aptasensors. Aptamers are artificial nucleic acid ligands (DNA or RNA) able to specifically recognize a target. Under optimized conditions, we are able to detect CAM with a high specificity. The peak current is corresponding to the CAM reduction. Moreover, the enhanced electrochemical response of aptamers towards CAM was observed in the presence of antibiotics with very similar structures to CAM , antibiotics that often appears in the same matrices as CAM, thus proving target specificity.

Research on ocular diseases is limited by the constraints on studying pathophysiologic processes in the postmortem human eye. As an alternative, animal models can provide valuable information for the analysis of disease progression and response to treatment. Histology is the standard investigative technique employed to characterize microstructural changes in enucleated eyes, but requires sacrificing animals at different time points. Recently, high-resolution cross-section imaging of tissue morphology in situ and in real time was achieved using Optical Coherence tomography (OCT). Using a non-invasive imaging technique to visualize inside the eye allows repeat measurements to track disease progression and assess therapeutic impact. However, OCT imaging in rodents is challenging due to the small size of the animal eye when compared with humans. In the present study, we assessed the potential of OCT to monitor the vitreo-retinal changes occurring after injection of therapeutic agents in the mouse eye. OCT imaging was performed with a contact lens to remove corneal refraction. OCT fundus images showing blood vessels and the optic nerve head were generated enabling precise registration of OCT images and measurements to fundus features. After imaging, the animals were humanely killed and the eyes were enucleated and immediately fixed in 4% paraformaldehyde, and processed for standard histological staining analysis. Vitreous detachment was evaluated on the OCT images and confirmed by microscopic analysis. We have demonstrated that high-resolution Spectralis OCT in the mouse is an effective means of detecting vitreolytic-induced posterior vitreous detachment and other responses to pharmacological treatment. The results clearly indicate that this method will be essential to assess preclinical therapeutic strategies for vitreo-retinal diseases.

Symptomatic vitreomacular adhesion (symptomatic VMA) is an eye condition characterized by decreased vision due to focal adhesion of the posterior vitreous gel to the retina resulting in traction. Ocriplasmin, a bio-active fragment of plasmin which induces posterior vitreous detachment (PVD) and is a very promising non-surgical treatment for symptomatic VMA In patients, proteolytic activity is initiated shortly after intravitreal injection of ocriplasmin, however the majority of the treated patients only achieve resolution of their VMA at 28 days post-treatment. Interestingly, ocriplasmin is rapidly inactivated and 3 days post-injection, ocriplasmin activity is no longer detected, implying that it might achieve its therapeutic function through the activation of downstream molecular targets. The objective of this study was to investigate the underlying molecular pathways of ocriplasmin-induced PVD by characterizing the degradome of ocriplasmin in the vitreous and the retina. A better understanding of the substrates of ocriplasmin will help define its mechanism of action and aid in the development of next generation treatments.. Proteomics screening techniques like 2-D DIGE (2-dimensional differential gel electrophoresis) and iTRAQ (isobaric tag for relative and absolute quantification) are excellent methods to identify the (in)direct molecular players involved in ocriplasmin-dependent PVD. We compared the protein patterns of ocriplasmin-treated and control mouse vitreous and retina samples by means of 2-D DIGE. In vitro experiments, in which the proteomes of mouse vitreous treated with ocriplasmin in vitro and untreated vitreous were analyzed, resulted in 43 differential spots that were directly or indirectly influenced by ocriplasmin. Subsequent in vivo comparison between ocriplasmin-injected and buffer-injected vitreous samples collected 1 day post-treatment, revealed 6 other differential spots. Given that the in vitro and in vivo experiments have resulted in different datasets, we conclude that it is of vital importance to perform proteomic screens in a complex and dynamic biological context in order to unravel the genuine substrates of ocriplasmin, which may play a crucial role in PVD. In addition, we also analyzed the in vivo effect of intravitreally injected ocriplasmin on the mouse retina. Analysis of retina samples from ocriplasmin-injected, buffer-injected and control eyes resulted in 104 differential spots. Further analysis and experimental validation of this data will reveal the substrates of ocriplasmin that are relevant for the induction of PVD in patients.

BACKGROUND – Parkinson’s Disease (PD) is a neurodegenerative disease. Current treatment is mainly pharmacological and focuses on relief of symptoms. Recent research shows positive results of exercise and physiotherapy for PD. Due to disease specific features, PD patients need to exercise very often, maintain this for a long period of time and train without inducing fall risk. AIM - The CuPiD project intends to develop a technological device with which PD patients can train at home. The training data will be transmitted through the internet to the patient’s physiotherapist so that feedback can be given and the training programs adjusted directly via the training platform. This platform will consist of: 1) body sensors to register the movements and give feedback; 2) a base station connected to a screen which receives data from the sensors wirelessly; and 3) a game-like interface for some of the training programs. At present it is unknown how PD patients perceive such a device and what they want from it. Therefore, we assessed PD patients’ attitudes and expectations towards technical devices, home training and telemedicine. METHODS – We conducted several focus group interviews on selected patients with severe (N=6), and less severe disease (N=6), aged between 52-77 years, two caregivers groups (N=4), and a professional group with physiotherapists specialized in PD (N=8). Cross-validation is envisaged with different patients and caregivers (N=5) to achieve data saturation. RESULTS – We found that despite the fact that the prototype was not yet available, future stakeholders could already give detailed and relevant information and imagine using the device. PD patients and their carers are very enthusiastic about a technological home exercise device which enables training of both motor and cognitive function, provided that the exercise can be done without enhancing fall risk. Patients are motivated to train with the device on a daily basis, especially when the exercises are incorporated in a game-like interface. Therapists reiterate the importance of being able to provide more variety of exercise. During home training, patients report to have no problem with wearing 3 sensors. However, when it is needed to wear the sensors all day for monitoring of physical activity levels, PD patients are prepared to only wear 1. Furthermore, they support the suggestion to have the sensors built into a cell phone (men) or a watch (women) to make it less visible to the outside world. Finally, patients indicate that telemedicine, involving a transfer of training results, is not perceived as a privacy issue as long as they know who the person on the receiving end is. CONCLUSION – We conclude that patients, caregivers and professionals have positive attitudes towards a technological platform for home rehabilitation. Hence, such a development may have potential for future progress in managing a chronic disease and prevent immobility.

Non-clinical safety pharmacology aims to identify adverse events of new drug candidates prior to administration to humans in clinical trials. In particular, the potential of new drug candidates to provoke Torsade de Pointes (TdP) is one of the major concerns for pharmaceutical companies, as this arrhythmia can be fatal and its low rate of occurrence make it very difficult to detect in both clinical and preclinical settings. Current regulatory guidelines therefore suggest assessing the effects of new drugs on the QT interval of the ECG as a surrogate marker for TdP risk; however this can lead to the unnecessary termination of the development of promising new medicines. Bio-Plus Safety Pharmacology is a start-up niche contract research organization (CRO) that specializes in cardiovascular safety pharmacology. Both the business and scientific approach of Bio-Plus Safety Pharmacology involved out-of-the-box thinking. In contrast to the traditional large CROs, which normally offer a wide battery of non-clinical tests, Bio-Plus Safety Pharmacology has chosen to focus on one single test- the anaesthetized guinea-pig model. Using this alternative business approach allows us to bring this model to the next level for cardiovascular safety screening with both scientific back-up and commercial impact. In order to meet our scientific goals, Bio-Plus Safety Pharmacology has over the last year, - with support from the IWT - heavily invested in an R&D program to improve the predictive value of its test platform in anaesthetized guinea-pigs. As a result of this R&D effort, left ventricular pressure measurements have been added to the model enabling direct monitoring of the cardiac pump function. Furthermore, a new risk marker, linking electrical and mechanical activity of the heart (the electro-mechanical window, BeatEM® technology) was established. This new marker has increased predictive power over currently used markers thereby bringing better predictive value to safety pharmacology screening. Finally, a new proarrhythmia model has been developed and validated that directly evaluates TdP risk (ChallengEM® technology). The intellectual property resulting from the R&D project has been protected by registration of trade names and by a patent application for the ChallengEM® technology. In conclusion, our innovations will help the pharmaceutical industry to make strategic decisions early-on and ultimately to focus resources on the development of safe drug candidates. In addition, these results illustrate the value of focused and innovative thinking as the cornerstone of sustained growth of a start-up research company.

Next generation sequencing has considerably increased the data throughput. High performance bioinformatics systems are required to process the vast amounts of data generated. The computationally challenging problem of assembling up to millions of reads is met by the Power Assembler, an assembly pipeline tool in the BioNumerics software, for managing high throughput sequence data. The features and possibilities of this tool will be illustrated using publicly available sequence reads from bacterial genomes. A power assembly project is essentially a series of actions, which together constitute a project pipeline. Besides a set of predefined actions for frequent manipulations, there is also the possibility to construct user-defined actions. The project results can be overviewed in summarizing reports, and represented as sequence curves displaying e.g. coverage or sequence quality information, summary graphs, or in an assembly view. There is an information flow path from the Power Assembler to the underlying BioNumerics database, allowing further analyses of the resulting contig sequences. The Chromosome Comparison module allows full genome comparisons and clustering for evolutionary and population genetic studies to be calculated. Both DNA-based chromosome comparisons and CDS-based chromosome comparisons can be performed. Moreover, the annotation of new genomes, mutation analysis and gene selection, and chromosome-wide comparisons can be performed to study the organization and structure of genomes.

Background: Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC) form the majority of primary hepatic tumours and are the third most common cause of cancer related deaths. These liver tumours rapidly outgrow their vascular supply and become hypoxic, resulting in the production of hypoxia inducible factors and triggering the angiogenic switch. Therefore, inhibiting angiogenesis has proved to be a valuable therapeutic strategy in hepatocellular carcinoma, yet less is known about its use in cholangiocarcinoma. Aims: In this study, we assess whether inhibiting the placental growth factor (PlGF) could offer a therapeutic option in mice with HCC and CC. PlGF is a homologue of the vascular endothelial growth factor (VEGF) that is only involved in pathological angiogenesis, therefore, its inhibition does not induce adverse effects. Methods: We have used a chemically induced transgenic mouse model in which both HCC and CC develop after 25 weeks and treated these tumours with murine monoclonal antibodies targeting PlGF. Tumour-lesions and non-tumour tissue were separately collected and snap frozen in liquid nitrogen for subsequent analysis using ELISA. Haematoxilin-eosin staining (H&E) was performed to evaluate the morphological changes inflicted by the treatment with αPlGF. Sirius Red staining was used to determine the metavir-score for fibrosis and to score the burden of CC-lesions. Stainings were done using standard histology protocols and evaluated by an experienced pathologist. Immunohistochemistry was used to quantify protein levels inside hepatic tumours and in surrounding non-tumour tissue. As a marker for angiogenesis, a monoclonal antibody was used targeting CD105 or Endoglin (R&D systems, ref AF1320, Abingdon, UK). Intercapillary distance (ICD) was used as a marker for microvessel density, by measuring the average distance between vessels in HCC-nodules on CD105-­tained slides. Macrophages were visualised using F4/80‐staining (AbD serotec, ref MCA497G, Dusseldorf, Germany). Results: This study shows for the first time that inhibiting PlGF decreases CC-burden, by affecting both angiogenesis and inflammation. Although treatment with αPlGF has shown in previous studies to decrease vascularisation of HCC-lesions, no significant difference was seen between αPlGF and IgG treated mice in this study. Yet αPlGF-treated HCC‐lesions showed a relevant trend towards lower vascularisation, measured both by determining the intercapillary distances as by the percentages of endoglin (CD105) staining inside the hepatocellular tumours, and were reduced both in size as in number compared to control treated lesions. Treatment with αPlGF significantly normalized the increased vascularisation of non‐tumour tissue surrounding the CC and HCC lesions. Conclusions: The use of monoclonal antibodies targeting PlGF could thus offer a potential systemic treatment for patients who suffer from primary liver tumours.

Matrix metallo-proteinase 2 (MMP-2) is involved in a number of physiological (morphogenesis, angiogenesis, wound healing) and pathological (arthritis, cancer/metastasis, obesity) processes. Using naïve, high-diversity scFv- and Fab-on-phage libraries (n-CoDeR®), we identified a series of fully human antibodies that bind to human MMP-2. The affinity (KD) of these antibodies as well as the on- and off-rates (kon, koff) characterizing the interaction with the antigen were measured by surface plasmon resonance in a high-throughput format assay in which the anti-MMP-2 antibodies were captured using an anti-human IgG surface and the interaction with MMP-2 was characterized from a single injection of the antigen. Our data show that over 50 % of the tested antibodies displayed single-digit nanomolar affinities, thereby demonstrating that potential therapeutic leads can be identified from our phage libraries without the need for affinity maturation.

Background: Scientists can buy a ‘complete’ human genome sequence through a sequence service provider with quick delivery of data within a few weeks at a reasonable cost. ‘Complete Genomics’ (abbreviated as CG; 1, 2), not only performs library preparation and NGS, but also developed a very consistent annotation and analysis pipeline to deliver high quality data to its customers. We used data from 69 open access healthy HAPMAP genomes released by CG to assemble a number of baseline ‘Tracks’ inspired from the universally adopted UCSC tables. Our Tracks are catalogs of specific features present in CG genomes, which allow substantial enrichment when analyzing private genomes, and are therefore an important resource to bring down validation costs of data obtained from newly sequenced genomes. Methods: Data presented here was obtained from 69 CG ‘var files’ available from the CG ftp repository (1). These files report results obtained by CG after mapping reads to the hg18 (hg19 also available) human reference and calling SNVs, short-insertions, -deletions, and -substitutions. We used cgatools ‘listvariants’ and ‘testvariants’ (v1.4; 1) to produce a global database file from the 69 ‘var files’. Various in-house developed parsing scripts were then applied to this large table to extract and reformat data for the different ‘Tracks’. Results: Our current Tracks include: * ‘Genome regions where non-ambiguous read mapping OR calling are impossible (no-calls)’ * ‘Systematic variants’ found in a majority of CG genomes and likely resulting from reference or mapping/calling issues. * The ’69-genome variome’ reporting all variations found in 69 genomes and their allele frequencies. * ‘The bit-called fraction’ from 69 genomes at each base position of the reference genome (experimental evidence of the CG ‘mappability’). Conclusions: Disease variant discovery is largely based on the ability to identify features specific to patient genomes. Experimental design has been shown to play a key role in the ability to pinpoint disease variants. However, easy access to Tracks of aggregate, pre-processed data from multiple genomes allows a reduction of the number of candidate variants to a list compatible with Sanger-sequencing validation. Combining novelty filtering with ‘Biologist-readable’ annotations will make full-genome data more accessible to expert biologists. References: 1. http://www.completegenomics.com/sequence-data/ (v1.10, hg18) 2. R. Drmanac, et. al. Science 327 (5961), 78. [DOI: 10.1126/science.1181498]

Sensorgram methodology is increasingly applied mainly via surface plasmon resonance (SPR) by the biotech world. For the first time, we show how to obtain information on adsorption/interaction rates and equilibrium constants of (bio)molecules using a potentiometric alternative method. Sensorgrams are obtained by injection of square concentration pulses of analyte molecules in a flow injection (FIA) setup equipped with a flow-through system. The compounds studied until now include metabolic acids, environmental contaminants, pharmaceutical drugs plus alkaloids, and oligonucleotides. Proteins and bioparticles are potential candidates. As potentiometric sensor coatings we use a soft, hydrophilic gelatin based coating and an ionically conductive rubber based coating. The “on” and “off” kinetic parameters kon and koff, and Kass values were obtained from sensorgrams studied as a function of analyte concentration. Langmuir adsorption is shown to be active. When soft gelatin coating materials are used in the sensor, we can study the interaction of metabolic acids with the collagen protein matrix. This gelatin (or other biopolymer) material can be easily doped with a (protein, DNA, aptamer…) biomolecule. Covalent attachment or ionic incorporation is more easy than covalent coupling to e.g. a gold layer (SPR). This allows qualitative and quantitative use for analyte/biomolecule and analyte/surface interaction studies. Electrode biopolymer coatings can be easily applied by the researcher, and biomolecules attached by well-known coupling reactions. The technique offers a practical and sensitive alternative to SPR, with easier coupling facilities and application in a much broader concentration range. The technology is developed by UA. Octens (www.octens.com) and Peira (www.peira.be) may be future partners for industrial applications.

The human intestinal flora is a highly diverse and complex microbial ecosystem. This microbiome changes drastically in CF patients due to the frequent and combined antibiotic usage. Moreover, the decreased release of digestive enzymes by the pancreatic duct leads to a different alimentary environment in which the microbial system resides. A shotgun metaproteomics approach is used to characterize the predominant members of the intestinal microbiota of a group of cystic fibrosis patients compared with the flora of siblings. The aim of this study was to evaluate a metaproteomics pipeline (gel-LC-ESI-FT-MS/MS) on secretome and proteome samples extracted from feces of a CF patient and its sibling. These two fractions, proteome and secretome were separated by 12.5 % SDS-PAGE. After reduction and alkylation, the complete lanes were cut from the gel and sliced into 6 bands per lane. Each band was in-gel digested as previously described (Vanrobaeys et al., 2005). Peptides were first separated on an Agilent 1200 chromatographic system (Agilent, Santa Clara, CA, USA) and on-line measured on a LTQ-FT Ultra mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Raw LC-ESI-FT-MS/MS data were searched against the NCBInr database and a decoy database using Mascot Daemon. The characterization of the predominant members of the intestinal microbiota was performed using a novel approach for identifying taxon-specific peptides (Unipept). Our preliminary data point to a decrease in species richness and in less unique peptides originating from Firmicutes, Proteobacteria and the Bacteroidetes/Chlorobium group in the microbiota of the CF patient compared to the sibling. Similar findings are also reported in a recent microbiological study (Duytschaever et al, 2011) on fecal samples of CF patients. Furthermore, the detection of unique peptides of Viridiplantae indicates incomplete digestion of food in the intestinal tract. The unique peptides from Sus scrofa in the CF patient can be explained by the presence of pancreatin, a mixture of the pancreatic enzymes lipase, amylase and protease extracted from pig (Sus scrofa) pancreas glands. Duytschaever, G., G. Huys, et al. (2011). "Cross-sectional and longitudinal comparison of the predominant fecal microbiota composition between a group of pediatric patients with cystic fibrosis and their healthy siblings." Appl Environ Microbiol 77(22):8015-24.

Biofilms, groups of microorganisms that stick together on different surfaces within the human body and escape conventional antibiotic treatment, are an increasing challenge in the medical field. These biofilms are typically found on medical devices like implants. The most recent generation of implants with open porosity enables fast osseointegration, but also presents an increased risk of microbial biofilm-associated infection. Biofilm-associated infections are responsible for 15-25% of implant failure, and necessitate burdensome and costly revision surgery. The latter is estimated to represent a supplementary medical cost of €800m/year in Europe without taking into account the pain and distress of the patients, indicating that any significant reduction of this type of implant failure is highly recommended. Until now, biocidal implant coatings have been developed that are based on either the release of silver ions, which are toxic upon accumulation, or on conventional antibiotics that have poor activity against microorganisms in biofilms. Therefore, the COATIM consortium, consisting of 5 SMEs and 5 universities/research institutes, was established with support from the European Commission (Seventh Framework Program – FP7; www.coatim.eu). COATIM aims to develop the next generation of implant coatings containing novel potent proprietary antibiofilm molecules (ABMs) with inhibitory activity against microbial biofilms. In COATIM, these ABMs are grafted or deposited on small titanium implant substrates, as a model for dental and orthopaedic implants. Next, the ABM-coated implants are evaluated for in vitro and in vivo activity in resisting microbial infection without compromising osseointegration. Finally, the ABM-coating is applied on complex orthopaedic and dental implants, allowing the exploitation of the results by industry. In parallel, the antibiofilm mode of action of the ABMs is unraveled.

The induction of potent cellular and humoral immune responses will be key for the development of the next generation of vaccines against global killers such as HIV, tuberculosis, malaria as well as for the cancer vaccines in view of anti-tumor immunotherapy. Unfortunately, the currently licensed adjuvants for human use largely fail in this task. By contrast, formulating vaccine antigens in particulate, rather than in soluble form, was demonstrated to dramatically boost the induction of cellular immunity, especially the generation of cytotoxic T cells (CTLs) that can recognize and eliminate infected or malignant cells. The underlying reason for this is that particulates (50 nm – 5 µm) are regarded by immune cells, especially dendritic cells, as being potentially pathogenic. This leads to different routes of cellular uptake, processing and presentation to T cells, compared to soluble antigen. All of which enhance generating cellular immunity. However, despite their high potential microparticulate vaccine formulations did not yet reach the market. The reason for this can be attributed to the harsh conditions to which antigen is exposed during formulation, as well as the requirement of multiple batch operations with inherent difficulties for up scaling. Here we present a versatile and efficient method to produce microparticulate antigen formulations, based on spray drying of an aqueous phase containing oppositely charged biodegradable polyelectrolytes, antigen and a sacrificial component. This process yields a dry powder, which is especially attractive for long-term storage of vaccines. Importantly, upon reconstitution in water, these microparticles keep the antigen stable encapsulated due to the formation of a network of oppositely charged polyelectrolytes. Key in this approach is the use of the sacrificial component that is extracted from the microparticles after spray drying and allows tailoring the porosity of the microparticles. The rationale for the design of porous microparticles is that porous structures, exhibiting a high surface to volume ratio should, facilitate intracellular processing of antigens. We speculate that this first step is crucial for eliciting potent cellular and humoral immune responses. We demonstrated that the formulation process allows high production efficiency while not affecting the stability of the antigen. In vivo results in murine models demonstrate potent humoral and cellular immune responses that are higher than those induced by antigen formulated with alum, which is currently the only FDA approved adjuvant. Our approach creates a highly versatile generic formulation platform as it allows formulation of a large variety of antigens as well as co-formulation of additional immune-stimulating compounds.

Anaerobic digestion technology is an upcoming technology for treatment of industrial wastewater, often described as a more sustainable process for wastewater treatment compaired to activated sludge systems. An important fraction of the formed biogas dissolves however into the digestate, leading to a 10 to 30 % loss of methane, an effective greenhouse gas, to the atmosphere. We propose a novel approach to treat such effluents using a coculture of methane oxidizing communities and microalgae, further indicated as methalgae, which would allow microbial methane oxidation with minimal CO2 emissions. Coculturing a methane oxidizing community with microalgae in sequence batch reactors under continuous lightning yielded a factor of about 1.6 more biomass relative to the control without microalgae. Moreover, 55 % less external oxygen supply was needed to maintain the methane oxidation, as oxygen was produced in situ by the microalgae. An overall methane oxidation rate of 171 ± 27 mg CH4 L-1 liquid phase d-1 was accomplished in a semi-batch set-up, while the excess CO2 production was lower than 1 mg CO2 L-1 d-1. Both nitrate and ammonium were feasible nitrogen sources for the methalgae. These results show that a coculture of microalgae and methane oxidizing communities can be used to oxidize dissolved methane under O2-limiting conditions, which could lead to a novel treatment for dissolved methane in anaerobic effluents.

In modern society, we cannot imagine life without personal care and life style products containing chemicals. However, chemical consumption may be associated with health risks such as sensitization, affecting over 20% of the adult population. In this context, VITOSENS has been developed as an in vitro assay to identify chemical allergens by quantifying gene expression changes specifically induced by these compounds. The research described here aims at valorizing VITOSENS in the development of additives with an anti-allergic effect, in a mechanistically relevant and animal-sparing approach. Hematopoietic stem cells were isolated from human cord blood and differentiated towards dermal dendritic cells. Multiple doses of a chemical allergen were co-administered to the cells together with molecules with a possible anti-allergic activity. qPCR analyses of the co-administration experiments revealed a shift in the sensitizing dose-response as opposed to exposure to the allergen alone. Assessment of the efficacy of possible anti-allergic substances indicates the use of VITOSENS for compiling cosmetics that may attenuate allergic reactions by a well-defined mechanism. We aim to further valorize the application of this human in vitro assay for toxicity as well as efficacy purposes. *Nathalie Lambrechts is supported by a fellowship from the Agency for Innovation by Science and Technology. This project is being performed in collaboration with ‘Basic Science Research Europe & Asia, Estée Lauder Companies, Belgium’.

According to the currently adopted definition by FAO/WHO (Food and Agriculture Organization/ World Health Organization), probiotics are: “Live microorganisms which when administered in adequate amounts confer a health benefit on the host”. The effect of probiotics can only be guaranteed if the bacteria survive both the production process and the passage through the digestive system. Probiotics are most commonly used in fermented dairy products, but today probiotic microorganisms are also used in other foods such as dry fermented sausages. The use of probiotics in dry fermented sausages is an interesting application because of the health benefit. Adding probiotics to dry fermented sausages is technically possible. In fact, no high temperatures in means of cooking or baking are comprised in the production process. It was decided to produce dry fermented sausages to which in addition to a starter culture also probiotics have been added. The following probiotics were used: Lyofast BGP 93 (containing Lactobacillus casei LC-01) and Lactobacillus paracasei LTH 2579. The molecular method Polymerase Chain Reaction (PCR) with specific primers can be used for a rapid and accurate identification of bacteria in all stages of the dry fermented sausage production. In this research is colony PCR, whether or not combined with a multiplex setting, evaluated. Colony PCR does not use extracted DNA as template DNA but intact bacterial cells are provided as the template DNA source. Colony PCR is a time saver compared to PCR for which DNA extraction is required. The goal of this research is to translate DNA-identification of the Lactobacillus species present in the starter culture (Lactobacillus sakei) or the probiotic microorganisms (Lactobacillus casei or Lactobacillus paracasei LTH 2579) by colony PCR into a useful tool.

Lung cancer is the second most common cancer in men and the third in women. Moreover, lung cancer is the leading cause of cancer death. In 2008, approximately 951.000 men and 427.000 women died from lung cancer worldwide. However, screening for lung cancer remains a challenge. Consequently, lung cancer is often diagnosed at a metastatic stage. New screening tools that would allow the detection of lung cancer at an early stage could possibly lead to a reduction in the mortality rate of this cancer. In this perspective, recent studies indicate that the determination of the metabolic profile of a blood sample by means of nuclear magnetic resonance spectroscopy has the potential for early disease detection. For several diseases, changes in metabolite concentrations have been shown to correlate with the presence of a certain disease. Identifying which changes in the metabolite concentrations correlate with the presence of lung cancer could allow us on the long-term to easily detect the presence of lung cancer in a simple blood sample. We hypothesize that metabolic phenotype analyses by NMR spectroscopy permit the detection of lung cancer and that the metabolic changes, detected in the blood of lung cancer patients, correlate with metabolic alterations at the tumor site. These metabolic changes in the tumor are measured by means of 18-fluorodeoxyglucose-positron emission tomography-computed tomography. Previous studies have shown that the uptake of 18F-FDG is substantially increased in most types of cancer, reflecting an elevated glucose consumption by tumor cells. Venous blood samples of 29 patients with confirmed lung cancer (before treatment or surgery) and 29 control subjects were collected. All patients had fasted for at least 6 hours and their blood glucose levels were lower than 200 mg/dl. To determine the correlation between metabolic changes in the blood and metabolic alterations at the tumor site, the patients in this study underwent a whole-body CT- and a PET-scan one hour after administration of 18F-FDG. The degree of tumoral 18F-FDG uptake was measured by the standardized uptake value, normalized for lean body mass (SUL). Application of supervised orthogonal partial least squares-discriminant analysis on a well-defined and selected panel of metabolites, allowed us to discriminate lung cancer patients from control subjects with a sensitivity of 93% and a specificity of 96%. These results are very promising, however, further validation with additional samples of lung cancer patients and controls is necessary. Furthermore, regression analysis between the SUL and all the metabolites present in the metabolic fingerprint will be performed to determine the correlation between the metabolic changes at the tumor site and changes in the blood.

Over the past decades, there has been a remarkable progress in the field of biosensing technologies. Biosensors are analytical devices that use biological components for target recognition and subsequently convert this molecular event into a physically detectable signal. One type of biosensor became particularly appealing for studying biomolecular interactions, namely surface plasmon resonance (SPR) biosensor. This is one of the most advanced label free, real time detection technologies that provides information on quantification, but also on the kinetics of the binding reaction. Moreover, it can be used for studying a diverse set of interactions between proteins, lipids, nucleic acids, or low molecular weight molecules such as drugs. Because of these qualities, SPR-based biosensors are lately becoming a competitive tool for various applications in life science, food analysis, environmental monitoring, medical diagnostics and drug discovery. However, despite this, commercially available SPR biosensors cover only limited area of the biochemical monitoring market, because they have to compete with existing technologies on the basis of factors such as low cost, ease of use, robustness and sensitivity. Although lately there has been a constant technical improvement in generating and amplifying the SPR signal, most of the commercially available SPR platforms are still bulky instruments with expensive optical components. Our research group has developed an innovative fiber optic (FO) SPR platform that has a great potential for becoming applicable outside the specialized research environment due to several features. First, the FO-SPR sensor consists of a low-cost light source, a spectrophotometer, a bifurcated optical fiber, and sensor tips integrated on a robotic arm that allows their easy manipulation in a microplate format. As such, this platform has a capacity for multichannel performance needed for high-throughput screening applications. Furthermore, the detection limits of our biosensor, and thus its sensitivity, are improved by incorporating nanoparticles into bioassays, which is not feasible with traditional, microfluidics based SPR systems. We have also shown that the FO-SPR sensor can be applied in an automated setup for both protein- and DNA-based bioassays, making it thus useful for different applications. For example, the DNA amplification/melting processes can be monitored in real-time, which has not been accomplished before with commercial SPR setups as they do not allow for fast thermocycling. By exploiting this concept, we are able to screen for single nucleotide polymorphisms in real-time, which makes FO-SPR ideally suited for fast genetic screening of short fragments. Moreover, in various proof-of-concept studies the sensor was successfully applied for pathogen detection in food, paratuberculosis detection in cattle, and allergenicity screening in blood serum, demonstrating thus its great potential.

The most studied diseases using C. elegans as a model are the neurodegenerative diseases such as Alzheimer’s, Parkinson’s and Huntington’s disease. Mutations in leucine-rich repeat kinase 2 (LRRK2) represent the most frequent cause of autosomal dominant Parkinson’s disease (both familial as late –onset) identified to date. The G2019S mutation within the kinase domain of LRRK2, which is associated with increased kinase activity, occurs more frequently and is definitively linked to PD. C. elegans contains one sole homolog of this kinase, namely LRK-1. In this study, we make a quantitative comparison of the protein profiles of null mutants of LRK-1 (tm1898, km41) to try to identify downstream effects of the absence of this protein. To do this, we used a label based quantitative proteomics approach: we digested all proteins of the extracts with trypsin and labeled all resulting peptides with isobaric tandem mass tags (TMT). Three biological replicates of both WT and mutants (6-plex) were pooled and measured on the LTQ Velos Orbitrap system. A threshold for differential proteins was set to 2-fold change. Proteins were only taken into account when identified by at least 2 medium or high confident peptides (FDR = 1%) over the technical replicates (n = 5). Using an reversed phase LC gradient of only 60 minutes (1D-LC), we could already identify 5 upregulated proteins, of which one has a human homolog that can be linked with Parkinson’s disease. Preliminary data on the further optimization of the LC shows that we can extend this list.

Given that the average cost of bringing a new pharmaceutical product to market is USD$1.3 billion and the average time to develop such product is 10-15 years, there is no denying that the drug development process is in need of reform. One of the major shortcomings in the current drug development process (the “Process”) is the technology transfer model, which fails to bridge the gap between early stage and pre-clinical research. Known as the “Valley of Death”, technologies that may be the next “blockbuster success” could potentially languish because of lack of funding and expertise to bring the technology through to proof of concept. Collaborative partnerships between industry, public research organizations, and foundations appear to be an obvious model to explore since none of the individual players have all the necessary skills to discover and develop products. The proof of concept centre (PoCC) is a collaborative model whereby the respective expertise of academia (in basic research and discovery) and industry (in the translational process) are brought together to de-risk the Process. By creating an international network of affiliate and partner institutions, PoCCs support project development by offering access to state of the art facilities, equipment, and resources, as well as technical expertise and business acumen in order to demonstrate proof of concept to support commercialization. By leveraging existing resources, PoCCs de-risk the development process by proving value in early stage innovations, making it easier to attract partners for commercialization. This session aims to discuss how various PoCCs around the world function and whether the existing legal frameworks support this type of international collaboration. Specifically, an unintended consequence of such research collaborations is the potential for a violation of competition law due to anticompetitive conduct arising from the intersection of competition law and intellectual property law. For example, on one hand, intellectual property rights act as an incentive to attract investment in R&D and innovation, thereby driving economic growth and social welfare. On the other hand, benefits offered by intellectual property protection and the exercise of those rights may facilitate the dominance of the rights-holder and the potential abuse of its position, thereby negating the original justification for engaging in innovation. The intersection between competition law and intellectual property rights is examined in an ex-post manner and fails to consider the same intersection ex-ante when the relevant parties are negotiating the delicate balance between incentive and allocation of risk. After being urged to compete, innovate, and become a solution to the drug development problem, should industry players be penalized if they gain dominant market power through legitimate competition?

Cell culture process development and optimization is critical for the commercially viable production of biologics. It impacts downstream processing and manufacturing processes including scalability and costs. It is thus a pre-requisite to understand and control the growth conditions (e.g feed strategy, accumulation of toxic metabolites) as early as possible. As parameters having influence on cell culture are numerous and complex, process development scientists generally engage in “brute force” approaches using different culture media and DoE to select a feed strategy. However, they don’t know whether the selected conditions generate the optimal process for scaling and whether there is more room for increasing yields. To fill up this gap, our scientists have developed an innovative NMR-based technology which provides rapid and reliable profiling of culture media to support cell culture development and upstream processing. This multiplex approach gives detailed information on these complex matrices by providing access to the concentration of over 50 compounds present in a medium in one single analysis. Thus, we can analyze feed components (e.g. saccharides, amino-acids), contaminants and metabolites (e.g. lactate, TCA cycle metabolites). This robust method has been shown to outperform LC-based method on all counts (rapidity, structural resolution, linearity, etc). Furthermore, we managed to reach a throughput (matter of minutes) and level of details allowing the application of the method to chemically non-defined media and to different cellular systems such as mammalian cells, yeast and bacteria. Besides the support to cell culture development, this technology provides a substantial input in building knowledge on complex cell media by profiling raw materials to prevent and troubleshoot performance variation issues in Bioproduction. Last but not least, this technology completes current analytical tools used to understand and control Bioprocesses, and is fully in line with Quality by Design.

Human and animal fungal infections are on the rise in modern society, due to the widespread use of antibiotics and chemoterapics in the clinical practice. Fluconazole is the most widely used antifungal, but its action is hampered by the fact that its action is fungistatic and not fungicidal. In addition, and probably as a consequence of its fungistatic nature, fungal resistance to fluconazole is a dramatic reality in hospitals around the world. Combination therapy with drugs that could make fluconazole more effective is therefore highly sought after. However, substances discovered to act synergistically with fluconazole, such as Cyclosporin A and FK506, or Geldanamycin and 17-AAG, are of limited practical use due to the fact that, while containing fungal infections, they hamper the human host’s antifungal defense. We recently discovered that a licensed antibiotic, with no intrinsic antifungal activity, synergizes with fluconazole against Candida albicans, one of the major human fungal pathogens. Combination with the antibiotic converts fluconazole from fungistatic to fungicidal and prevents the onset of drug resistance in vitro. Synergism appears to be mediated by iron sequestration by the antibiotic, thus following an as yet unexplored route for containment of fungal growth. Preliminary results indicate that iron depletion may lead to the loss of mitochondrial membrane potential, and to death of the fungal cells as a consequence. Assessment of the possible in vivo antifungal potential of the fluconazole+antibiotic combination, an exciting perspective of our studies, is going to be tested soon in the mouse model. At the same time, we are devoting efforts to shed light on the fine mechanism of action of this drugs combination, using the powerful molecular tools of the yeast Saccharomyces cerevisiae. On the one hand, we are screening a complete array of yeast mutants carrying deletion mutations in all non-essential genes (~4800 mutants), to identify strains capable of growth in the presence of the drugs. On the other hand, we are complementing this analysis with the identification of strains that become resistant as a result of single genes’ overexpression. Preliminary results are revealing interesting, so far unidentified links between different aspects of fungal cell biology, which may possibly open new avenues for therapeutic intervention against fungal pathogens.

Objective: Atherosclerotic plaque rupture remains the leading cause of acute cardio- and cerebrovascular events. Current animal models flaw because they merely generate an unstable plaque phenotype without spontaneous rupture and clinical sequels. Therefore, we aimed to develop mouse model of spontaneous atherosclerotic plaque rupture with hard clinical endpoints. Findings: Elastin fragmentation of the vessel wall – as seen in humans during aging – led to increased vascular stiffness and vessel dilation in fat fed apolipoprotein E deficient mice (ApoE-/-) that have a mutation (C1039G+/-) in the fibrillin-1 (Fbn1) gene. In contrast to standard ApoE-/- mice, atherosclerotic plaques were larger, highly unstable and showed neovascularization and hemorrhage, important features of human vulnerable plaques. Furthermore, we observed acute plaque rupture, stroke, myocardial infarction and sudden death in ApoE-/- Fbn1C1039G+/- mice. Conclusion: These findings show that elastin fragmentation plays a key role in plaque destabilization and rupture. ApoE-/- Fbn1C1039G+/- mice are the first mouse model that develops spontaneous plaque rupture with hard clinical endpoints, offering the possibility to investigate novel plaque stabilizing therapies.

Introduction ChondroCelect® is a cellular therapy product presented as a sterile suspension of autologous chondrocytes in excipient medium. It is indicated for the treatment of cartilage lesions of the knee. As an approved medicinal product, strict production and regulatory requirements apply to the ChondroCelect® preparation process. Herein, we describe a series of studies conducted to support the introduction of a cryopreservation step in the routine manufacturing of ChondroCelect®, while meeting scientific, practical and regulatory challenges. Approach To define a suitable cryopreservation approach, a series of studies were first performed to evaluate the parameters critical during cryopreservation, including cryopreservation media composition, cell concentration at freezing, methods of freezing and thawing, and cell processing after cryopreservation. Next, critical process ranges were established with a view to the practical implementation of the process in a clean room and GMP compatible environment. In the last phase, a final study was performed to validate the complete process, and to demonstrate the comparability of the final cellular product prepared with a cryopreservation step versus without cryopreservation. Results and Discussion The cryopreservation studies outlined above demonstrated the feasibility of cryopreserving chondrocytes efficiently, and showed that a range of cryopreservation approaches could be used effectively. The final selected procedure yielded good post-thaw cell viability (average >90%) and minimal cell loss (average <10%). It was also observed that cells required a recovery period post-thaw, as direct processing of thawed cells to final formulation had an impact on final product characteristics. In the process validation and product comparability study, the complete process with cryopreservation was shown to be comparable to the process without cryopreservation based on extensive in vitro and in vivo characterization of the respective ChondroCelect® products. Conclusions Overall, the results of the described studies demonstrated that a suitable approach for a cryopreservation step in the commerical manufacturing process could be developed, which was both defined and practically feasible in routine manufacturing. The quality and functional capacity of the final products which underwent cryopreservation was equivalent to those produced by the manufacturing process without cryopreservation.

EC², The European Centre for Chirality (www.chiralitycentre.eu) is a starting spin off company from the University of Ghent and Antwerp , offering its high expertise service in analysing the structure of small molecules and recently also of biologicals. Knowledge of the absolute configuration of small molecules is proven to be of vital importance in Pharmaceutical industry, as many of the current blockbusters drugs are chiral and it is now required that the chiral structures be registered because molecules with the wrong absolute configuration may have reduced or even dentrimental biological activity (cfr. Softenon) EC² is today the expertise centre in Vibrational Circular Dichroïsm (VCD) and Raman Optical Activity (ROA), which are proven to be highly valued “information rich” techniques in chiral related structural determinations. When complexity of the molecules transcend the in-house possibilities of your current techniques, EC² is THE lab extension and THE outsourcing solution for all challenges related to chiral structure determinations. As the emergent area in drug development lies within biotech companies, the need to understand how structure and function interplay is also of vital importance and registration of higher order structures is highly advised by regulatory bodies such as the FDA. It is also recognized by the industry itself that having detailed higher order information will help build opportunities such as improved drug efficacy, quality, product differentiation and IP. ROA establishes higher order structure data of proteins and other biologically important compounds in water and allows, amongst others: - Conformational changes “ formulation studies” with various excipients - Elucidating the secondary and tertiary structure of peptides and biopharmaceuticals - Studying post-translational modification and degradation studies of biological. - Indentifying glycosylation and fucosylation patterns in peptides, antibodies, etc. - Establishing spectral markers that can be attributed to individual amino acid side chains - Comparison studies with so-called biosimilars or biobetters The European of Centre of Chirality want to be the first to bring the ROA know how to biopharmaceutical developments, helping to discover what improvements and growth it can generate for the Biotech industry.

In this abstract, we present digital lab-on-a-chip technology as an innovative diagnostic platform and a successful life science research tool. The concept of lab-on-a-chip (or micro total analysis system) technology involves the integration of all required lab operations on one single miniaturized device. Fluid manipulations in these miniaturized systems can occur in two different ways. Continuous microfluidics refers to a continuous fluid flow through microchannels whereas digital microfluidics refers to the transport of individual droplets on a chip, based on the ‘electrowetting-on-dielectric’ (EWOD) principle. By the application of an electric field over electrodes, covered by a dielectric, the contact angle between the droplets and the dielectric changes. As a result, individual droplets (in the nL-µL range) are generated, transported, mixed and merged on a matrix of microelectrodes. Hence, these droplets can be considered as individual microreactors, perfectly suited for low volume, high-throughput biotechnological applications. In first instance, this technology was evaluated for the implementation of homogeneous and heterogeneous bioassays. As a proof-of-principle study for the first assay type, an enzymatic assay for D-glucose was integrated and executed completely autonomously on-chip. Due to a software based approach, the assay-variability could be lowered to CV-values <2%. As an illustration, the chip was successfully used to measure glucose concentrations in blood serum samples. Since more complex applications, such as immunoassays and cell based assays, require the immobilization of the biomolecules (antibodies, cells,…), two innovative methodologies were elaborated to implement these heterogeneous bio-assays on chip. The first methodology deals with the manipulation and actuation of biofunctionalized magnetic particles, in combination with integrated magnetic actuation. This approach allows the extraction of the particles from the droplet and creates the possibility to execute very efficient washing steps in e.g., bead based immunoassays. Due to this washing step, the overall assay variability for an IgG immunoassay was reduced to CV-values below 3%. The second methodology involves the spatially controlled chip surface biofunctionalization with the creation of local hydrophilic zones in the top layer of the chip. A cytotoxicity study was executed, involving immobilized HeLa cells and droplets, containing different concentrations of doxorubicin. Similar results were obtained as in the reference study executed in microtiterplates, but with lower analysis volumes in the nanoliter range. The aforementioned studies indicate the potential of digital lab-on-a-chip technology for the accurate execution of a versatile range of bioassays. In addition, the technology offers a very flexible platform for different biotechnological applications in the low µL-range, such as PCR or other biocatalytic processes.

A common problem hindering biomedical progress today is finding or keeping up with the knowledge relevant to research in the shear amount of available literature and data, especially when that information is functionally only indirectly connected and distributed among various resources. BioGraph (http://biograph.be) is a data mining platform that allows easy extraction of available information relevant to research questions. Without requiring prior domain knowledge from its users, BioGraph offers prioritizations of research targets, supported by comprehensible, literature-endorsed functional hypotheses. The technology is based on the integration of a wide range of heterogeneous biomedical databases in a central knowledge base, covering annotated relations among biomedical concepts such as diseases, genes, compounds, pathways, ontology annotations, etc. Through the use of innovative data mining techniques, the service can extract non-obvious but relevant functional hypotheses linking potential research subjects with targets. By assessing the relevance of these hypotheses and because the method is fast, one potential application is to prioritize genes with respect to a disease of interest, for example in omics experiments. Our method is capable of retrospectively confirming and successfully identifying presumed research targets based on valid functional support. The high performance of the method is achieved primarily by the underlying data mining algorithm, which is based on avoiding unspecific concepts during the construction of the paths. The resulting hypotheses are, consequently, highly specific and relevant to the linked concepts. Because of the diversity of the integrated databases, the hypotheses comprise diverse types of biomedical relations specific to the linked concepts, with generic applications in various biomedical domains. The proposed methodology offers a range of significant improvements over leading platforms for the identification of susceptibility genes. (1) Highly ranked disease genes are grounded in valid, putative functional hypotheses, consisting of paths of refereed relations, verifiable by their references in the literature. (2) Since our method is based on the integration of a large set of heterogeneous knowledge sources, the generated hypotheses offer rich semantics compared to related data mining efforts. (3) In contrast with other methods, our approach is unsupervised and does not require pre-existing domain knowledge from the user, as such removing possible user biases and problems with prediction robustness in supervised alternatives. (4) Tests on published gene prioritization benchmarks show that our prioritization method considerably outperforms leading technologies built specifically for this task. (5) The presented methodology is generic, i.e., the framework offers a range applications in the prioritization of genes, drugs, compounds, annotations, etc. in the context of diseases, pathways, gene sets, etc.

Personalized medicine, in which a therapy is tailored to an individual's need, is a topic of vastly increasing societal, scientific and economic interest. In this context simple and cheap genotyping methods are important since knowledge on disease-related mutations is growing fast. The simplest techniques suited for genotyping are based on DNA hybridization, i.e. the formation of a stable double helix between two complementary DNA strands. These techniques are easy to miniaturize and parallelize, as done e.g. in microarrays, hence also suited for use in lab-on-chip implementations. In a collaboration between KULeuven and VITO a new methodology has been developed for the design and analysis of hybridization experiments [1]. It puts physico-chemical knowledge of DNA hybridization on top of a sound statistical approach and lifts the possibilities of this molecular technique to a much higher level of accuracy than obtained so far. This innovation opens new opportunities for genotyping and variation analysis with current and future hardware. Recently the practical relevance of the methodology was shown in a case study on HIV diagnostics in collaboration with Virco BVBA. Correct information on the HIV genome sequence is of crucial importance in anti-viral therapy, but due to the extreme genetic variability it poses a most challenging diagnosis problem. In this study the hybridization approach was applied, the sequence variations around HIV RT codon 184 were determined in real clinical samples and compared with sequencing data. The successful handling of HIV amplicons and advantages of the methodology for mixed samples will be presented.

Alpha-synuclein (a-SYN) is considered as a key player in Parkinson’s disease (PD), but the exact relationship between a-SYN aggregation and pathogenesis remains incompletely resolved. Testing of new therapeutic strategies targeting alpha-synucleinopathy is currently hampered by the lack of a robust and reproducible a-SYN-based animal model that reproduces the hallmark features of PD. We have optimized a viral vector-based rat model based on overexpression of a-SYN using adeno-associated viral (AAV) vectors. An AAV serotype 2/7 vector encoding A53T mutant a-SYN was stereotactically injected into rat substantia nigra and the effect was determined by histology, non-invasive imaging and behavioural analysis. Immunohistochemical staining for tyrosine hydroxylase showed a reproducible and progressive loss of approximately 80% of dopaminergic neurons at 4 weeks post-injection. Monitoring of dopamine transporter binding with micro-PET imaging revealed the highest rate of dopaminergic degeneration between day 7 and day 21 post-injection. This dopaminergic cell loss resulted in impaired spontaneous motor behaviour assessed by cylinder test, which could be reversed by administration of L-DOPA. From a therapeutic perspective, we have recently shown that FK506 binding protein 12 (FKBP12), an enzyme with peptidylprolyl isomerase activity, accelerates a-SYN aggregation and neurotoxicity in vitro and in cell culture (Gerard M, et al. J. Neurosci. 2010; Deleersnijder et al., J. Biol. Chem., 2011). This effect was counteracted by FK506, a specific FKBP inhibitor. To validate this finding in vivo, we have tested the effect of systemic administration of FK506 in the a-SYN AAV rat model. At the 4-week time point, a 2.5-fold higher survival of dopaminergic neurons was demonstrated in the rats treated with FK506 compared with placebo control. These data put FKBP12 forward as a potential new drug target for alpha-synucleinopathy. In conclusion, we have developed a robust rat model for alpha-synucleinopathy, which allows rapid evaluation of new therapeutic strategies or compounds for PD in vivo.

Deregulation of biotech plant products requires the submission of a scientific dossier to regulatory authorities around the world. Building this dossier requires input from multiple scientific disciplines : DNA and protein analysis, toxicology and allergenicity safety assessments as well as agronomic and environmental impact work. Experts from these disciplines need to work together often over several years to achieve a common goal of ensuring product suitability and safety. This means that scientists from very diverse disciplines, often located in different areas of the world need to work closely together. Bringing all these disciplines together at the appropriate time requires project management skills and increasingly strong evidence of laboratory and field quality assurance processes. The challenge of finding staff with scientific competencies and other skill sets such as quality assurance and project management is currently very high, and could be a future focus for education programmes.

The WCRF Report (2007) claims that consumption of red and processed meat increases the risk of developing colorectal cancer. Most epidemiological studies can not confirm this and there is also a lack of scientific research. Our research studies the toxicity of food on an in vitro cell culture model of colon epithelia. Therefore we optimized an in vitro gastrointestinal digestion method. Following digestions were used: pure digestive juices, fresh meat (beef, pig, chicken) and processed meat (beef, pig). Before digestion, processed meat was pickled with only a salt solution (2% or 6% NaCl) or with a solution of salt (2% or 6% NaCl) and 150ppm nitrite (NaNO2). The cytotoxicity was analyzed by incubating Caco-2 cells with samples in a 96-well plate (24h, 48h). Before analysis, samples were pretreated in different manners: no pretreatment; centrifugation (95’, 21382 g); filtration (22µm) or dialysis (24h, 10kDa). After incubation, cytotoxicity was quantitatively analyzed by MTS or through staining with neutral red. Immunofluorescence and flow cytometry with annexin-V and propidium iodide was used to distinguish between apoptosis and necrosis. The optimal pretreatment was centrifugation, best incubation time was 24h and cytotoxicity was preferably analyzed by MTS. The most favorable dilution for pure digestive juices was 243x, while meat showed unclear outcomes. The cytotoxicity by fresh meat was 5 to 30% lower than salted meat, which was 10 to 50% lower than meat prepared with salt and nitrite. Preparations with 6% salt caused the highest cytotoxicity. White meat caused more cytotoxicity than red meat. Keeping in mind similarities and contradictions in literature, further research is necessary to elucidate the influence on the cytotoxicity of Caco2 cells.

Although quantification of biomolecules such as DNA, RNA and proteins, based on ultraviolet-visible (UV-Vis) absorption spectroscopy is straightforward, the traditional approaches suffer from a lack of specificity, in particular in complex, unpurified samples. As a consequence, colorimetric or fluorescent-dye based alternative methods have been developed and commercialized. However, the strength of delivering specific quantification does come with a significant price in respect to convenience because it requires additional dispensing, mixing and incubation steps, and there is the need to run standard curves in parallel. Trinean has developed a breakthrough platform that offers the convenience of the direct UV-Vis spectroscopy on microliter volume samples (DropSense®96) in combination with state-of-the-art mathematical analysis power (cDrop™) to deliver adequate specificity even for applications that were deemed impossible before. A proprietary model-driven approach unravels the measured spectra into the relevant molecular components that contribute to the absorption, thereby not only reporting a more specific concentration of the molecule of interest but also estimating the content of the interfering or contaminants. In the presentation we will discuss the performance and limitations of this approach for a.o.: - Specific quantification of dsDNA in mixtures with RNA, nucleotides, proteins and extraction components (e.g. phenol) - RNA-quantification in diluents after RNA-extraction - Post-PCR amplicon quantification in the unpurified reaction mix - Total protein-quantification in crude cell lysates The impact of this new methodology on typical laboratory workflows will be illustrated based on real-life case studies. Finally we will touch upon Trinean’s vision on the future developments that will further expand the capabilities of the platform.

The optimal recruitment of patients for participation in clinical trials is of major importance for hospitals and study sponsors. When a new drug or treatment is evaluated in a clinical trial, all included patients have to meet pre-specified inclusion- and exclusion-criteria. The process of screening patients for inclusion often occurs manually by reviewing individual patient files. However, studies have shown that up to 60% of eligible patients may be missed using this approach, prolonging the time required to meet the specified target. Prior to the start of a trial, feasibility surveys are often conducted by the study sponsor to assess the number of patients that could be recruited at a potential site. Fast response times to these surveys are sometimes required. In a typical large hospital, several hundreds of clinical trials are initiated annually, signifying a need to be able to describe the inclusion- and exclusion-criteria of a study unambiguously through a set of reusable components. Therefore, an automated platform for the selection of eligible patients for clinical trials was developed that serves two partially overlapping objectives. Firstly, eligible patients are identified in semi real-time when entering the hospital. Secondly, feasibility surveys are answered more efficiently both in terms of workload and overall time duration. The system is designed such that it meets both these objectives and is composed of the following components: 1) Topic Maps representation of clinical trial inclusion and exclusion criteria; 2) an evaluation engine for formal evaluation of these criteria; 3) a connection with the hospital information system; 4) an integration layer with hospital databases; 5) a user interface for clinicians allowing follow up of evaluated patients. Topic Maps was chosen as the technology to describe the inclusion- and exclusion criteria of a clinical trial. Topic Maps (ISO/IEC 13250:2003) is a knowledge representation technology for describing data and information. It consists of a set of standards describing the Topic Maps Data Model, several representation formats, a constraint language and a query language (under development). Compared to similar semantic technologies, Topic Maps focuses on a more natural way to capture knowledge by providing varying levels of formality. Describing clinical trials as topic maps allowed the creation of easily reusable resources that serve as building blocks for future trials. Furthermore, semantic information and annotations stored using Topic Maps, allows for general reusability and large-scale data federation through standardized merging and querying capabilities. The pilot system was applied retrospectively for two different clinical trial settings. Experiments confirmed that overall workload is decreased, that the number of participants can be increased by means of an automated rule based system and that the response time regarding prospective questions can be lowered significantly.

The BioMiMedics consortium is a cross-border collaboration in the Maas-Rijn Euregion that aims at the development of new biodegradable polymers. These new polymers are envisioned with applications ranging from new wound dressing materials to 3D scaffolds for tissue engineering and controlled drug delivery. Expertise of research groups in Maastricht (UM), Aachen (RWTH, INB), Liège (ULg: CERM, CEIB) and Hasselt (UH: IMO-IMOMEC, BIOMED) is combined to successfully achieve this goal. BIOMED is together with UM involved in the in vitro studies of the newly synthesized material. This is an essential step before proceeding to further in vivo studies. Besides the obligatory cell viability assays, special attention is devoted to the cell-material interaction. At BIOMED, label-free microscopy is applied to investigate this interaction. In this way, potential deleterious effects of fluorescent probes can be excluded and live cell imaging can reliably be performed. The techniques involved are second harmonic imaging microscopy, two-photon excitation microscopy and fluorescence lifetime imaging microscopy. Current examples include the imaging of biological cells on meshes of flax fiber, cells in a collagen matrix and cells in contact with newly synthesized polymers.

Toll-like receptor (TLR)7 and TLR8 recognize single stranded viral RNA in the endosomes. Upon engagement, these receptors signals through MyD88 and the NF-kB/IRF/AP-1 pathway to intitiate immune responses. Various pro-inflammatory cytokines are secreted, most importantly IFN-α, switching infected and neighboring cells into an antiviral state. TLR7 and TLR8 can also be engaged by small chemical molecules, referred to as immunomodulators, which are known to induce the same arsenal of cytokines. For example, imiquimod (R837) has been shown to activate NF-κB through TLR7 while resiquimod (R848) has been shown to activate NF-κB through TLR7 and TLR8. In order to find new TLR7/8 immunomodulators, an assay was set-up that is amenable to high-throughput screening (HTS). This assay was used to screen 400K compound library. Briefly, HEK-293 cells were co-transfected with two plasmids in order to equip them with a more sensitive mutant of TLR7 (pCMV-hTLR7dLRR2) and a luciferase-based reporter harboring a NFkB-responsive promoter (pNFkB-TA-Luc) are a suitable tool to detect activation of the TLR7 pathway. The assay was adapted for a high throughput platform to screen a 400K compound library while reducing the cost significantly. Using this automated reporter assay, a 400K library of small molecules was tested at 1 concentration (10 µM, n=2), resulting in the selection of 12490 hits. The activity of 401 compounds could be confirmed using the same assay. Subsequently, these 401 confirmed hits were tested in a counterscreen to distinguish TLR7-dependent from TLR7–independent NFĸB-inducers. Only one compound demonstrated TLR7 specificity while all other compounds induced NF-ĸB through another pathway. This one hit (LEC 1.29 µM) was similar in activity to imiquimod (LEC 0.99 µM) and seems to interact with the same domain on TLR7 as other agonists. The activity of the hit was confirmed in an assay using a wild type hTLR7 plasmid (LEC 5.46 µM) and its ability to induce IFN-α in freshly isolated human peripheral blood mononuclear cells (PBMC) was demonstrated (LEC 1µM). Further evaluation on HEK-293 cells transfected with other TLRs (TLR1/2, TLR3, TLR4, TLR5, TLR6/2, TLR8 and TLR9) revealed additional activity on TLR8. In conclusion, an assay using HEK-293 cells, co-transfected with human TLR7 and a reporter construct was adapted for high throughput screening of a 400K compound library. One hit with dual TLR7/8 activity was identified demonstrating potency similar to imiquimod and capable of inducing IFN-α in human PBMC. The hit identified in this HTS will be the starting point of a parallel medicinal chemistry program.

GenomeComb: a package for the analysis of whole-genome data Peter De Rijk, Anthony Liekens, Maarten Van Den Bossche, Jurgen Del-Favero As prices for bulk sequencing are plummeting, whole genome sequencing is becoming more and more available to researchers, and the potential applications in genomic medicine are enormous. The computational analysis however remains a challenge. The huge lists of resulting variants contain a substantial number of genotyping errors. Validation of genome sequencing results can easily become the most time-consuming and expensive part of the analysis. In the course of a study comparing the genomes of a mono-zygotic twin discordant for schizophrenia, we developed GenomeComb to find the very small number of true differences caused by somatic mutations among a much larger number of differences caused by sequencing errors. The package can compare multiple whole genomes, both on the level of variants and sequenced regions in the genome. Variants can be extensively annotated with information influencing sequence quality, as well as functional information such as the effect on known or predicted genes or the presence in known regulatory or conserved elements. Powerful queries on variant and genome region data files allow adaptive filtering based on quality as well as functional annotation . In the twin study, the tool allowed a 290-fold reduction in error-rate by using strict quality-based filtering, bringing the validation step to a workable number. GenomeComb also supports validation by providing automation in primer design for Sanger or Sequenom validation directly from the variant files. This allowed detection of the first 2 validated SNVs between twins by whole genome comparison. The strict quality filtering did of course remove a considerable fraction of the genome (32%) from consideration. For other types of studies, filter combinations and settings can be used that remove less from the genome, at the expense of a smaller reduction in error-rate, combined with functional filters Being designed for handling variants and regions from whole genomes, the tool has of course no problem analysing exome or targeted region data. GenomeComb is available at genomecomb.sf.net.

Toll like receptor (TLR)7 or TLR8 agonists induce production of various cytokines in particular type I interferon (IFN). A 400K compound screening with HEK-293 cells transfected with a TLR7 plasmid and a NF-κB reporter construct, resulted in a hit displaying TLR7 and 8 activity. In order to confirm its dual TLR7/8 activity, the hit was profiled ex vivo alongside TLR7, TLR7/8 and TLR 8 reference compounds on human peripheral blood mononuclear cells (PBMC) or isolated plasmacytoid dendritic cells (pDC), monocytes, myeloid DC (mDC), B cells and Natural killer (NK) cells. The activation profiles were determined by flowcytometry and cytokines were measured by luminex after stimulation of the different cell types with the reference compounds and the hit for 24/48 hours. PBMC stimulated with TLR7 agonists like imiquimod mainly induce production of IFN-α. In addition, they induce maturation and proliferation of B cells. The same effect on B cells was observed for a TLR7/8 compound like resiquimod. For both compounds, the lowest effective concentration (LEC) resulting in IFN- α production is comparable with the LEC that induces the emergence of maturation markers. This was not the case for a selective TLR8 compound which could not induce any B cell maturation/proliferation. Stimulation of PBMC with TLR8 or TLR7/8 agonists like resiquimod resulted in the production of IFN- but more prominently of pro-inflammatory cytokines like IFN-γ, IL-10, IL-12p70 and IL1-β. Using intracellular flowcytometry, it was shown that NK cells were accountable for IFN-γ. The hit induced B cell maturation/proliferation and the LEC for IFN- α production and maturation were comparable. Its cytokine pattern corresponded to a TLR7/8 signature when used to stimulate PBMC. Further characterization of the hit was performed by using isolated pDC, monocytes and mDC. Selective TLR7 and dual TLR7/8 agonists showed maturation and secretion of IFN-α by pDC, whereas it was not the case for selective TLR8 compounds. TLR7/8 and 8 agonists induced maturation and secretion of pro-inflammatory cytokines by mDC and monocytes. Here as well, the hit behaves as TLR7/8 agonist, inducing maturation markers on pDC, mDC and monocytes while eliciting IFN-α and pro-inflammatory cytokines from pDC and mDC/monocytes, respectively. In conclusion, the identified TLR7/8 agonist behaves as expected in direct ex vivo assays on human PBMC and isolated subtypes. It could induce TLR7 specific maturation and proliferation of B cells and pDC similarly to imiquimod. The TLR8 activity of this compound was characterized by detecting pro-inflammatory cytokines. These assays can be used to further characterize and rank analogues of the hit in a more relevant setting.

In current wet-lab practice high-throughput experiments are becoming a standard to identify leads or genetic causes underlying complex phenotypes. High-throughput expression profiling techniques are used to find components of response pathways. Genetic screenings identify genetic hits of which manipulation alters the phenotype of the individual cell. With the availability of next-gen sequencing, complete genotyping in combination with expression analysis (eQTL) analysis allows finding genetic loci that underlie interesting phenotypic traits. Each of the experiments mentioned above eventually results in a list of candidate genes or loci that all relate to the same phenotype of interest. However, most computational analysis are restricted to a simple functional enrichment analysis to elucidate the potential pathways affected by a phenotype, but the interactions between the identified candidate genes or their role in the global molecular network of the cell remains unknown. Such insights can only be obtained by exploiting the large body of publicly available "omics" datasets in addition to own experimental data. In our work we developed an ensemble of tools that allow analyzing dedicated in-house experimental expression data and interrogating them with publicly available omics data and provided a proof of concept of our analysis flow on prokaryotic and eukaryotic model systems. To be able to maximally exploit the large body of publicly available expression data, we can generate expression compendia consisting of the expression value of all genes for a given organism over thousands of conditions. Our query tools allow identifying in such compendium the genes coexpressed with a given geneset of interest. Thus, they help identifying the larger context of pathways and conditions under which the genes of interest showed to be active (modules). Coclustering strategies or explanatory models help finding regulatory programs that can explain the expression profiles in these modules. Our integrative inference procedures can combine information from expression data to other sources of information such as regulatory motifs or ChIP-seq data. They also allow identifying regulatory programs consisting of regulators of which the expression profile is not necessarily related to that of their targets. To detect to what extent identified modules are conserved across species we developed a coclustering strategy that combines homology relations and coexpression to identify conserved modules. Lastly we developed a network based approach that allows overlaying coexpressed modules with a molecular interaction network of the organism of interest (work in progress).

Both biological sciences and clinical medicine are currently overwhelmed by vast amounts of complex data, thereby being increasingly dependent on information technology to handle and interpret these datasets. To address these growing computational needs and to enable new interdisciplinary cross-fertilization, a single research and expertise center was established by University Antwerp and the Antwerp University Hospital at the intersection of bioinformatics, medical informatics and translational medicine. The core research activity is the application of computational techniques for integration and analysis of clinical and 'omics data. Through its embedding in the Antwerp University Hospital, biomina is able to combine fundamental research with innovative prototyping of clinical applications, thereby occupying a unique position within Flanders. The center aims to facilitate research and to foster synergies among research groups by offering computational infrastructure and services within the University of Antwerp / Antwerp University Hospital and to external partners. Biomina operates in a dual model through direct participation in research projects and by offering consultancy to research groups for research prototyping, bioinformatics and medical informatics. Ongoing research activities are focused on two overlapping key areas: 1) bioinformatics: analysis, interpretation, data mining and integration of heterogeneous 'omics data (genome, transcriptome, proteome, metabolome), and algorithmic modeling of biological systems; 2) Medical informatics: applied machine learning, computational data integration, advanced data visualization, advanced prototyping. Current projects include clinical decision support models for rare disease diagnosis, automated patient recruitment for clinical trials, reconstruction of post-translational protein modification networks, pattern mining in large peptide mass spectrum libraries, development of new metabolomics data handling workflows, setup of next generation sequencing infrastructure (Galaxy, Spotfire, Taverna, …), setup of a text mining framework (UZA) and pattern mining for real-time clinical decision support systems (Intensive care, UZA).

A lot of effort is being undertaken in pharmaceutical and biomedical research to develop nano-sized systems that can perform challenging tasks such as delivering therapeutical drugs to the right location in the human body. In order to tailor the properties of these drug delivery systems, it is indispensable to study their mobility inside the biological environment where they have to operate, for example in the blood circulation. Single particle tracking (SPT) is the ideal technique for this purpose, it employs video microscopy to characterize the motion of individual fluorescent particles [1]. In SPT experiments, the sample is typically illuminated with a broad light beam in order to generate fluorescence. All particles outside the focus of the microscope objective lens thus also emit fluorescent light, which degrades the contrast of the visible particles in focus. The obvious solution to this problem is called sheet illumination, where only the particles in focus are illuminated, providing superior contrast. However, creating sheet illumination with conventional optics is technically challenging and requires bulky set-ups, only a few groups have managed so far [2]. We propose a new way of achieving sheet illumination by using a planar waveguide. Light exiting such a waveguide preserves its shape over a certain distance, thus producing sheet illumination. Besides reducing complexity, the major advantage of this approach is that it can be integrated on a chip that contains microfluidic channels for transport of the sample. This can allow for automated SPT measurements, requiring only small sample volumes. If material cost is low enough, the device can be made disposable, a feature of great added value in the pharmaceutical context where contamination should be avoided. The polymer SU-8 (MicroChem) was used for the fabrication of the planar waveguide. A microfluidic channel for transport of the sample was obtained inside the SU-8 waveguide by photolithography. Laser light was coupled into the waveguide, resulting in sheet illumination inside the channel. Fluorescent microspheres (Invitrogen) diffusing in water were sent through the channel, a fluorescence microscope equipped with an EMCCD camera was used for imaging. A significant contrast improvement over conventional illumination was seen. The sheet of light was indirectly observed by counting the number of visible microspheres at different heights in the channel. We demonstrated that sheet illumination can be obtained on chip using a planar waveguide with microfluidic channels inside. In a first version of the device, there was a substantial contrast improvement compared to conventional illumination. We foresee several biomedical and pharmaceutical applications, ranging from drug development to diagnostic tests. [1] Braeckmans et al., Nano Letters 2010, 10:4435-4442 [2] Ritter et al., Optics Express 2008, 16:7142-7152

Background: Combined analysis of cerebrospinal fluid (CSF) biomarkers ß-amyloid1-42 (Aß1-42), total tau (T-tau) and hyperphosphorylated tau (P-tau181P) reduces the uncertainty associated with clinical dementia diagnosis. These CSF biomarkers can be analyzed using three single-analyte tests or one multi-analyte test. Neuropathology is currently still recognized as gold standard in dementia diagnosis and, therefore, biomarker testing should be evaluated in comparison to neuropathology. Previously, the single-analyte tests were evaluated accordingly. Since absolute values depend on the method, the multi-analyte test should undergo the same evaluation. Methods: The present study evaluated the diagnostic accuracy of CSF biomarker concentrations obtained with a multi-analyte Luminex test (INNO-BIA AlzBio3) in comparison to neuropathology and compared the diagnostic performance of concentrations measured using single-analyte ELISA tests (INNOTEST) versus multi-analyte Luminex test. Results: Data from 51 Alzheimer’s disease (AD) and 15 non-AD patients (neuropathologically confirmed) and 95 controls were retained for statistical analysis. Mean biomarker concentrations differed between groups with both methods. Discrimination of dementia patients from controls using Aß1-42 and T-tau yielded a diagnostic accuracy of 87% and 90% for INNO-BIA and INNOTEST, respectively. Discriminating AD patients from controls, the diagnostic accuracy was 90 and 93% for INNO-BIA and INNOTEST, respectively. Optimal discrimination of AD and non-AD patients was achieved by combining Aß1-42 and P-tau181P (diagnostic accuracy=86% for INNO-BIA and INNOTEST). Conclusion: The clinical value of AD biomarkers in CSF is independent of the method by which the concentrations are determined and, based on these data, none of these methods is to be favoured over the other.

Nanomaterials present great opportunities for innovative products and technological solutions, but also raise health concerns. In contact with living organisms the physico-chemical characteristics of nanomaterials influence their fate and the resulting biological response. It is thus critical to advance our knowledge on nano-bio interactions to support technological growth without impacting on human health. The research project aims at understanding the interaction of manufactured nanomaterials with biomolecules from the microenvironment of human defense cells and its impact on biological responses, to enable the safe and efficacious design of nanomaterials for consumer or medical applications. A set of manufactured, functionalized nanoparticles that vary in one parameter (e.g. surface charge or size) will be used for in vitro exposure of human primary cells that are representative of the human defense system. The physico-chemical properties of these nanoparticles will be thouroughly characterized in their native state, as well as in the biological medium. High-tech molecular techniques, such as proteomics, will be used to investigate the nature and abundancy of biomolecules that attach to the nanoparticles from the biological matrix surrounding the cells. Modulation of the cellular response will be measured and connected to the dose, the physico-chemical properties and the biomolecule corona of the nanoparticles. Classical effect markers related to innate or adaptive immune responses will be measured in the first place. In addition, novel, more specific markers of immune activation or suppression induced by nanoparticle exposure will be identified using microarray technology. Insight into the role of molecular interactions in nanoparticles’ fate in a biological environment may promote development of high-value functional, complex materials and devices.

Background As genetic variation data is being generated at an unprecedented scale, assessment of functional consequences of the variants in a given patient or patient cohort is a challenging task, both from a computational as from a data management perspective. It is expected that in this new era of personalised genomics, a clinical sample may need to be re-annotated repeatedly as new annotation information on the genome becomes available and new insights on variant interpretation accumulate. While various initiatives emerge to collect the overwhelming amount of genomic variants currently generated, a central system to manage and store the annotation of genomic variants as well as determining the functional effects is still missing. Results Here we present our efforts to create a one stop solution to next generation sequencing data analysis. The ‘NeXT-generation Variant Annotation Tracker’ or ‘NXTVAT’ webtool is a frontend to a highly scalable cloud based analysis platform. Combining this web based frontend with an object oriented shardable database and fully distributed analysis pipelines allows us to scale this application to virtually any required size. A “plug in” style organisation of the variant annotation pipelines makes updating and extending variant annotation easy. Currently variant annotation and effect prediction is done using the Ensembl API (1), the polyphen2 algorithm (2) and genesplicer (3). The user friendly web interface enables submission of variants in various formats, including the emerging standard format VCF version 4.0 from the 1000 genomes consortium (4,5). Moreso a full next generation sequenicg data analysis can be triggered from data off any kind of next generation sequenicng data available over the internet. 1. Mclaren W, Pritchard B, Rios D, Chen Y, Flicek P, Cunningham F. Deriving the consequences of genomic variants with the Ensembl API and SNP Effect Predictor. Bioinformatics. 2010 Aug. 15;26(16):2069–2070. 2. Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, et al. A method and server for predicting damaging missense mutations. Nat Meth. 2010 Apr. 1;7(4):248–249. 3. Pertea M, Lin X, Salzberg SL. GeneSplicer: a new computational method for splice site prediction. Nucleic Acids Res. 2001 Mar. 1;29(5):1185–1190. 4. Danecek P, Auton A, Abecasis GR, al E. The Variant Call Format and VCFtools. …. 2011; 5. Durbin RM, Altshuler DL, Durbin RM, Abecasis GR, Bentley DR, Chakravarti A, et al. A map of human genome variation from population-scale sequencing. Nature. 2010 Oct. 28;467(7319):1061–1073.

Today’s healthcare is subject to an exponential growth in technology and knowledge, but also to a shrinkage in available budgets. In this way, medicine is in transformation towards a more cost-efficient care model. One of the hot items which fits well in this paradigm is translational medicine, and more precisely individualized medicine where the concept ‘biomarker’ has been key. Nowadays, we become aware that this concept is also moving to a ‘biomarker platform’ in order to gain more specificity concerning certain diseases and in casu cancer. Moreover, including more biomarkers results in a significant risk reduction concerning non-responsiveness in the medical treatment. Providing individualized therapy in case of a false-positive biomarker is life-threatening, because the patient is lacking the adequate conventional chemotherapy in time. In line with today’s diagnostic commercially available molecular platforms which are focusing on one biomarker per test (f.e. EGFR, KRAS, ALK-EML4, BRAF), the reimbursement is also provided per test and limited to one or maximal two biomarkers per patient. In this point of view, Pathlicon wants to use multi-biomarker molecular platforms per disease type. Next to this, Pathlicon is strongly believing that diseases, and more specifically oncologic diseases, will not be defined as histopathological entities in the near future, but as (epi-) genetic abbarative entities which are drugable in an ideal situation. In other words, new screening and sensitive technology must be developed to follow. Moreover, patient samples available for molecular screening are becoming smaller in view of the more frequent usage of minimal invasive techniques like for example needle punctions. Pathlicon is an unique player in this field. It functions as a real hybrid between histopathology and a molecular biotechnology laboratory, each with its own and specific expertises, knowledge and resources. Currently, Pathlicon is designing high-sensitive and high specific molecular tests which fit in the multimarker molecular detection platform concept for certain types of drugable cancer. By use of these types of platforms, sensitivity of the cancer to most of the current available individualized chemotherapies can be tested in ‘one glance’ in almost any routine molecular laboratory. In this way, Pathlicon is able to provide the ‘full monty’ in a diagnostic setting for cancer patients while one still has to face shortages of the conventional and current ongoing molecular ‘individualized’ medicine test settings. In parallel, by offering more- cost-effective tests, more patients will have access to personalized healthcare especially in case of scarce tissue material in view of minimal invasive techniques.

Micro-engineering technologies have the potential to offer interesting tools for in vitro cell culturing as they allow mimicking of physiologically relevant in vivo microenvironments. One research area taking advantage of these emerging technologies is the differentiation of stem cells. It is widely believed that diverse chemical and physical cues (e.g. mechanical strains, electrical fields, fluidic shear stress) significantly contribute to the differentiation process. There is thus an obvious need for platforms which allow investigation of the role of the diverse cues in the differentiation process separately, but also simultaneously (as is the case in vivo). Stretchable microelectrode arrays (SMEAs) are of particular interest as they allow simultaneous electrical and mechanical actuation and monitoring of cell cultures. We have developed a technology allowing fabrication of SMEAs composed of gold metallic conductors (and electrodes), patterned in meandering shapes and embedded in a soft, conformable biomedical grade elastomer such as polydimethylsiloxane (PDMS). Apart from their obvious use as a tool to assist stem cell research, SMEAs also offer opportunities in cardiovascular physiology or allow studying the mechanisms of pathologies such as traumatic brain injury. Microfluidics is another field of particular interest as it allows the close replication of in vivo microenvironments and the control of fluidic shear stresses by continuous perfusion, but also enables high-throughput screening. Within the frame of the IWT-SBO funded project HEPSTEM, we are currently developing 3-D PDMS-based microfluidic bioreactors which have an architecture closely resembling that of the liver in vivo as it is hypothesized that these microenvironments could improve differentiation of stem cells towards liver specific cells (e.g. hepatocytes). Ideally, this bioreactor could confine different cell populations to diverse microfluidic channels which are separated by porous membranes, allowing ‘natural’ (physical and chemical) interaction between the different cell populations. In conclusion, two multivariable platforms are being developed which offer new opportunities to help understand stem cell behavior and differentiation. Although these platforms have not been used in combination with cell cultures, evaluation is planned in the near future.

Introduction Multiple sclerosis (MS) is a progressive disease of the central nervous system. Over the last years, the role of imaging in both the diagnosis and follow-up of MS patients has increased, especially focussing on magnetic resonance imaging (MRI) (Barkhof 2009, Filippi 2010). MS imaging is quickly evolving from a qualitative tool to a quantitative tool or imaging biomarker. However, to obtain quantitative imaging biomarkers, the measurement variability, caused by differences in acquisition protocol, analysis protocol and human observers, needs to be reduced. One way to reduce the variability is to evolve from human to automated observers. Automated observers not only allow applying identical protocols independent of the site and operator. They also can significantly reduce the workload associated with meticulous manual measurements. This abstract focuses on an automated method for the measurement of total MS lesion load, i.e. the total volume of MS lesions present in a multi-modal brain scan. Total lesion load is a promising biomarker for MS diagnosis and follow-up, and a very active field of research (Lladó 2011). The proposed method is validated on simulated images with MS lesions (Collins 1998). Methods The EMS framework, as proposed by Van Leemput et al. (VanLeemput 2001) performs intensity-based segmentation of the white-matter, gray-matter and cerebro-spinal-fluid. MS lesions are simultaneously detected as outliers that are not sufficiently explained by the model, defined by the Mahalanobis threshold. We have evaluated two possible extensions to this method: atlas initialisation by nonrigid registration, and a multi-resolution approach, in which MS lesions detected on coarse scale are used to initialise the final fine segmentation. To evaluate the segmentations, we calculated the Dice Similarity Coefficient (DSC) compared to the ground truth segmentation. Overall, a DSC 0.80 is considered to express good agreement. Results For the optimal Mahalanobis threshold, the multi-resolution approach improves the DSC from 0.816 to 0.818. A non-rigid initialisation, contrary to our expectations, causes the DSC to slightly decrease. Visually comparing the ground-truth with the multi-resolution approach, a very good agreement between both segmentations is achieved. While small differences can be noticed, the multiresolution approach shows promising results that potentially could be applied in clinical practice. This will have to be evaluated by a more in-depth validation. Conclusions The proposed multi-resolution EMS based lesion segmentation method outperform the traditional approach on the evaluation data, with a DSC of almost 0.82. While small differences still exist between the automated segmentation and ground-truth, these are comparable to the inter-observer differences between multiple manual delineations. Moreover, the automated approach has the additional benefits that it can be consistently applied to multiple datasets.

Diabetes currently affects more than 330 million people. For patients with type 1 diabetes, and to a lesser extent for type 2 diabetes, the final curative therapy is replacement of lost beta-cells. Over the last decade, many labs have attempted to generate functional beta-cells from embryonic stem cells (ESCs), as donor pancreata or islets are very scarce. Unfortunately until now, it has remained impossible to reverse the hyperglycemia of diabetic mice immediately after transplantation of differentiated hESC. Therefore there is still a need to develop better and more reproducible differentiation methods to generate mature and well-defined beta-cells suitable for cell therapy. However, most differentiation protocols yield mixed progeny and in addition most insulin-expressing cells generated in vitro are poly-hormonal cells, while when transplanted in mice, these cells tend to preferentially differentiate into mature glucagon-producing cells. We hypothesized that it might be better to generate homogenous populations of endocrine precursors and allow them subsequently to mature towards beta-cells in vivo or ex vivo. The endocrine pancreas progenitor cell is characterized by the expression of NGN3, a gene known to be important in the development of all endocrine cell types of the pancreas. We have generated a hESC cell line carrying a drug selectable cassette as well as a GFP gene at the 3’ end of the NGN3 gene by using zinc finger nucleases. This now makes it possible to detect endocrine progenitors from day12 to day16 of differentiation, and allows us to isolate them by a combination of drug selection and FACS. Preliminary results showed that these cells express NGN3, PDX1, NEUROD1 and RFX6, but no NKX6.1. The prevalence of an NGN3-expressing cell in our differentiation culture is estimated at 1.5 in 1000 cells. We are now planning to further characterize the NGN3+ cell population for gene and protein expression profile and for functional differentiation towards beta-cells in vitro and/or in vivo.

Molecular in vivo imaging holds promise for refined monitoring of inflammation and cancer, not only providing information about the amount of inflammation, but also on the type of inflammation and on cells and/or receptors involved, thus offering perspectives for in-depth preclinical testing and mode-of-action analysis of new disease-modifying compounds as well as clinical applications. An important requisite hereby is however the availability of proper biomarkers and specific probes to target these. Myeloid cells such as macrophages and dendritic cells play a crucial role in a range of pathological and physiological processes. Due to the high degree of plasticity and versatility of these cells upon activation and/or differentiation, myeloid cells and the activation states they acquire in response to various triggers represent potential in vivo sensors for the status of the immune system during inflammation and cancer. Nanobodies are single-domain antigen-binding fragments derived from heavy-chain antibodies occurring naturally in camelids. Cloned as fragments of affinity-matured antibodies from immunized animals, they offer the high binding affinity and specificity of antibodies. As we have recently reviewed (1), these small-sized (15 kD) antigen-binding proteins are excellent probes for non-invasive bio-imaging of cancer cells, featuring high signal-to-noise ratios as early as 1 h after tracer injection. Recently, we have also reported the use of Nanobodies for imaging the biodistribution of myeloid cells in naive animals (2). To provide a proof-of-principle for tracking myeloid cells during inflammation and cancer using bio-imaging with Nanobodies, we have built on the finding that tumor-associated macrophages in various pre-clinical tumor models contain distinct subsets differing in angiogenic properties and intra-tumoral localization (normoxic/perivascular tumor areas versus hypoxic regions), with Macrophage Mannose Receptor (MMR) being highly expressed on strongly pro-angiogenic tumor-associated macrophages residing in hypoxic tumor areas (3). Using pinhole Single-photon emission computed tomography (SPECT)/Micro-CT, we have shown that Nanobodies raised against MMR specifically target the receptor in vivo. When using anti-MMR Nanobodies, zones of positive signals are detected in the subcutaneous tumor and co-localize with zones of hypoxia. Finally, although MMR is expressed on cells in different tissues and organs throughout the body, we have developed a method to obtain specific visualization of MMR-positive cells in tumors. Other ongoing research has validated Nanobodies against selected myeloid cell markers as imaging probes for tracking vulnerable plaques in atherosclerosis (4) or inflammatory foci in rheumatoid arthritis. (1) Vaneycken et al., Curr Opin Biotechnol, 22:877, 2011 (2) De Groeve et al., J Nucl Med, 51:782, 2010 (3) Movahedi et al., Cancer Res, 70:5728, 2010 (4) Broisat et al., Circ Res, 110:927, 2012.

The injection of air-bubbles into the bloodstream seems somewhat contradictory, but in the world of ultrasound imaging this is common practice. These tiny (1-10 µm) air-bubbles or microbubbles are used as contrast agents and more recently they have become widely studied as drug-delivery vehicles. The mechanism of action of these microbubbles is based on their specific interaction with the ultrasound waves. Ultrasound consists of different pressure cycles that induce compression and rarefaction in the microbubbles. Depending on the intensity of these pressure cycles this will translate in an oscillating or collapsing microbubble. The oscillations are responsible for the echogenic properties of the bubbles that make them suitable as an imaging contrast agent. However, microbubble collapse on the other hand is a real advantage for drug delivery. If a drug, that can be packaged in a nanoparticle, is attached to a collapsing bubble, it will be locally released. This allows local (e.g.: in tumors) image guided drug delivery because the microbubbles still have their properties as imaging agents. Local drug uptake is also facilitated with these systems by a process called “sonoporation”, i.e.: a transient permeabilization of the cell membrane. These concepts first emerged in the scientific world at the end of the 90s, and since then the number of publications grew exponentially. However, little effort was done in transforming this concept into a tangible device applicable in clinical practice. In this respect we took the challenge to transform the drug-loaded microbubbles into something which was more than just another promising concept. We’ve developed a material that can self-assemble just by shaking a simple vial containing the appropriate chemicals, drug-loaded nanoparticles and gas. This allows us to produce a sterile product with a convenient shelf-life. To further improve these materials we’ve attached an antibody that targets a cancer specific antigen to the surface of our constructs. This would allow an increase in target-site bubble concentration and it would keep the microbubbles in the vicinity of the tumor. Adding a targeting moiety to our system also allows us to combine specific cell type delivery with molecular imaging. The insights obtained in our research can enable these materials to overcome the still existing barriers associated with microbubble-enhanced drug-delivery and maybe these systems will, in a few decades time, become widely used in clinical centres worldwide.

Protein-protein interactions (PPI) play a key role in virtually all biological and disease related pathways. Although PPIs were thought to be undruggable, in recent years a considerable progress has been made in the design and discovery of small molecule PPI inhibitors (SMPPIIs). The binding of von Willebrand factor (VWF) with the glycoprotein (GP)Ibα receptor on blood platelets is a PPI of particular importance in maintaining haemostasis. Upon vascular injury, circulating VWF adheres to subendothelial collagen through its A3 domain and under high shear conditions conformational changes in the VWF occur to unmask the cryptic binding site for GPIbα within the VWF-A1 domain. This interaction allows initial tethering of blood platelets and the formation of a haemostatic plug that prevents excessive blood loss. Low VWF results in a bleeding disorder known as von Willebrand’s disease, whereas high VWF levels increase the risk for acute coronary syndromes and ischemic stroke. To date two inhibitors of the VWF-GPIbα interaction are under clinical development, however their size and/or protein nature hampers oral administration. We now aimed to develop a small molecule modulator of the VWF-GPIb interaction through a combinational approach involving in silico computational modeling based on crystal structures and de novo drug design including fragment based screening through saturation transfer difference NMR (STD-NMR). For the computational modeling approach putative small molecule binding pockets on the surface of VWF-A1 and GPIbα were determined using site finding algorithms and molecular dynamics. Next, through in silico high-throughput molecular docking of commercially available compound libraries into the predicted pockets, a number of compounds were selected and tested for their in vitro effect on VWF-GPIbα complex formation. This approach identified a compound that, by targeting GPIbα, stimulated the VWF-GPIbα binding and increased platelet adhesion in whole blood to collagen under arterial shear rate, but that in contrast inhibited ristocetin-induced platelet aggregation. In the second approach, binding of fragments from the Maybridge fragment library to the VWF-A1 and homologous VWF-A3 domain was determined using STD-NMR. A pharmacophore based analogues search for the two hits that bound uniquely to VWF-A1, identified fragments that caused broadening of the 1H NMR spectrum of VWF-A1, but not of GPIbα, indicating their specificity towards VWF-A1. Surprisingly, these fragments stimulated VWF-GPIbα binding. Computational docking, mutagenesis and structure activity relationship showed that these compounds likely bind to VWF-A1 in a defined pocket, from where they form an electrostatical bridge with GPIb and as such are stabilizing complex formation. We are now expanding the fragments in order to cause sterical hindrance for the GPIbα binding, and by this make the molecular glue-like fragments evolve into fully active SMPPIIs.

Hepatotoxicity remains one of the primary reasons for clinical adverse events, contributing significantly to both the withdrawal of marketed pharmaceuticals and the failure of drug candidates in clinical trials. Hepatotoxicity assays with better predictive value are therefore needed earlier in the drug discovery and development process. Although advances have recently been made with the development of hepatocyte-based toxicity screens, such as the high-content HepG2 assay, such in vitro assays detect only cell-autonomous toxicities. However, many in vivo hepatotoxities involve drug absorption, distribution, metabolism and excretion, or other mechanisms and tissues than cannot easily be modeled in vitro, as in the case of bile duct hypertrophy. For these reasons, in vivo models should be re-considered for early-stage hepatotoxicity screening. Zebrafish embryos and larvae offer numerous advantages for this purpose, including their small size (1-4 mm, compatible with 96-well plates), their rapid development (livers develop within 2-3 days post-fertilization), and their appreciable anatomical, physiological, and molecular similarity with humans. We have developed an in vivo, early-stage hepatotoxicity assay based on the expression analysis the liver-specific fatty acid-binding protein fabp10a. We have evaluated this assay with a training set of 10 drugs known to induce liver toxicity in humans and 5 non-hepatotoxic drugs. The known hepatotoxic compounds caused a reduction in liver size and/or a decrease in fabp10a expression in a dose-dependent manner, suggesting the loss of viable liver tissue as a result of drug treatment. These data have been confirmed by histopathology analysis. Based on these results, this zebrafish-based assay may be useful as an additional tool for early-stage hepatotoxicity screening in drug discovery.

GLPG0634 is an orally-available, selective inhibitor of Janus kinase 1 (JAK1). JAKs are critical components of signaling mechanisms utilized by a number of cytokines and growth factors, including those involved in rheumatoid arthritis (RA). Other nonselective JAK inhibitors have shown long-term efficacy in RA trials with an early onset of action, but doses and thereby efficacy are limited by side effects. By specifically targeting JAK1, treatment with GLPG0634 is predicted to result in a cleaner safety profile. A double-blind, placebo-controlled clinical Proof-of-Concept Phase IIA trial in patients with active RA, showing an insufficient response to the standard-of-care treatment with methotrexate (MTX), was conducted to evaluate the efficacy and safety of GLPG0634. Three groups of 12 patients with moderate to severe disease received either a 200 mg once-daily (q.d.) or a 100 mg twice-daily (b.i.d.) dose regimen of GLPG0634 or placebo, for a period of four weeks, while continuing to take their stable background therapy of MTX. Patient characteristics were similar for all three dose groups, and in each dose group, 11 out of 12 patients were females. GLPG0634 dosed as oral capsules was well exposed in RA patients, with essentially the same pharmacokinetics as previously observed in healthy volunteers. GLPG0634 was found well tolerated and safe with a high level of efficacy. Considering the small sample size, no relevant differences were observed among the 100 mg b.i.d. and the 200 mg q.d. dose regimens. GLPG0634 met the primary endpoint of significant improvement in ACR20 response rate. Overall ACR20 responses were observed in 83% of patients receiving GLPG0634 vs. 33% of patients receiving placebo (p<0.01). GLPG0634 showed impressive results in secondary efficacy endpoints such as the disease activity score DAS28 and in reductions of serum C-reactive protein (p<0.001 vs. placebo). All patients completed the trial and no treatment-emergent safety signals were reported. No severe adverse events were reported in patients receiving GLPG0634. In contrast to observations with non-selective JAK inhibitors, no anemia or increases in LDL/cholesterol were observed in this trial. These early clinical results, which are among the best currently reported for JAK inhibitors in RA, are the first to demonstrate that selective inhibition of JAK1 is efficacious and safe for treatment of inflammatory and autoimmune conditions such as RA. GLPG0634 is now being progressed to an extended dose-range finding study to further define the optimal doses for efficacy and safety for longer term studies in patients.

Introduction Most traits of industrial importance in yeast are polygenic, therefore complex and difficult to study. In our laboratory we have developed a new technology, called ‘pooled segregant sequence analysis'. In this study, the trait of interest was high ethanol tolerance, which is a trait of major industrial importance. High ethanol tolerance is important for enhancing the fermentation rate in the later stages of the fermentation and for complete attenuation of the sugar. In the bio-ethanol production industry, high ethanol tolerance is a prerequisite for obtaining high levels of ethanol, thereby reducing distillation costs, contamination levels and lowering liquid volumes in the factory. The aim of this study was to identify causative mutations underlying high ethanol tolerance. Methods VR1-5B, a segregant of the former Brazilian bioethanol production strain, VR1, was crossed with the BY lab strain for genetic mapping. From a total of 5974 segregants phenotyped for high ethanol tolerance, two pools were assembled. Pool 1 contained 136 segregants tolerant to at least 16% ethanol, while pool 2 contained 31 segregants from the first pool tolerant to at least 17% ethanol. The DNA of the pools and the parents was extracted and sequenced by Illumina sequencing. The nucleotide frequency of the SNPs in the pooled DNA was plotted against the chromosomal location. Reciprocal hemizygosity analysis (RHA) was used to identify the causative genes in the QTL. Results and discussion Three QTL were identified, located on chromosome V, X and XIV. Several additional minor QTL appeared in the second pool, which was selected under the more stringent condition of 17% ethanol tolerance. The three loci were validated by statistical analysis of the segregation of distinct SNPs in individual segregants from the pools. The causative genes in the two loci with the strongest linkage have been identified using RHA: URA3 on chr. V and MKT1, SWS2 and APJ1 on chr. XIV. Further analysis showed that all auxotrophic mutations compromise tolerance to high ethanol levels to some extent, presumably because of impaired uptake of the auxotrophic compound. Deletion of MKT1 and SWS2 reduced ethanol tolerance. Interestingly, the BY allele of SWS2 was superior to the VR1 allele and the superior character of the VR1 allele of APJ1 is likely due to a lower expression level. Deletion of APJ1 improved ethanol tolerance in both BY and VR1-5B. Conclusions Our results reveal that pooled segregant sequencing is a promising, powerful technology for QTL analysis of complex traits. We are also applying this technology for polygenic analysis of other commercially important traits like maximal ethanol accumulation, higher yield, thermotolerance and acetic acid tolerance. In each case, all QTL have been mapped and new causative genes identified. Superior alleles identified are now being transferred to industrial yeast strains to improve their performance under commercial conditions.

Early 1900’s, Kastner, a German brewing scientist, reported a strong foam and beer overflow after opening normal beer bottles without shaking. This unwanted phenomenon known as gushing was also described later in other CO2 supersaturated beverages (champagne, sparkling wine, cider, soda, mineral water) but seems to be most relevant in beer. Gushing represents severe economic losses and bad brand image. In the 1960-1970s, some causes of this phenomenon were proposed and a fungal contamination of raw material by molds such as Fusarium sp. was suggested but molecules responsible remained unknown. In the 1990s an amphiphilic peptide produced by filamentous fungi (Ascomycetes) was discovered and was called hydrophobin by Wessels et al. on the base of its amino-acids sequence. At the end of the 1990s hydrophobins were tackled as the major responsible molecules for primary gushing by a research group in Finland but the mechanisms remained unexplained. More practically, Casey (1996) submitted the hypothesis that some organic materials formed a solid pellicle around CO2 gas without explaining how and why. Still later research was intensified in Germany, Japan, Finland and other countries. Due to agricultural practice (grain humidity at harvest) and malting practice (germination temperature), the economic damages caused by gushing become more and more frequent (from one or two times pro decade earlier till three or four times pro decade now) . Finally, the missing piece of the puzzle was added by Deckers et al.(2010) making the link between gaseous CO2 (hydrophobic gas) and hydrophobins (hydrophobic patch) and by introducing the concept of “nanobombs”. Hydrophobins form a crystalline layer around CO2 nanobubbles resulting in the appearance of surpressurized nanobubbles in a closed bottle. At opening of the bottle these surpressurized nanobubbles will explode due to their contact with the atmospheric pressure. This explosion generates the energy required for the formation of nucleation sites allowing some dissolved CO2 to escape under an uncontrolled bubble form. Considering the physico-chemical properties of both hydrophobins and CO2 at atmospheric pressure allowed us to develop a new predictive method for primary gushing applicable to all the production steps, starting from barley till bottled beer (2011). The understanding of this mechanism allows us to bring a definitive solution to a worldwide beer sickness.

Plants synthesize an overwhelming variety of triterpene saponins with an enormous range of biological activities relevant for the pharmaceutical and chemical industry. Saponins are synthesized by multiple glycosylations of sapogenin building blocks, which in turn are produced by multiple modifications (e.g. hydroxylations) of basic sapogenin backbones such as β-amyrin, lupeol, and dammarenediol. These diverse backbones are generated by specific cyclizations of 2,3-oxidosqualene, which is also an intermediate in the synthesis of membrane sterols. The structural complexity of the sapo(ge)nins hampers chemical synthesis and harvesting from the natural plant sources is often inefficient. Therefore, a synthetic biology program in a convenient heterologous host, like Saccharomyces cerevisiae, might be a potent alternative for the cost-effective production of high value sapogenins or other triterpene building blocks. A pre-requisite for the development of a versatile heterologous sapogenin production system is the availability of a library of saponin biosynthesis plant genes. Using a gene discovery platform based on jasmonate elicitation, targeted metabolite profiling and genome-wide transcript profiling, we could isolate several putative novel saponin biosynthetic genes from the saponin-producing medicinal plants Panax ginseng, Glycyrrhiza glabra, Bupleurum falcatum, Maesa lanceolata, and the model legume Medicago truncatula. A collection of 140 candidate genes, including oxidosqualene cyclases, cytochrome P450s (CytP450), oxidases, reductases, glycosyltransferases, other transferases (acyl-, methyl-, malonyl-, etc), esterases and transcription factors, has been cloned. Exploiting this collection, we have engineered S. cerevisiae strains capable of accumulating high levels of β-amyrin and lupeol by modifying flux through the endogenous ergosterol pathway. Next, we super-transformed the β-amyrin producing yeast strain with CytP450 genes and identified 4 functionally active CytP450s. One of these CytP450s, encoded by a novel gene from B. falcatum, promotes accumulation of erythrodiol, a sapogenin possessing a wide range of biological activities including antiproliferative, apoptotic and vasorelaxant activities. Overexpression of CYP716A12, a characterized CytP450 from M. truncatula promoted accumulation of oleanolic acid, a sapogenin with potential hepatoprotective, anti-inflammatory, antioxidant, and anticancer activities. In nature, this array of genes is physically spread out in different plant species resulting in structurally and functionally diverse species-specific saponins. In the future, we will assemble these genes in new-to-nature combinations within our engineered S. cerevisiae strain. This combinatorial biochemistry approach may ultimately result in the synthesis of novel triterpene products with enhanced or novel biological activities.

The objective of our interdisciplinary research consortium is the development of anti-biofilm agents, with a special interest for agents that specifically target biofilms without affecting the bacterial growth. These specific anti-biofilm agents have the advantage of being less prone to the development of resistance than classical biocides or antibiotics. The anti-biofilm agents have potential to be used in a preventive fashion (e.g. as coatings on medical implants and devices) or to eradicate biofilms (possibly in combination with antibiotics or disinfectants). S. Typhimurium, P. aeruginosa and E. coli are the model organisms, against which the anti-biofilm agents are being developed. Our development strategy consists of (i) in vitro and in silico screenings of compound libraries for anti-biofilm agents, (ii) chemical synthesis of analogues of the identified hits, (iii) structure-activity relationship studies and structure-toxicity relationship studies, (iv) mode of action (MOA) studies of the lead compounds combining wet lab (transcriptomics, gene reporter fusion libraries and/or artificial evolution) and bioinformatics approaches and (v) further evaluation and development of the lead compounds (e.g. synergy studies, use of more applied test systems, effect on multispecies biofilms …). Results: *We synthesized a library of 25 3-alkyl-5-methylene-2(5H)-furanones and 3-alkyl-maleic anhydrides, by using known and newly developed chemistry, and tested their effect against Salmonella biofilms. The most active furanones have IC50 values around 1 µM. The furanones were found to interfere with the synthesis of flagella by Salmonella. Interestingly, pretreatment with furanones rendered Salmonella biofilms more susceptible to antibiotic treatment. *An in silico screening revealed a 2N-subsituted 2-aminoimidazoline as inhibitor of biofilm formation. Analogues (~250) with either a 2-aminoimidazoline or a 2-aminoimidazole scaffold were synthesized by using newly developed chemistry. (4)5-phenyl-2-amino-1H-imidazole was found to have a moderate inhibitory activity against Salmonella and Pseudomonas biofilm formation. However, substitution of the N1-position, the 2N-position and the 4(5)-phenyl ring enhanced the activity up to 100 times (IC50 ~1 µM). MOA studies revealed two possible cellular targets of the imidazoles, which are currently under further investigation. An experimental evolution experiment is being performed to identify additional targets. The 2-aminoimidazoles have been introduced in (i) a battery of challenge tests and (ii) the EU-project COATIM on anti-biofilm coatings for implants. *A high throughput screening of a library of >20,000 very diverse small-molecules (CD3) resulted in the identification of 140 anti-biofilm agents. Three compound families have been selected for which an early SAR study has been performed and MOA studies are currently being performed.

The ever increasing prices of petroleum have made the production of renewable fuels and materials from plants cost competitive. Second generation biofuels are produced from lignocellulosic energy crops like fast growing trees (e.g., Poplar and Eucalyptus) and grasses (e.g., Energy cane and Switchgrass). During this process, the cellulose of the plant cell wall is enzymatically converted to glucose that is subsequently used as substrate in fermentation reactions, for instance, to produce bio-ethanol. Second generation biofuels are much more attractive than their first generation counterparts (e.g., bio-ethanol from corn or sugar beet) for several reasons: 1) the substantial higher yields per acre, 2) the highly beneficial net energy balance, 3) the reduced need for fertilizer and water, 4) the relatively low production costs and (5) the reduced green-house gas emissions. In addition, (6) their feedstock does not compete directly with food production. After successful tests with pilot plants, several biofuel factories that will run on lignocellulosic biomass are under construction in the USA and expected to be fully operational in 2013. Both lignin and hemicellulose form important obstacles to use plant cell walls as a source for biofuels and biomaterials. Consequently, the enzyme cocktails to hydrolyze the complex hemicellulose polymers as well as the chemical and/or mechanical pretreatments of the biomass to remove the recalcitrant lignin form major costs in the production of fermentable sugars from lignocellulosic biomass. Lowering lignin concentrations in energy crops or changing the lignin and hemicellulose composition via genetic optimization, is expected to largely reduce pretreatment costs and thus the overall process cost and time. The Bio-energy lab is performing both fundamental and applied research on lignin and hemicellulose and their corresponding biosynthetic pathways. Basically, the lab focuses on engineering the lignin and hemicellulose polymers via genetic modifications. Therefore, candidate genes are selected using systems biology approaches (amongst others). These genes are subsequently screened for their role in lignification via reversed genetics combined with state-of-the-art metabolic profiling techniques (GC-MS and UPLC hyphenated to qTOF, IT or FTICR-MS) and semi-throughput lignin quantification and saccharification assays. Furthermore, orthologs of the positive hits are searched for in poplar and maize. Field trials with genetically modified poplar and maize (VIB and ILVO) are performed in the frame of UGent’s flagship project ‘Biotechnology for a Sustainable Economy’.

Introduction Second generation bioethanol production, which uses non-food energy crops, agricultural wastes and forest residues as substrates, is advancing to commercial production scale. One of the biggest hurdles for economically competitive ethanol production from lignocellulosic biomass remains the availability of microorganisms that efficiently convert all the sugars present in the lignocellulose hydrolysates into ethanol under industrial conditions. The yeast Saccharomyces cerevisiae remains the best organism for industrial ethanol production However, it is unable to metabolize pentose sugars, particularly xylose, which represent up to 35% of total sugars in second generation feedstocks. Thus, efficient utilization of xylose is required for cost effective and sustainable production of ethanol from lignocellulosic material. Objective Our general objective was to construct a robust yeast strain that efficiently converts both hexose and pentose sugars into ethanol and is tolerant to inhibitors present in lignocellulosic hydrolysates. Methods An industrial bio-ethanol production yeast strain (Ethanol Red) with both xylose (Clostridium phytofermentans Xylose Isomerase based) and arabinose metabolism gene cassettes integrated into the genome (H. Dietz, E. Boles, Goethe University of Frankfurt) was used. Despite the presence of all the genes that are required for xylose and arabinose utilization in the genome, this strain hardly ferments both xylose and arabinose to ethanol. We used a combination of random mutagenesis, genome shuffling and subsequent evolutionary engineering to improve the xylose fermentation performance. Results Using the above methods, we constructed a robust xylose utilizing yeast strain that is able to efficiently utilize the major pentose sugar, xylose. When characterized for fermentation performance in synthetic medium containing xylose and glucose, the strain completely consumed all 36g/L glucose and 38g/L xylose in less than 22 hours. The yield of ethanol from the mixture of sugars was 0.48gram/gram sugars; that is equivalent to 95.5% of the maximum theoretical ethanol yield. We also evaluated the performance of our strain in hydrolysates of three industrially relevant feedstocks: Arundo donax (giant reed) (Chemtex, Italy), spruce (SEKAB, Sweden) and a mixture of hay/wheat straw (KaHo Sint-Lieven, Ghent). In spite of the presence of various inhibitory compounds in these acid pre-treated raw materials, the strain exhibited a fast rate of fermentation, producing up to 88% of the maximum theoretical ethanol yield in less than 4 days. Conclusion Since ethanol yield from the biomass is a major factor in the productivity of the bioethanol plant, efficient xylose fermentation will significantly reduce the overall cost of ethanol production. Our new yeast strain is therefore very promising for cost effective industrial bio-ethanol production from second generation feedstocks.

Since the foundation of BASF Plant Science (BPS) in 1998, constant innovation has been our key to success. With currently more than 750 employees worldwide, BPS has developed key leading technology platforms for plant biotechnology and is committed to continue to strive for innovation in the future. Under the motto of ‘INNOVATION YIELDS RESULTS’, we want to emphasize to the world our innovativeness. INNOVATION: As the technology specialists for gene discovery and integration, we excel in developing innovations that lead to superior crop performance. YIELDS: With our innovative spirit and our unique technology platform, we work closely with partners in all relevant crops to develop the complex traits they need for superior yield. RESULTS: The result is maximized success for our partners in an ‘environment’ of stewardship, respect and trust. Today we want to offer a glimpse of how innovation is implemented at BASF Plant Science, to lead to great results. An overview of company initiatives to foster innovation within our company will be presented together with an overview of our technology partners and examples from our product pipeline.

Post-translational modifications of proteins constitute a crucial level of molecular regulation in living systems. The most extensively studied post-translational modification, phosphorylation, is carried out by a large and diverse group of enzymes called kinases. They play essential regulatory roles in diverse processes such as growth and development, and lie at the core of many human diseases. Although the fact that protein phosphorylation performs similar and equally important functions in plants makes it an interesting target for biotechnology, compared to other model organisms our understanding of system-wide phosphorylation networks in higher plants is still limited. Whereas we do know that the kinases of the model plant Arabidopsis thaliana are more diverse than human kinases, we do not have sufficient experimentally confirmed phosphorylation data to generate a system-wide network, or to build computational plant kinase specific substrate prediction models. In the work presented here we employ insights in phosphorylation in other, well-studied organisms, functional information and the limited plant phosphorylation data available to computationally generate a large-scale candidate kinase-substrate network for the target organism Arabidopsis thaliana. The core of this integrative bioinformatics method is a computational model based on a sequence labeling approach called “Conditional Random Fields”. The model is learnt from the protein structures surrounding known phosphorylation sites in several reference organisms. Extensive internal and external validation showed that the method outperforms previous efforts in the prediction of novel phosphorylation sites. Kinase-specific phosphorylation prediction models constructed from well-studied (”reference”) organisms are then transferred to the ”target” organism through orthology mapping, and merged with global plant phosphorylation prediction models. The resulting predictions are significantly enriched with confirmed phosphorylation sites and confirmed kinase-substrate interactions, respectively. Gene ontology analysis demonstrates significant functional coherence between predicted kinases and their putative substrates. This functional coherence is further used to filter the network to obtain a small high-confidence predicted network that is interpreted with regards to literature evidence. Statistical, functional and literature-based analyses of the predicted network demonstrate that the prediction and orthology mapping method yields a biologically relevant network. The resulting predicted network is a valuable resource for further biological and/or computational analyses and will further expand our current understanding of plant complexes and pathways. In addition, the methods developed can be adapted to reconstruct phosphorylation networks in other organisms of interest. Finally, they provide a basis for the reconstruction of other post-translational modification networks.

Introduction: Cancer is one of the major causes of death in the industrialized world. As it is believed that conventional therapies such as surgery and chemotherapy have reached the end of their product life cycle, immunotherapy is regarded as the most promising new direction. This strategy is based on the ability of the immune system to discriminate between healthy cells and tumor cells based on the expression of tumor associated antigens (TAAs). One strategy to educate T cells to recognize and kill cancer cells is the use of dendritic cells (DCs), the professional antigen-presenting cells of our immune system. The success of DC-based vaccines is dependent on several parameters of which the DC maturation status and the route of vaccine delivery play an important role. To address the maturation status, we developed a strategy to deliver not only TAAs but also molecules necessary to enhance the T cell stimulatory potential of DCs and this by activation of the DCs with mRNA encoding the immune modulating molecules CD40 Ligand (CD40L) and a constitutive active Toll-like receptor 4 (caTLR4) together with the co-stimulatory molecule CD70. As the lymph node is a critical site for immune activation and harbours a lot of DCs, direct intranodal vaccination is regarded as an important route of vaccination. Aim: In the current study, we evaluated the use of mRNA as an off-the-shelf vaccine for the in vivo modification of DCs by intranodal delivery of different mRNA molecules encoding tumor antigens and TriMix. Results: Using fluorescence microscopy, as well as in vivo bioluminescence imaging on CD11c depleted mice, we demonstrated selective uptake and translation of the mRNA in vivo by lymph node resident CD11c+ DCs. We further demonstrated that this process was hampered by the co-delivery of classical maturation stimuli such as lipopolysaccharide but not by TriMix mRNA. Importantly, we demonstrated by flow cytometry that the intranodal delivery of TriMix mRNA results in DCs with a mature phenotype. Moreover, it was demonstrated in an immune array that mRNA encoding TriMix induced a T cell attracting and stimulatory environment. Importantly, we could show an enhanced induction of antigen-specific CD4+ and CD8+ T cells against the model antigen ovalbumin. We further demonstrated enhanced induction of cytotoxic T lymphocytes (CTLs) against several tumor antigens, including Trp2, WT1 and tyrosinase. Encouraged by these results we compared the immunization with antigen and TriMix mRNA to ex vivo modified DCs for the induction of CTLs and in therapy. We demonstrated that the mRNA vaccine is as efficient as the DC-vaccine, resulting in a reduced tumor growth hence prolonged survival of tumor-bearing mice. Conclusion: Intranodal administration of TAA mRNA together with mRNA encoding immune modulatory molecules is a very promising vaccination strategy.

Misfolding of the naturally unfolded protein tau causes its aggregation into oligomers and fibrils of as yet unknown structure. Formation of larger aggregates, i.e. neurofibrillary tangles in neuronal somata and neuropil threads in neuronal processes, constitute the typical post-mortem pathological signature in primary tauopathies. In Alzheimer's disease (AD), the same tau pathology is co-diagnostic with amyloid pathology, but their respective roles and relative contributions remain debated and open for analysis. In addition, the actual synaptotoxic and neurotoxic forms of amyloid and tau remain to be identified and characterized molecularly. Effective therapy for AD based on classical pharmacology is still lacking, emphasizing the need for additional treatment options, including immunotherapy. As opposed to vaccination against amyloid in experimental studies, tau-based immunotherapy must yet demonstrate its potential as an interesting strategy to alleviate the impact of tauopathy. Despite the many hurdles from the medical point of view, pre-clinical studies are needed and will also contribute to unravel the role of tau in the patho-physiology of primary and secondary tauopathies, including AD. We designed, generated and explored experimentally in vivo, phosphopeptide-liposomes and tested them as active vaccines in Tau.P301L mice, our validated pre-clinical model for tauopathy. Liposome-based vaccines developed as described for amyloid (Muhs et al,2007) were configured to display synthetic phospho-peptides that mimic different important “patho-topes” of human tau. The vaccines elicited robust and specific antisera responses in wild-type and Tau.P301L mice. The specificity of the polyclonal antisera for pathological tau was visualized by TAUPIR on brain sections from Tau.P301LxGSK3β mice with proven forebrain tauopathy (Terwel et al,2008). The abundant labeling of neurofibrillary tangles in hippocampus and cortex was corroborated biochemically by western blotting on mouse brain extracts and on isolated hP-Tau preparations (Vandebroek et al,2005). Specific ELISA demonstrated high and stable antibody titers against synthetic phospho-tau peptides as opposed to weak or negative reaction with the same non-phosphorylated peptides. Long-term regimes of vaccination demonstrated beneficial effects of the novel vaccines: reduction of tau-related pathological parameters and improved clinical condition and survival of Tau.P301L mice, without obvious side-effects. In addition to the promising active vaccination strategy, some of the vaccinated mice were used to generate monoclonal antibodies by classic methods from splenocytes. By suitable selection of monoclonal antibodies, these can be developed for the diagnosis of biomarkers and for passive vaccination, while other options are being investigated. Muhs A, et al. (2007) PNAS 104: 9810-9815 Terwel D, et al. (2008) Am J Pathol 172: 786-798 Vandebroek T, et al. (2005) Biochemistry 44: 11466-75

Nanobodies are therapeutic proteins based on the smallest functional fragments of naturally occurring heavy chain only antibodies. Due to their unique nature they can be formatted to achieve specific features such as high potency while retaining characteristic physico-chemical properties allowing, in the case of ALX-0171, direct delivery to the airways by nebulisation. We developed Respiratory Synyctial Virus (RSV) neutralising Nanobodies directed against different antigenic sites. Upon formatting into trivalent Nanobodies, consisting of either three identical building blocks (one epitope) or two different building blocks (different epitopes), in vitro potency against both RSV serotypes increased 2000-7000 fold. Finally ALX-0171, consisting of three identical building blocks, was selected as lead Nanobody for pre-clinical development. The antiviral activity of ALX-0171 was compared with that of palivizumab at 40 microgram/mL in a plaque reduction assay with 61 recently isolated RSV clinical strains. ALX-0171 reduced viral replication below the detection limit more efficiently than palivizumab (84% of strains versus 20%). ALX-0171 formulated at 50mg/ml was readily nebulised using a vibrating mesh nebuliser without affecting potency and with only minor product quality changes. Delivered as a nebulised drug, ALX-0171 was shown to be effective in vivo in a therapeutic RSV cotton rat model. A two week repeated dose toxicity study by nebulisation was conducted in rats, achieving maximal feasible dose levels without dose limiting toxicity. Immunogenicity, as measured in plasma, appeared not to be enhanced compared to intravenous administration. A Phase I clinical trial is currently ongoing to assess tolerability and safety in man.

Background. Therapeutic antibodies (Abs) nowadays have a prominent position in medicine, being the strongest performing molecule type with an annual growth rate outpacing small molecules, therapeutic proteins and vaccines. Special effort is being put in the development of fully human Abs because of their superior biological characteristics. In this field, phage display and human immunoglobulin transgenic mice represent the current state-of-the-art technologies. The availability of immunodeficient mice with functional human immune cells (also termed humanized mice) offers a powerful and innovative alternative for the generation of fully human Abs. Aim. In human hematopoietic stem cell (HSC) transferred immunodeficient mice, we aim to induce de novo human antibody responses against virtually any human or non-human antigen. Methodology. CD34+ HSC are isolated from cord blood with informed consent of donors and subsequently transferred to newborn NOD.SCID.IL2RγcKO (NSG) animals. Mice with stable human/mouse chimerism were subsequently immunized with commercially available human vaccines as model antigens. Results. Intrahepatic transfer of CD34+ HSC (1.73 ± 0.16 x 105) into newborn NSG mice (N=132) leads to rapid, stable and reproducible human/mouse chimerism both in blood and lymphoid tissues. Six weeks after HSC transfer, 49.1 ± 25.1 % of blood leukocytes are of human origin (hCD45+). Among these, the majority of cells (79.7 ± 9.54 %) belong to the B cell lineage. CD4+ helper T cells represent 15.0 ± 6.84 % of circulating human leukocytes. In the spleens of HSC humanized mice, follicular-like structures are observed containing human B cells but virtually all cells are arrested in the CD27-IgM+IgD-/+ T1/T2 transitional stage. Human IgM (range 5.31 – 36.4 µg/ml) and IgG (range 0.98 – 13.9 µg/ml) immunoglobulins were detectable in serum of respectively 79.1 % and 24.2 % HSC transferred NSG mice. Low affinity antigen-specific human antibody responses (IgM isotype) were detected in 3/51 mice that were immunized with a commercially available human vaccine containing tetanus, diphtheria and polio antigens. Conclusion. Stem cell humanized mice with functional human immune cells provide an interesting research platform allowing de novo generation of human antibody responses. Nevertheless, to exploit this model for therapeutic human antibody generation, several limitations of the currently available models have to be overcome.

Polytopic membrane proteins such as GPCRs are dynamic proteins that exist in an ensemble of functionally distinct conformational states. Crystallogenesis typically traps the most stable low energy states, making it challenging to obtain agonist bound active-state structures of GPCRs. Stabilization of an active conformation of a GPCR can be achieved in different ways. The most physiologic approach is to use a native signaling partner such as a G protein. An alterative to using a G protein is to identify another binding protein that can stabilize the same conformational state. Antibodies evolved to bind to a diverse array of protein structures with high affinity and specificity. Last year, we generated Nanobodies that selectively recognize an active state of the human beta2 adrenergic receptor (b2AR). Such Nanobodies that faithfully mimic the effects of G protein binding were used to obtain diffraction quality crystals and to solve the very first structure of an active agonist-bound state of the human beta2 adrenergic receptor(2). More interesting, we also identified nanobodies that stabilize the b2AR-Gs complex. One of these nanobodies was used to obtain the high-resolution crystal structure of this complex, providing the first view of transmembrane signaling by a GPCR (3). Our work illustrates the power of the Nanobody platform for GPCR research. Nanobodies are the small (15 kDa) and stable single domain fragments harboring the full antigen-binding capacity of the original heavy chain only antibodies that naturally occur in Camelids. Because of their unique three-dimensional structure, nanobodies have access to cavities or clefts on the surface of proteins. The nanobody platform has the competitive advantage to other recombinant scaffold libraries in that large numbers (109) of fragments harboring the full antigen-binding capacity of genuine in vivo matured antibodies can be screened for high affinity binders in a couple of days, allowing one to fully exploit the humoral response of large mammals against native antigens. For all information see: http://steyaertlab.structuralbiology.be 1. Steyaert, J & Kobilka, B.K. (2011) Nanobody stabilization of G protein coupled receptor conformational states. Current Opinion Structural Biology 22, 567-572. 2. Rasmussen, S.G.F., et al. (2011) Structure of a nanobody-stabilized active state of the β2 adrenoceptor. Nature 363, 446–448. 3. Rasmussen, el al. (2011) Crystal Structure of the 2 Adrenergic Receptor-Gs protein complex. Nature 477, 549–555

Long non-coding RNAs (lncRNAs) represent an important class of regulatory transcribed elements. Research involving these lncRNAs is rapidly emerging in the field of cancer research, because of their implications in important cellular processes, through new modes of action. lncRNAs most likely constitute a novel class of powerful biomarkers and expression profiling of this underexplored class of RNAs may lead to disease specific gene signatures. The lack of a high-throughput platform to reliably quantify lincRNAs has hampered their study so far. On top of that, lincRNA are generally low abundant, which is why there was a genuine need for a platform with ultimate sensitivity superseding microarray analysis. To accommodate this need, Biogazelle has developed a qPCR-based platform that allows high-throughput screening of 1718 lncRNAs per sample. Assays have been designed and successfully validated for 1666 human intergenic lncRNAs (lincRNAs) and 52 human lncRNAs. All assays underwent thorough in silico quality control, followed by extensive empirical validation according to international qPCR standards. All qPCR reactions are performed in triplicate using Wafergen’s SmartCycler platform. The power of the newly developed platform has been successfully demonstrated in several proof-of-concept studies. One of these studies involved lincRNA expression profiling in a TP53 gene perturbation model using the anti-cancer nutlin-3 compound in neuroblastoma cells. This study revealed numerous lncRNAs under the control of TP53. Recently, the newly developed platform was also used in a large regulatory network discovery study using 3-way integration of high-dimensional mRNA, miRNA and lncRNA expression data from the entire NCI60 cancer cell line panel. The data offer numerous opportunities towards a better understanding of complex regulatory networks in cancer. In conclusion, Biogazelle has developed a high-throughput, low-volume qPCR platform for the quantitative detection of lincRNAs. Biogazelle now offers lincRNA profiling in service, which offers a unique way to investigate the expression patterns of lncRNAs in health and disease.

Multiplicom is a fully integrated molecular diagnostics company that enables clinical laboratories to perform cost-effective testing for personalized medicine. Multiplicom designs, develops, manufactures and commercializes leading edge and easy-to-use assays, based on the latest molecular diagnostic technologies. These assays, termed MASTR (Multiplex Amplification of Specific Targets for Resequencing) assays, are highly multiplexed PCR assays developed using Multiplicom’s technology platform in combination with a proprietary MultiplexerTM algorithm allowing straightforward primer design for multiplex PCR amplification. Multiplicom’s MASTR assays are compatible with all commercially available massive parallel sequencing (MPS) platforms and allow simultaneous detection of single nucleotide variants (SNVs), insertion-deletion variants (indels) and copy number variants (CNVs). While current PCR reactions typically amplify one or a few target DNA sequences, Multiplicom’s approach allows amplification of up to 100 target sequences in one standard PCR reaction. The Multiplicom assay design pipeline allows robust, rapid and cost effective development of novel MASTR assays for diagnostic massive parallel sequencing. Resulting MASTR assays substantially reduce processing cost and front-end workload. The latter is demonstrated by the fact that clinical diagnostic labs using Multiplicom’s MASTR assays, were able to reduce their turnaround time from ~ 6 months to ~ 6 weeks. This is due to the fact that (i) only 5 robust PCR reactions are required to amplify all coding exons of BRCA1/2 compared to ~ 100 reactions in a simplex PCR approach and (ii) multiple DNA samples can be processed simultaneously (16 DNA samples can be processed per 96-well plate). Additionally, processing cost is reduced by a factor 2-5, compared to Sanger based sequencing resulting from the combination of highly specific amplification (> 96% of resulting MPS reads map back to BRCA1/2 reference sequence) and a very narrow spread factor (i.e. average number of all obtained MPS reads divided by the number of reads of the lowermost amplicon) of 2,5. Both parameters enable efficient use of MPS platform resources and hence enable significant cost reduction over other MPS based resequencing technologies. Furthermore, several independent studies performed using the BRCA MASTR assay by diagnostic labs showed an accuracy of > 99,999% compared to Sanger sequencing. Multiplicom has currently four MASTR assays on the market: for breast cancer (BRCA1 and BCRA2; IVD-CE label pending); for colon cancer: FAP (APC and MUTYH) and HNPCC (MLH1, MSH2, MSH6, PMS2, 3’ UTR of EPCAM) and a Cystic Fibrosis assay (CFTR). Several other assays are under development including MASTR assays for Marfan syndrome, cardiovascular diseases, renal diseases, targeted cancer therapy (on FFPE derived DNA) and non-invasive trisomy testing.

Diabetes is a common chronic disease that, despite current treatment, reduces quality of life and increases the risk for life threatening complications. In both the type 1 and the type 2 form, the key failure is located at the functional beta cell mass, consisting of an inadequate number and function of the insulin-producing beta cells, be it at varying proportions. It is generally accepted that a restoration of the number of beta cells is essential for the cure of type 1 diabetes but will also be beneficial for type 2 patients. In order to restore of the number of beta cells in a patient, two strategies are developed, one consisting in transplanting an adequate number of beta cells, the other in inducing regeneration of beta cells in their natural site, the pancreas. Clinical proof-of-concept has been achieved for the cell transplantation route, both by our multicenter program and by centers abroad. Further development is however limited by shortage in human donor organs and their low yield of functional beta cells. This led us to search for alternative sources of beta cells, such as the perinatal pig pancreas and stem cells. Irrespective of the source, it will be important to have agents available that induce duplication of the beta cells and maintain their survival. These compounds will not only be important for generating beta cell transplants, but also for sustaining their metabolic role in vivo. They are also expected to help regenerate the beta cell mass in the pancreas. In view of this therapeutic perspective with broad application, we have developed a platform for screening compounds that induce proliferation of beta cells, sustain their survival and regulate their insulin production activity. Our novel assay combines the ability of analyzing the number and function of purified single cells during prolonged serum-free culture, and simultaneously assessing their viability and degree of differentiation. Its image-based, high-content screening (HCS) component has been validated on rat and porcine beta cells. Conditions have been identified that reproducibly double the number of fully functional beta cells over a two-week culture period. The assay also allows detection of compounds that suppress functions or reduce the number of beta cells. It is now extended to the use of human beta cells. In collaboration with Beta-Cell NV (S. Andreasson, CEO), we are now establishing a BetaScreen platform that can perform compound screening for industrial and clinical use. The goal is to translate the assay to an industrial format and apply it to HCS screening, characterization and (pre)-clinical development of diabetes drugs, in partnership with pharmaceutical companies or institutes that can provide selected compound libraries or drug candidates. BetaScreen is expected to open a novel therapeutic window in the field of diabetes, joining knowledge and expertise at our diabetes research center with industry-directed approaches to novel drugs.