Experimental validation of computerised models of clustering of platelet glycoprotein receptors that signal via tandem SH2 domain proteins.

The clustering of platelet glycoprotein receptors with cytosolic YxxL and YxxM motifs, including GPVI, CLEC-2 and PEAR1, triggers activation via phosphorylation of the conserved tyrosine residues and recruitment of the tandem SH2 (Src homology 2) domain effector proteins, Syk and PI 3-kinase. We have modelled the clustering of these receptors with monovalent, divalent and tetravalent soluble ligands and with transmembrane ligands based on the law of mass action using ordinary differential equations and agent-based modelling. The models were experimentally evaluated in platelets and transfected cell lines using monovalent and multivalent ligands, including novel nanobody-based divalent and tetravalent ligands, by fluorescence correlation spectroscopy. Ligand valency, receptor number, receptor dimerisation, receptor phosphorylation and a cytosolic tandem SH2 domain protein act in synergy to drive receptor clustering. Threshold concentrations of a CLEC-2-blocking antibody and Syk inhibitor act in synergy to block platelet aggregation. This offers a strategy for countering the effect of avidity of multivalent ligands and in limiting off-target effects.

Mini-batch optimization enables training of ODE models on large-scale datasets.

Quantitative dynamic models are widely used to study cellular signal processing. A critical step in modelling is the estimation of unknown model parameters from experimental data. As model sizes and datasets are steadily growing, established parameter optimization approaches for mechanistic models become computationally extremely challenging. Mini-batch optimization methods, as employed in deep learning, have better scaling properties. In this work, we adapt, apply, and benchmark mini-batch optimization for ordinary differential equation (ODE) models, thereby establishing a direct link between dynamic modelling and machine learning. On our main application example, a large-scale model of cancer signaling, we benchmark mini-batch optimization against established methods, achieving better optimization results and reducing computation by more than an order of magnitude. We expect that our work will serve as a first step towards mini-batch optimization tailored to ODE models and enable modelling of even larger and more complex systems than what is currently possible.

Climate change influences chlorophylls and bacteriochlorophylls metabolism in hypersaline microbial mat.

This study aimed to determine the effect of the climatic change on the phototrophic communities of hypersaline microbial mats. Ocean acidification and warming were simulated alone and together on microbial mats placed into mesocosms. As expected, the temperature in the warming treatments increased by 4 °C from the initial temperature. Surprisingly, no significance difference was observed between the water pH of the different treatments despite of a decrease of 0.4 unit pH in the water reserves of acidification treatments. The salinity increased on the warming treatments and the dissolved oxygen concentration increased and was higher on the acidification treatments. A total of 37 pigments were identified belonging to chlorophylls, carotenes and xanthophylls families. The higher abundance of unknown chlorophyll molecules called chlorophyll derivatives was observed in the acidification alone treatment with a decrease in chlorophyll a abundance. This change in pigmentary composition was accompanied by a higher production of bound extracellular carbohydrates but didn't affect the photosynthetic efficiency of the microbial mats. A careful analysis of the absorption properties of these molecules indicated that these chlorophyll derivatives were likely bacteriochlorophyll c contained in the chlorosomes of green anoxygenic phototroph bacteria. Two hypotheses can be drawn from these results: 1/ the phototrophic communities of the microbial mats were modified under acidification treatment leading to a higher relative abundance of green anoxygenic bacteria, or 2/ the highest availability of CO2 in the environment has led to a shift in the metabolism of green anoxygenic bacteria being more competitive than other phototrophs.

A proposal for the future of scientific publishing in the life sciences.

Science advances through rich, scholarly discussion. More than ever before, digital tools allow us to take that dialogue online. To chart a new future for open publishing, we must consider alternatives to the core features of the legacy print publishing system, such as an access paywall and editorial selection before publication. Although journals have their strengths, the traditional approach of selecting articles before publication ("curate first, publish second") forces a focus on "getting into the right journals," which can delay dissemination of scientific work, create opportunity costs for pushing science forward, and promote undesirable behaviors among scientists and the institutions that evaluate them. We believe that a "publish first, curate second" approach with the following features would be a strong alternative: authors decide when and what to publish; peer review reports are published, either anonymously or with attribution; and curation occurs after publication, incorporating community feedback and expert judgment to select articles for target audiences and to evaluate whether scientific work has stood the test of time. These proposed changes could optimize publishing practices for the digital age, emphasizing transparency, peer-mediated improvement, and post-publication appraisal of scientific articles.

Long term effects of cigarette smoke extract or nicotine on nerve growth factor and its receptors in a bronchial epithelial cell line.

Long-term exposure to cigarette smoke induces severe injuries to respiratory system through several mechanisms, some of them are well defined, but many others are not yet elucidated. Beside its classical role in nervous system, we have previously shown that Nerve Growth Factor (NGF) and its receptors have a crucial role in airway inflammatory diseases, such as Chronic Obstructive Pulmonary Disease. To expand our knowledge about the relevance of NGF and its receptors in airway diseases induced by cigarette smoking, we exposed for 16 weeks the bronchial epithelial cell line BEAS-2B to sub-toxic concentrations of whole cigarette smoke extract or pure nicotine. Viability, cell cycle gene expression, cell morphology and migration ability were tested and compared to NGF release and gene expression. Modulation of its receptors TrKA and p75NTR was also analyzed. The present study shows that long term exposure of BEAS-2B cells to cigarette smoke extract or nicotine induces: (A) differences: in cell viability, in the expression of cell cycle-related genes, in NGF release and in gene expression of NGF and its receptors; (B) similarities: in morphology and migration ability. Taken together, our data provide new insights about the biological role of NGF and its receptors in airway diseases induced by long-term cigarette smoking and, finally, our data evidence the opportunity not to use nicotine lozenges or e-cigarettes as anti smoking replacement therapy in patients with a previous airway disease according to the ability of nicotine to increase the amount of the pro-inflammatory cytokine NGF into the bronchial environment.

Models of Models: A Translational Route for Cancer Treatment and Drug Development.

Every patient and every disease is different. Each patient therefore requires a personalized treatment approach. For technical reasons, a personalized approach is feasible for treatment strategies such as surgery, but not for drug-based therapy or drug development. The development of individual mechanistic models of the disease process in every patient offers the possibility of attaining truly personalized drug-based therapy and prevention. The concept of virtual clinical trials and the integrated use of in silico, in vitro, and in vivo models in preclinical development could lead to significant gains in efficiency and order of magnitude increases in the cost effectiveness of drug development and approval. We have developed mechanistic computational models of large-scale cellular signal transduction networks for prediction of drug effects and functional responses, based on patient-specific multi-level omics profiles. However, a major barrier to the use of such models in a clinical and developmental context is the reliability of predictions. Here we detail how the approach of using "models of models" has the potential to impact cancer treatment and drug development. We describe the iterative refinement process that leverages the flexibility of experimental systems to generate highly dimensional data, which can be used to train and validate computational model parameters and improve model predictions. In this way, highly optimized computational models with robust predictive capacity can be generated. Such models open up a number of opportunities for cancer drug treatment and development, from enhancing the design of experimental studies, reducing costs, and improving animal welfare, to increasing the translational value of results generated.

Cancer Precision Medicine: Why More Is More and DNA Is Not Enough.

Every tumour is different. They arise in patients with different genomes, from cells with different epigenetic modifications, and by random processes affecting the genome and/or epigenome of a somatic cell, allowing it to escape the usual controls on its growth. Tumours and patients therefore often respond very differently to the drugs they receive. Cancer precision medicine aims to characterise the tumour (and often also the patient) to be able to predict, with high accuracy, its response to different treatments, with options ranging from the selective characterisation of a few genomic variants considered particularly important to predict the response of the tumour to specific drugs, to deep genome analysis of both tumour and patient, combined with deep transcriptome analysis of the tumour. Here, we compare the expected results of carrying out such analyses at different levels, from different size panels to a comprehensive analysis incorporating both patient and tumour at the DNA and RNA levels. In doing so, we illustrate the additional power gained by this unusually deep analysis strategy, a potential basis for a future precision medicine first strategy in cancer drug therapy. However, this is only a step along the way of increasingly detailed molecular characterisation, which in our view will, in the future, introduce additional molecular characterisation techniques, including systematic analysis of proteins and protein modification states and different types of metabolites in the tumour, systematic analysis of circulating tumour cells and nucleic acids, the use of spatially resolved analysis techniques to address the problem of tumour heterogeneity as well as the deep analyses of the immune system of the patient to, e.g., predict the response of the patient to different types of immunotherapy. Such analyses will generate data sets of even greater complexity, requiring mechanistic modelling approaches to capture enough of the complex situation in the real patient to be able to accurately predict his/her responses to all available therapies.

Impaired Planar Germ Cell Division in the Testis, Caused by Dissociation of RHAMM from the Spindle, Results in Hypofertility and Seminoma.

Hypofertility is a risk factor for the development of testicular germ cell tumors (TGCT), but the initiating event linking these pathologies is unknown. We hypothesized that excessive planar division of undifferentiated germ cells promotes their self-renewal and TGCT development. However, our results obtained from mouse models and seminoma patients demonstrated the opposite. Defective planar divisions of undifferentiated germ cells caused their premature exit from the seminiferous tubule niche, resulting in germ cell depletion, hypofertility, intratubular germ cell neoplasias, and seminoma development. Oriented divisions of germ cells, which determine their fate, were regulated by spindle-associated RHAMM-a function we found to be abolished in 96% of human seminomas. Mechanistically, RHAMM expression is regulated by the testis-specific polyadenylation protein CFIm25, which is downregulated in the human seminomas. These results suggested that spindle misorientation is oncogenic, not by promoting self-renewing germ cell divisions within the niche, but by prematurely displacing proliferating cells from their normal epithelial milieu. Furthermore, they suggested RHAMM loss-of-function and spindle misorientation as an initiating event underlying both hypofertility and TGCT initiation. These findings identify spindle-associated RHAMM as an intrinsic regulator of male germ cell fate and as a gatekeeper preventing initiation of TGCTs. Cancer Res; 76(21); 6382-95. ©2016 AACR.

Predictive Modeling of Drug Treatment in the Area of Personalized Medicine.

Despite a growing body of knowledge on the mechanisms underlying the onset and progression of cancer, treatment success rates in oncology are at best modest. Current approaches use statistical methods that fail to embrace the inherent and expansive complexity of the tumor/patient/drug interaction. Computational modeling, in particular mechanistic modeling, has the power to resolve this complexity. Using fundamental knowledge on the interactions occurring between the components of a complex biological system, large-scale in silico models with predictive capabilities can be generated. Here, we describe how mechanistic virtual patient models, based on systematic molecular characterization of patients and their diseases, have the potential to shift the theranostic paradigm for oncology, both in the fields of personalized medicine and targeted drug development. In particular, we highlight the mechanistic modeling platform ModCell™ for individualized prediction of patient responses to treatment, emphasizing modeling techniques and avenues of application.

Network and systems biology: essential steps in virtualising drug discovery and development.

The biological processes that keep us healthy or cause disease, as well as the mechanisms of action of possible drugs are inherently complex. In the face of this complexity, attempts at discovering new drugs to treat diseases have alternated between trial-and-error (typically on experimental systems) and grand simplification, usually based on much too little information. We now have the chance to combine these strategies through establishment of 'virtual patient' models, centred on a detailed molecular characterisation of thousands or even, in the future, millions of patients. In doing so, we lay the foundations for truly personalised therapy, as well as a far-reaching virtualisation of drug discovery and development in oncology and other areas of medicine.

Correlation of rheoencephalogram and intracranial pressure: results of a rat study.

Measuring brain electrical impedance (rheoencephalography-REG) is a potential technique for noninvasive, continuous neuro-monitoring. Typically, intracranial pressure (ICP), an invasive monitoring modality, is used in brain monitoring. Our hypothesis was that both modalities would reflect cerebrovascular reactivity. In the present study we compared results of REG to results of ICP measurement. Rats were used under anesthesia ([Formula: see text]; 36 control and 59 vinpocetine infusions). REG was measured by two bipolar REG amplifiers; time constants (Tc) were 3 and 0.3 s. The vinpocetine injection caused a transient decrease in systemic arterial pressure (SAP) and a simultaneous increase in ICP and REG pulse amplitude. SAP decrease was 25% ± 14%; ICP was 28% ± 16%; REG pulse amplitude increase was 209% ± 17% (Tc 3) and 107% ± 68% (Tc 0.3). ICP increase correlated with REG pulse amplitude increase. Area under the receiver operating characteristic curve was 0.9481 for ICP-REG time constants 3 and 0.9335 for ICP-REG time constants 0.3; both with [Formula: see text]. The fact that both REG and ICP reflect cerebrovascular reactivity indicates the usefulness of REG as a potential technique for noninvasive, continuous neuro-monitoring. The Tc of REG amplifier requires optimization for continuous monitoring of pressure reactivity index.

Cardiovascular parameters in a mixed-sex swine study of severe decompression sickness treated with the emulsified perfluorocarbon Oxycyte.

Intravenous perfluorocarbons (PFC) have reduced the effects of decompression sickness (DCS) and improved mortality rates in animal models. However, concerns for the physiological effects of DCS combined with PFC therapy have not been examined in a balanced mixed-sex population. Thirty-two (16 male, 16 female) instrumented and sedated juvenile Yorkshire swine were exposed to 200 feet of seawater (fsw) for 31 min of hyperbaric air. Pulmonary artery pressure (PAP), cardiac output (CO), and systemic arterial pressure (SAP) were monitored before (control) and after exposure. Animals were randomized to treatment with Oxycyte (5 ml/kg; Oxygen Biotherapeutics, Inc., Morrisville, NC) vs. saline (control) with 100% oxygen administered upon DCS onset; animals were observed for 90 min. Parameters recorded and analyzed included PAP, CO, and SAP. In all animals PAP began to rise prior to cutis marmorata (CM) onset, the first sign of clinical DCS, generally peaking after CM onset. Female swine, compared with castrated males, had a more rapid onset of CM (7.30 vs. 11.46 min postsurfacing) and earlier onset to maximal PAP (6.41 vs. 9.69 min post-CM onset). Oxycyte therapy was associated with a sustained PAP elevation above controls in both sexes (33.41 vs. 25.78 mmHg). Significant pattern differences in PAP, CO, and SAP were noted between sexes and between therapeutic groups. There were no statistically significant differences in survival or paralysis between the PFC and control groups during the 48-h observation period. In conclusion, Oxycyte therapy for DCS is associated with a prolonged PAP increase in swine. These species and sex differences warrant further exploration.

Xenograft of microencapsulated Sertoli cells for the cell therapy of type 2 diabetes mellitus in spontaneously diabetic nonhuman primates: preliminary data.

Insulin resistance in type 2 diabetes mellitus (T2DM) may be due to a chronic inflammation of the visceral adipose tissue (VAT) leading to local and systemic increases in proinflammatory cytokines. Microencapsulated porcine Sertoli cells (MC-pSC), by provision of immunomodulatory and trophic factors, have been successfully used to reduce such inflammation in rodent animal models of type 1 diabetes with no complications or deleterious side effects. Herein, we have begun to investigate this novel and safe therapeutic approach in the spontaneously obese nonhuman primate with spontaneous, insulin-dependent T2DM. After MC-pSC intraperitoneal injection we have evaluated, throughout a 6-month follow-up period, daily ad libitum fed glucose levels, daily exogenous insulin supplementation, biweekly body weight measurements, periodic fasting blood glucose concentrations, glycated hemoglobin (HbA1c) levels, glucose tolerance tests (GTT), and fluorescence-activated cell sorting cytometry (FACS) assessment of peripheral blood mononuclear cells. Very preliminarily, we have observed a slight reduction in fasting (FPG) and mean nonfasting (NF) plasma glucose levels. We found minimal changes, only in 1 animal, in daily exogenous insulin requirements and HbA1c levels. Flow cytometric analysis was associated with decrease in CD8(+) cells only in 1 recipient with a reduction in mean regulatory T Cells (Treg), whereas interestingly, decrease of B lymphocytes was observed in both animals. These results may suggest that this novel MC-SC-based transplantation protocol might possibly impact the metabolic status of T2DM in higher mammals that are close to humans.

Microparticle-loaded neonatal porcine Sertoli cells for cell-based therapeutic and drug delivery system.

Neonatal porcine Sertoli cells (NPSC) are immune privileged cells showing innate phagocytic and antibacterial activities. NPSC have been shown capable of immunoaltering the body's response and possess lung homing capacity. These properties encourage investigation of NPSC as functional components of cell-based therapeutic protocols to treat lung infections and related complications. In this work, for the first time, NPSC were tailored to carry an antibiotic drug loaded into poly(d,l lactic) acid microparticles (MP). A loading protocol was developed, which afforded 30% drug uptake and high stability over time, with little or no effects on NPSC viability, morphology, reactive oxygen species production and DNA integrity. FSH receptor integrity, and TGFß (transforming growth factor ß) and AMH (anti-Müllerian hormone) expressions were unchanged after 1month of cryopreservation. Protein tyrosine kinase activation due to phagocytosis may have had resulted in changes in inhibin B expression. The activity of MP-loaded or NPSC alone against Pseudomonas aeruginosa was maintained throughout 1month of storage. NPSC couple an innate antibacterial activity with the capacity to embody drug loaded MP. We showed for the first time that engineered NPSC can be cryopreserved with no loss of their basic properties, thereby possibly representing a novel approach for cell-based therapeutic and drug delivery system.

Toxicity of cadmium on Sertoli cell functional competence: an in vitro study.

Cadmium (Cd), an ubiquitous environmental metal, mainly used for industrial purposes, may be toxic at level of the reproductive system. Testis tubular-based Sertoli cells (SC), play a major role in constituting the blood-testis barrier and provide a unique microenvironment for the genesis and differentiation of germ cells. Hence SC strictly control sperm qualitative and quantitative parameters. We aimed to assess whether exposure to Cd would adversely affect superior mammal SC viability and function. We isolated and purified SC from pre-pubertal pig testes according to our method and incubated the retrieved cells with three different Cadmium chloride concentrations (5-10-15 microM). Parameters of SC function such as inhibin B and anti-Mullerian hormone (AMH) were depressed by Cd exposure, contrary to what observed in untreated controls. No impairment of the FSH receptor integrity on the SC, as assessed by 17-beta-estradiol production, upon stimulation with FSH, was observed in either 5 microM Cd-treated or untreated controls. Differences, on the contrary, were observed for higher Cd concentrations (10 and 15 mM), in terms of FSH receptor integrity, that was altered, as compared to untreated controls, in terms of lower production of 17-beta-estradiol. In addition, the apoptotic test showed a significant increase of early (ANNEXIN V-/Propidium Iodide+) (AV-/PI+) and late apoptotic cells (AV+/ PI+) in all Cd -treated SC conditions as compared to controls. In conclusion, the Cd -related toxicity on SC, clearly demonstrated by our study, even at low concentrations, is expected to damage spermatogenesis that directly is dependent upon retention of SC viability and function.

LGALS3BP regulates centriole biogenesis and centrosome hypertrophy in cancer cells.

Centrosome morphology and number are frequently deregulated in cancer cells. Here, to identify factors that are functionally relevant for centrosome abnormalities in cancer cells, we established a protein-interaction network around 23 centrosomal and cell-cycle regulatory proteins, selecting the interacting proteins that are deregulated in cancer for further studies. One of these components, LGALS3BP, is a centriole- and basal body-associated protein with a dual role, triggering centrosome hypertrophy when overexpressed and causing accumulation of centriolar substructures when downregulated. The cancer cell line SK-BR-3 that overexpresses LGALS3BP exhibits hypertrophic centrosomes, whereas in seminoma tissues with low expression of LGALS3BP, supernumerary centriole-like structures are present. Centrosome hypertrophy is reversed by depleting LGALS3BP in cells endogenously overexpressing this protein, supporting a direct role in centrosome aberration. We propose that LGALS3BP suppresses assembly of centriolar substructures, and when depleted, causes accumulation of centriolar complexes comprising CPAP, acetylated tubulin and centrin.

Silica particle size and shape: in vitro effects on extracellular matrix metabolism and viability of human bronchial epithelial cells.

Crystal micro-morphology and dimension of silica particles could be responsible for the high prevalence of silicosis as recently found among goldsmiths. In the present study we investigated two samples of silica particles with different surface sizes and shapes for their capacity to induce changes in ECM component production. In addition we investigated if their different effects could be related to cytotoxicity and apoptotic effects. Human bronchial epithelial cells were cultured with or without a sample of Silica used for casting gold jewellery, named in our experiments Silica P or a commercial sample of Silica with different physical and chemical properties, named in our experiments Silica F. After 48 h of exposure PCR analysis determined levels of several matrix components. As induction of the apoptosis cascade, annexin assay, caspase 3 activity and cellular cytoxicity by MTT assay were assayed. Silica F promoted fibronectin, MMP12, tenascin C and Integrins b5 gene expressions more than Silica P. Silica P stimulated more TGFß1 and its TGFßR1 receptor than Silica F. Cytotoxic effects were induced by the two samples of Silica. On the contrary, no alteration in classic apoptotic marker protein expression was observed in presence of either Silica F or Silica P, suggesting silica particles affect ECM production and metalloproteases through a mechanism that does not involve apoptotic activation. Different Silica micromorphology and TGFß signal pathway are linked to lung fibrotic effects but the potential role Silica in apoptotic and toxic reaction remains to be ascertained.

Functional analysis of centrosomal kinase substrates in Drosophila melanogaster reveals a new function of the nuclear envelope component otefin in cell cycle progression.

Phosphorylation is one of the key mechanisms that regulate centrosome biogenesis, spindle assembly, and cell cycle progression. However, little is known about centrosome-specific phosphorylation sites and their functional relevance. Here, we identified phosphoproteins of intact Drosophila melanogaster centrosomes and found previously unknown phosphorylation sites in known and unexpected centrosomal components. We functionally characterized phosphoproteins and integrated them into regulatory signaling networks with the 3 important mitotic kinases, cdc2, polo, and aur, as well as the kinase CkIIß. Using a combinatorial RNA interference (RNAi) strategy, we demonstrated novel functions for P granule, nuclear envelope (NE), and nuclear proteins in centrosome duplication, maturation, and separation. Peptide microarrays confirmed phosphorylation of identified residues by centrosome-associated kinases. For a subset of phosphoproteins, we identified previously unknown centrosome and/or spindle localization via expression of tagged fusion proteins in Drosophila SL2 cells. Among those was otefin (Ote), an NE protein that we found to localize to centrosomes. Furthermore, we provide evidence that it is phosphorylated in vitro at threonine 63 (T63) through Aurora-A kinase. We propose that phosphorylation of this site plays a dual role in controlling mitotic exit when phosphorylated while dephosphorylation promotes G(2)/M transition in Drosophila SL2 cells.

DIPSBC--data integration platform for systems biology collaborations.

BACKGROUND: Modern biomedical research is often organized in collaborations involving labs worldwide. In particular in systems biology, complex molecular systems are analyzed that require the generation and interpretation of heterogeneous data for their explanation, for example ranging from gene expression studies and mass spectrometry measurements to experimental techniques for detecting molecular interactions and functional assays. XML has become the most prominent format for representing and exchanging these data. However, besides the development of standards there is still a fundamental lack of data integration systems that are able to utilize these exchange formats, organize the data in an integrative way and link it with applications for data interpretation and analysis. RESULTS: We have developed DIPSBC, an interactive data integration platform supporting collaborative research projects, based on Foswiki, Solr/Lucene, and specific helper applications. We describe the main features of the implementation and highlight the performance of the system with several use cases. All components of the system are platform independent and open-source developments and thus can be easily adopted by researchers. An exemplary installation of the platform which also provides several helper applications and detailed instructions for system usage and setup is available at http://dipsbc.molgen.mpg.de. CONCLUSIONS: DIPSBC is a data integration platform for medium-scale collaboration projects that has been tested already within several research collaborations. Because of its modular design and the incorporation of XML data formats it is highly flexible and easy to use.