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G6b-B is a megakaryocyte lineage-specific immunoreceptor tyrosine-based inhibition motif–containing receptor, essential for platelet homeostasis. Mice with a genomic deletion of the entire Mpig6b locus develop severe macrothrombocytopenia and myelofibrosis, which is reflected in humans with null mutations in MPIG6B. The current model proposes that megakaryocytes lacking G6b-B develop normally, whereas proplatelet release is hampered, but the underlying molecular mechanism remains unclear. We report on a spontaneous recessive single nucleotide mutation in C57BL/6 mice, localized within the intronic region of the Mpig6b locus that abolishes G6b-B expression and reproduces macrothrombocytopenia, myelofibrosis, and osteosclerosis. As the mutation is based on a single-nucleotide exchange, Mpig6bmut mice represent an ideal model to study the role of G6b-B. Megakaryocytes from these mice were smaller, displayed a less-developed demarcation membrane system, and had a reduced expression of receptors. RNA sequencing revealed a striking global reduction in the level of megakaryocyte-specific transcripts, in conjunction with decreased protein levels of the transcription factor GATA-1 and impaired thrombopoietin signaling. The reduced number of mature MKs in the bone marrow was corroborated on a newly developed Mpig6b-null mouse strain. Our findings highlight an unexpected essential role of G6b-B in the early differentiation within the megakaryocytic lineage.

Background Antigen-specific neuroinflammation and neurodegeneration are characteristic for neuroimmunological diseases. In Parkinson’s disease (PD) pathogenesis, α-synuclein is a known culprit. Evidence for α-synuclein-specific T cell responses was recently obtained in PD. Still, a causative link between these α-synuclein responses and dopaminergic neurodegeneration had been lacking. We thus addressed the functional relevance of α-synuclein-specific immune responses in PD in a mouse model. Methods We utilized a mouse model of PD in which an Adeno-associated Vector 1/2 serotype (AAV1/2) expressing human mutated A53T-α-Synuclein was stereotactically injected into the substantia nigra (SN) of either wildtype C57BL/6 or Recombination-activating gene 1 (RAG1)\(^{-/-}\) mice. Brain, spleen, and lymph node tissues from different time points following injection were then analyzed via FACS, cytokine bead assay, immunohistochemistry and RNA-sequencing to determine the role of T cells and inflammation in this model. Bone marrow transfer from either CD4\(^{+}\)/CD8\(^{-}\), CD4\(^{-}\)/CD8\(^{+}\), or CD4\(^{+}\)/CD8\(^{+}\) (JHD\(^{-/-}\)) mice into the RAG-1\(^{-/-}\) mice was also employed. In addition to the in vivo studies, a newly developed A53T-α-synuclein-expressing neuronal cell culture/immune cell assay was utilized. Results AAV-based overexpression of pathogenic human A53T-α-synuclein in dopaminergic neurons of the SN stimulated T cell infiltration. RNA-sequencing of immune cells from PD mouse brains confirmed a pro-inflammatory gene profile. T cell responses were directed against A53T-α-synuclein-peptides in the vicinity of position 53 (68–78) and surrounding the pathogenically relevant S129 (120–134). T cells were required for α-synuclein-induced neurodegeneration in vivo and in vitro, while B cell deficiency did not protect from dopaminergic neurodegeneration. Conclusions Using T cell and/or B cell deficient mice and a newly developed A53T-α-synuclein-expressing neuronal cell culture/immune cell assay, we confirmed in vivo and in vitro that pathogenic α-synuclein peptide-specific T cell responses can cause dopaminergic neurodegeneration and thereby contribute to PD-like pathology.

Treatment options for NAFLD are still limited. Bariatric surgery, such as Roux-en-Y gastric bypass (RYGB), has been shown to improve metabolic and histologic markers of NAFLD. Glucagon-like-peptide-1 (GLP-1) analogues lead to improvements in phase 2 clinical trials. We directly compared the effects of RYGB with a treatment using liraglutide and/or peptide tyrosine tyrosine 3-36 (PYYObese male Wistar rats (high-fat diet (HFD)-induced) were randomized into the following treatment groups: RYGB, sham-operation (sham), liraglutide (0.4 mg/kg/day), PYYRYGB and liraglutide+PYYThe combination therapy of liraglutide+PYY

TO THE EDITOR: Recently, biallelic (“double hit”) TP53 inactivation, occurring in 2% to 4% of newly diagnosed multiple myeloma (MM) patients, was identified as an ultimate high-risk feature of MM, being associated with median survival of

Identification of novel antibiotics remains a major challenge for drug discovery. The present study explores use of phenotypic readouts beyond classical antibacterial growth inhibition adopting a combined multiparametric high content screening and genomic approach. Deployment of the semi-automated bacterial phenotypic fingerprint (BPF) profiling platform in conjunction with a machine learning-powered dataset analysis, effectively allowed us to narrow down, compare and predict compound mode of action (MoA). The method identifies weak antibacterial hits allowing full exploitation of low potency hits frequently discovered by routine antibacterial screening. We demonstrate that BPF classification tool can be successfully used to guide chemical structure activity relationship optimization, enabling antibiotic development and that this approach can be fruitfully applied across species. The BPF classification tool could be potentially applied in primary screening, effectively enabling identification of novel antibacterial compound hits and differentiating their MoA, hence widening the known antibacterial chemical space of existing pharmaceutical compound libraries. More generally, beyond the specific objective of the present work, the proposed approach could be profitably applied to a broader range of diseases amenable to phenotypic drug discovery., Scientific Reports, 9 (1), ISSN:2045-2322

Damage to thinly myelinated and unmyelinated nerve fibers causes small fiber pathology, which is increasingly found in pain syndromes such as small fiber neuropathy (SFN) and fibromyalgia syndrome (FMS). The peripheral nerve endings of the small nerve fibers terminate within the epidermis, where they are surrounded by keratinocytes that may act as primary nociceptive transducers. We performed RNA sequencing of keratinocytes obtained from patients with SFN, FMS, and healthy controls. We found 141 deregulated protein coding genes between SFN patients and healthy controls and no differentially expressed genes between patients with FMS and healthy controls. When comparing patients with SFN with patients with FMS, we detected 167 differentially expressed protein coding genes (129 upregulated and 38 downregulated). Further analysis revealed enriched inflammatory pathways. Validation of selected candidates in an independent cohort confirmed higher expression of the proinflammatory mediators interleukin-8, C-X-C motif chemokine 3, endothelin receptor type A, and the voltage-gated sodium channel 1.7 in SFN compared with patients with FMS. We provide a diverse keratinocyte transcriptome signature between patients with SFN and patients with FMS, which may hint toward distinct pathomechanisms of small fiber sensitization in both entities and lay the basis for advanced diagnostics.

A major challenge for RNA-seq analysis of gene expression is to achieve sufficient coverage of informative nonribosomal transcripts. In eukaryotic samples, this is typically achieved by selective oligo(dT)-priming of messenger RNAs to exclude ribosomal RNA (rRNA) during cDNA synthesis. However, this strategy is not compatible with prokaryotes in which functional transcripts are generally not polyadenylated. To overcome this, we adopted DASH (depletion of abundant sequences by hybridization), initially developed for eukaryotic cells, to improve both the sensitivity and depth of bacterial RNA-seq. DASH uses the Cas9 nuclease to remove unwanted cDNA sequences prior to library amplification. We report the design, evaluation, and optimization of DASH experiments for standard bacterial short-read sequencing approaches, including software for automated guide RNA (gRNA) design for Cas9-mediated cleavage in bacterial rDNA sequences. Using these gRNA pools, we effectively removed rRNA reads (56%–86%) in RNA-seq libraries from two different model bacteria, the Gram-negative pathogen Salmonella enterica and the anaerobic gut commensal Bacteroides thetaiotaomicron. DASH works robustly, even with subnanogram amounts of input RNA. Its efficiency, high sensitivity, ease of implementation, and low cost (∼$5 per sample) render DASH an attractive alternative to rRNA removal protocols, in particular for material-constrained studies where conventional ribodepletion techniques fail.

Background: Treatment options for NAFLD are still limited. Bariatric surgery, such as Roux-en-Y gastric bypass (RYGB), has been shown to improve metabolic and histologic markers of NAFLD. Glucagon-like-peptide-1 (GLP-1) analogues lead to improvements in phase 2 clinical trials. We directly compared the effects of RYGB with a treatment using liraglutide and/or peptide tyrosine tyrosine 3-36 (PYY3-36) in a rat model for early NAFLD. Methods: Obese male Wistar rats (high-fat diet (HFD)-induced) were randomized into the following treatment groups: RYGB, sham-operation (sham), liraglutide (0.4 mg/kg/day), PYY3-36 (0.1 mg/kg/day), liraglutide+PYY3-36, and saline. After an observation period of 4 weeks, liver samples were histologically evaluated, ELISAs and RNA sequencing + RT-qPCRs were performed. Results: RYGB and liraglutide+PYY3-36 induced a similar body weight loss and, compared to sham/saline, marked histological improvements with significantly less steatosis. However, only RYGB induced significant metabolic improvements (e.g., adiponectin/leptin ratio 18.8 ± 11.8 vs. 2.4 ± 1.2 in liraglutide+PYY3-36- or 1.4 ± 0.9 in sham-treated rats). Furthermore, RNA sequencing revealed a high number of differentially regulated genes in RYGB treated animals only. Conclusions: The combination therapy of liraglutide+PYY3-36 partly mimics the positive effects of RYGB on weight reduction and on hepatic steatosis, while its effects on metabolic function lack behind RYGB.

The development of improved, innovative models for the detection of toxicity of drugs, chemicals, or chemicals in cosmetics is crucial to efficiently bring new products safely to market in a cost-effective and timely manner. In addition, improvement in models to detect toxicity may reduce the incidence of unexpected post-marketing toxicity and reduce or eliminate the need for animal testing. The safety of novel products of the pharmaceutical, chemical, or cosmetics industry must be assured; therefore, toxicological properties need to be assessed. Accepted methods for gathering the information required by law for approval of substances are often animal methods. To reduce, refine, and replace animal testing, innovative organotypic in vitro models have emerged. Such models appear at different levels of complexity ranging from simpler, self-organized three-dimensional (3D) cell cultures up to more advanced scaffold-based co-cultures consisting of multiple cell types. This review provides an overview of recent developments in the field of toxicity testing with in vitro models for three major organ types: heart, skin, and liver. This review also examines regulatory aspects of such models in Europe and the UK, and summarizes best practices to facilitate the acceptance and appropriate use of advanced in vitro models.

CEA TCB is a novel T-cell-bispecific (TCB) antibody targeting the carcinoembryonic antigen (CEA) expressed on tumor cells and the CD3 epsilon chain (CD3e) present on T cells, which is currently in Phase 1 clinical trials (NCT02324257) for the treatment of CEA-positive solid tumors. Because the human CEA (hCEA) binder of CEA TCB does not cross-react with cynomolgus monkey and CEA is absent in rodents, alternative nonclinical safety evaluation approaches were considered. These included the development of a cynomolgus monkey cross-reactive homologous (surrogate) antibody (cyCEA TCB) for its evaluation in cynomolgus monkey and the development of double-transgenic mice, expressing hCEA and human CD3e (hCEA/hCD3e Tg), as a potential alternative species for nonclinical safety studies. However, a battery of nonclinical in vitro/ex vivo experiments demonstrated that neither of the previous approaches provided a suitable and pharmacologically relevant model to assess the safety of CEA TCB. Therefore, an alternative approach, a minimum anticipated biological effect level (MABEL), based on an in vitro tumor lysis assay was used to determine the starting dose for the first-in-human study. Using the most conservative approach to the MABEL assessment, a dose of 52 μg was selected as a safe starting dose for clinical study.

The use of human pluripotent stem cells for in vitro disease modelling and clinical applications requires protocols that convert these cells into relevant adult cell types. Here, we report the rapid and efficient differentiation of human pluripotent stem cells into vascular endothelial and smooth muscle cells. We found that GSK3 inhibition and BMP4 treatment rapidly committed pluripotent cells to a mesodermal fate and subsequent exposure to VEGF-A or PDGF-BB resulted in the differentiation of either endothelial or vascular smooth muscle cells, respectively. Both protocols produced mature cells with efficiencies exceeding 80% within six days. On purification to 99% via surface markers, endothelial cells maintained their identity, as assessed by marker gene expression, and showed relevant in vitro and in vivo functionality. Global transcriptional and metabolomic analyses confirmed that the cells closely resembled their in vivo counterparts. Our results suggest that these cells could be used to faithfully model human disease.

Small molecule splicing modifiers have been previously described that target the general splicing machinery and thus have low specificity for individual genes. Several potent molecules correcting the splicing deficit of the SMN2 (survival of motor neuron 2) gene have been identified and these molecules are moving towards a potential therapy for spinal muscular atrophy (SMA). Here by using a combination of RNA splicing, transcription, and protein chemistry techniques, we show that these molecules directly bind to two distinct sites of the SMN2 pre-mRNA, thereby stabilizing a yet unidentified ribonucleoprotein (RNP) complex that is critical to the specificity of these small molecules for SMN2 over other genes. In addition to the therapeutic potential of these molecules for treatment of SMA, our work has wide-ranging implications in understanding how small molecules can interact with specific quaternary RNA structures., Nature Communications, 8 (1), ISSN:2041-1723

The initial step of bone digestion is the adhesion of osteoclasts onto bone surfaces and the assembly of podosomal belts that segregate the bone-facing ruffled membrane from other membrane domains. During bone digestion, membrane components of the ruffled border also need to be recycled after macropinocytosis of digested bone materials. How osteoclast polarity and membrane recycling are coordinated remains unknown. Here, we show that the Cdc42-guanine nucleotide exchange factor FGD6 coordinates these events through its Src-dependent interaction with different actin-based protein networks. At the plasma membrane, FGD6 couples cell adhesion and actin dynamics by regulating podosome formation through the assembly of complexes comprising the Cdc42-interactor IQGAP1, the Rho GTPase-activating protein ARHGAP10, and the integrin interactors Talin-1/2 or Filamin A. On endosomes and transcytotic vesicles, FGD6 regulates retromer-dependent membrane recycling through its interaction with the actin nucleation-promoting factor WASH. These results provide a mechanism by which a single Cdc42-exchange factor controlling different actin-based processes coordinates cell adhesion, cell polarity, and membrane recycling during bone degradation.

Due to their broad differentiation potential, pluripotent stem cells (PSCs) offer a promising approach for generating relevant cellular models for various applications. While human PSC-based cellular models are already advanced, similar systems for non-human primates (NHPs) are still lacking. However, as NHPs are the most appropriate animals for evaluating the safety of many novel pharmaceuticals, the availability of in vitro systems would be extremely useful to bridge the gap between cellular and animal models. Here, we present a NHP in vitro endothelial cell system using induced pluripotent stem cells (IPSCs) from Cynomolgus monkey (Macaca fascicularis). Based on an adapted protocol for human IPSCs, we directly differentiated macaque IPSCs into endothelial cells under chemically defined conditions. The resulting endothelial cells can be enriched using immuno-magnetic cell sorting and display endothelial marker expression and function. RNA sequencing revealed that the differentiation process closely resembled vasculogenesis. Moreover, we showed that endothelial cells derived from macaque and human IPSCs are highly similar with respect to gene expression patterns and key endothelial functions, such as inflammatory responses. These data demonstrate the power of IPSC differentiation technology to generate defined cell types for use as translational in vitro models to compare cell type-specific responses across species.

Background In the past decade the Göttingen minipig has gained increasing recognition as animal model in pharmaceutical and safety research because it recapitulates many aspects of human physiology and metabolism. Genome-based comparison of drug targets together with quantitative tissue expression analysis allows rational prediction of pharmacology and cross-reactivity of human drugs in animal models thereby improving drug attrition which is an important challenge in the process of drug development. Results Here we present a new chromosome level based version of the Göttingen minipig genome together with a comparative transcriptional analysis of tissues with pharmaceutical relevance as basis for translational research. We relied on mapping and assembly of WGS (whole-genome-shotgun sequencing) derived reads to the reference genome of the Duroc pig and predict 19,228 human orthologous protein-coding genes. Genome-based prediction of the sequence of human drug targets enables the prediction of drug cross-reactivity based on conservation of binding sites. We further support the finding that the genome of Sus scrofa contains about ten-times less pseudogenized genes compared to other vertebrates. Among the functional human orthologs of these minipig pseudogenes we found HEPN1, a putative tumor suppressor gene. The genomes of Sus scrofa, the Tibetan boar, the African Bushpig, and the Warthog show sequence conservation of all inactivating HEPN1 mutations suggesting disruption before the evolutionary split of these pig species. We identify 133 Sus scrofa specific, conserved long non-coding RNAs (lncRNAs) in the minipig genome and show that these transcripts are highly conserved in the African pigs and the Tibetan boar suggesting functional significance. Using a new minipig specific microarray we show high conservation of gene expression signatures in 13 tissues with biomedical relevance between humans and adult minipigs. We underline this relationship for minipig and human liver where we could demonstrate similar expression levels for most phase I drug-metabolizing enzymes. Higher expression levels and metabolic activities were found for FMO1, AKR/CRs and for phase II drug metabolizing enzymes in minipig as compared to human. The variability of gene expression in equivalent human and minipig tissues is considerably higher in minipig organs, which is important for study design in case a human target belongs to this variable category in the minipig. The first analysis of gene expression in multiple tissues during development from young to adult shows that the majority of transcriptional programs are concluded four weeks after birth. This finding is in line with the advanced state of human postnatal organ development at comparative age categories and further supports the minipig as model for pediatric drug safety studies. Conclusions Genome based assessment of sequence conservation combined with gene expression data in several tissues improves the translational value of the minipig for human drug development. The genome and gene expression data presented here are important resources for researchers using the minipig as model for biomedical research or commercial breeding. Potential impact of our data for comparative genomics, translational research, and experimental medicine are discussed. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-2119-7) contains supplementary material, which is available to authorized users.

The long-tailed macaque, Macaca fascicularis, is a preferred animal model in biomedical and drug-safety research because of its close genomic and physiological relationship to humans. This genome sequence is an important resource for genetic assays such as expression profiling, single nucleotide polymorphism discovery, or determination of copy number variation (CNV) at specific genomic loci. Macaques utilized for biomedical research and breeding programs are caught in the wild, mainly in peninsular Southeast Asia, the Philippines, and Mauritius. Since captive breeding colonies of M. fascicularis are derived from one or more of these three geographic subpopulations, genetic polymorphisms are frequent and affect the outcome of biomedical studies in which this species is an important animal model. In this chapter, CNVs and their possible effect on biomedical research for macaques originating from Southeast Asia, Mauritius, and the Philippines are described.

Vascular leakage is a serious side effect of therapies based on monoclonal antibodies or cytokines which may lead to life-threatening situations. With the steady increase of new drug development programs for large molecules, there is an urgent need for reliable tools to assess this potential liability of new medicines in a rapid and cost-effective manner. Using human umbilical vein endothelial cells (HUVECs) as a model for endothelium, we established an impedance-based assay measuring the integrity of the endothelial cell monolayer in real time. We could demonstrate that the HUVEC monolayer in our system was a relevant model as cells expressed major junctional proteins known to be responsible for maintaining tightness as well as receptors targeted by molecules known to induce vascular leakage in vivo. We assessed the time-dependent loss of barrier function using impedance and confirmed that signals obtained corresponded well to those from standard transwell assays. We assayed a series of reference molecules which led to the expected change of barrier integrity. A nonspecific cytotoxic effect could be excluded by using human fibroblasts as a nonresponder cell line. Finally, we could show reversibility of vascular permeability induced by histamine, IL-1β, or TNF-α by coincubation with established antagonists, further demonstrating relevance of this new model. Taken together, our results suggest that impedance in combination with HUVECs as a specific model can be applied to assess clinically relevant vascular leakage on an in vitro level.

The long-tailed macaque, also referred to as cynomolgus monkey (Macaca fascicularis), is one of the most important nonhuman primate animal models in basic and applied biomedical research. To improve the predictive power of primate experiments for humans, we determined the genome sequence of a Macaca fascicularis female of Mauritian origin using a whole-genome shotgun sequencing approach. We applied a template switch strategy that uses either the rhesus or the human genome to assemble sequence reads. The sixfold sequence coverage of the draft genome sequence enabled discovery of about 2.1 million potential single-nucleotide polymorphisms based on occurrence of a dimorphic nucleotide at a given position in the genome sequence. Homology-based annotation allowed us to identify 17,387 orthologs of human protein-coding genes in the M. fascicularis draft genome, and the predicted transcripts enabled the design of a M. fascicularis–specific gene expression microarray. Using liver samples from 36 individuals of different geographic origin we identified 718 genes with highly variable expression in liver, whereas the majority of the transcriptome shows relatively stable and comparable expression. Knowledge of the M. fascicularis draft genome is an important contribution to both the use of this animal in disease models and the safety assessment of drugs and their metabolites. In particular, this information allows high-resolution genotyping and microarray-based gene-expression profiling for animal stratification, thereby allowing the use of well-characterized animals for safety testing. Finally, the genome sequence presented here is a significant contribution to the global “3R” animal welfare initiative, which has the goal to reduce, refine, and replace animal experiments.

Bone digestion occurs when osteoclasts adhere onto bone surfaces and polarize to form acidic, hydrolase-rich resorption lacunae. For this process, they condense their actin-rich podosomes in tight belts to establish sealing zones, which segregate their basal membranes from those facing resorption lacunae. This polarization process remains poorly understood. Here, we combined quantitative proteomics and gene silencing to identify new substrates of the Src tyrosine kinase, a key regulator of osteoclast function. We now report that a depletion of the ARF GTPase-activating protein GIT2, which localizes to sealing zones upon Src phosphorylation, or a lack of GTP hydrolysis on ARF6 impairs sealing zone formation and polarized membrane traffic. Surprisingly, the Rho guanine nucleotide exchange factors α and β PIX, which usually coordinate ARF and Rho signaling, were found to be dispensable. We conclude that the Src-dependent localization of GIT2 is essential for down-regulating ARF6 activity at sealing zones, and thus for maintaining osteoclast polarity.

Transfer of glutamine between astrocytes and neurons is an essential part of the glutamate–glutamine cycle in the brain. Transport of glutamine was investigated in primary cultures of astrocytes and neurons and compared to glutamine transport in cell lines with glial and neuronal properties. Glutamine uptake in astrocytes was mainly mediated by general amino acid transporters with properties similar to ASCT2, LAT1, LAT2, SN1 and y+LAT2. In cultured neurons, transport activities were detected consistent with the presence of LAT1, LAT2 and y+LAT2, but the most prominent activity was a novel Na+-dependent glutamine transporter that could be inhibited by d -aspartate. The mRNA for system A isoforms ATA1 and ATA2 was detected in both neurons and astrocytes, but system A activity was only detected in neurons. ASCT2 on the other hand appeared to be astrocyte-specific. The cell lines F98 and 108CC-15, having astroglial and neuronal properties, respectively, expressed sets of glutamine transporters that were unrelated to those of the corresponding primary culture and are thus of limited use as models to study transfer of glutamine between astrocytes and neurons.

Summary Direct transdifferentiation of somatic cells is a promising approach to obtain patient-specific cells for numerous applications. However, conversion across germ-layer borders often requires ectopic gene expression with unpredictable side effects. Here, we present a gene-free approach that allows efficient conversion of human fibroblasts via a transient progenitor stage into Schwann cells, the major glial cell type of peripheral nerves. Using a multikinase inhibitor, we transdifferentiated fibroblasts into transient neural precursors that were subsequently further differentiated into Schwann cells. The resulting induced Schwann cells (iSCs) expressed numerous Schwann cell-specific proteins and displayed neurosupportive and myelination capacity in vitro. Thus, we established a strategy to obtain mature Schwann cells from human postnatal fibroblasts under chemically defined conditions without the introduction of ectopic genes., Graphical Abstract, Highlights • Direct conversion of human fibroblasts into Schwann cells • No introduction of ectopic genes • Induction of conversion by multikinase inhibitor, Here, Thoma and colleagues developed a strategy to convert human fibroblasts into Schwann cells solely by chemical treatment. Using a multikinase inhibitor, fibroblasts are transdifferentiated into transient neural precursors differentiating into cells with molecular and functional characteristics of Schwann cells. This study shows that cellular conversion can be achieved solely by small molecules and presents an approach to generate human Schwann cells without genetic modifications.