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The tumor microenvironment (TM), consisting of the extracellular matrix (ECM), fibroblasts, endothelial cells, and immune cells, might affect tumor invasiveness and the outcome of standard chemotherapy. This study investigated the cross talk between germ cell tumors (GCT) and surrounding TM cells (macrophages, T-lymphocytes, endothelial cells, and fibroblasts) at the transcriptome and secretome level. Using high-throughput approaches of three-dimensional (3D) co-cultured cellular aggregates, this study offers newly identified pathways to be studied with regard to sensitivity toward cisplatin-based chemotherapy or tumor invasiveness as a consequence of the cross talk between tumor cells and TM components. Mass-spectrometry-based secretome analyses revealed that TM cells secreted factors involved in ECM organization, cell adhesion, angiogenesis, and regulation of insulin-like growth factor (IGF) transport. To evaluate direct cell-cell contacts, green fluorescent protein (GFP)-expressing GCT cells and mCherry-expressing TM cells were co-cultured in 3D. Afterward, cell populations were separated by flow cytometry and analyzed by RNA sequencing. Correlating the secretome with transcriptome data indicated molecular processes such as cell adhesion and components of the ECM being enriched in most cell populations. Re-analyses of secretome data with regard to lysine- and proline-hydroxylated peptides revealed a gain in proteins, such as collagens and fibronectin. Cultivation of GCT cells on collagen I/IV- or fibronectin-coated plates significantly elevated adhesive and migratory capacity, while decreasing cisplatin sensitivity of GCT cells. Correspondingly, cisplatin sensitivity was significantly reduced in GCT cells under the influence of conditioned medium from fibroblasts and endothelial cells. This study sheds light on the cross talk between GCT cells and their circumjacent TM, which results in deposition of the ECM and eventually promotes a pro-tumorigenic environment through enhanced migratory and adhesive capacity, as well as decreased cisplatin sensitivity. Hence, our observations indicate that targeting the ECM and its cellular components might be a novel therapeutic option in combination with cisplatin-based chemotherapy for GCT patients., (© 2022 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Acute myocardial infarction (MI) induces an extensive sterile inflammation, which is dominated in the early phase by invading neutrophils and monocytes/macrophages. The inflammatory response after MI critically affects infarct healing and cardiac remodeling. Therefore, modulation of cardiac inflammation may improve outcome post MI. Insulin-like growth factor 1 (IGF1) treatment reduces infarct size and improves cardiac function after MI via IGF1 receptor mediated signaling in myeloid cells. Our study aimed to investigate the effect of IGF1 on neutrophil phenotype both in vitro and in vivo after MI. We show that IGF1 induces an anti-inflammatory phenotype in bone marrow derived neutrophils. On the molecular and functional level IGF1 treated neutrophils were indistinguishable from those induced by IL4. Surprisingly, insulin, even though it is highly similar to IGF1 did not create anti-inflammatory neutrophils. Notably, the IGF1 effect was independent of the canonical Ras/Raf/ERK or PI3K/AKT pathway, but depended on activation of the JAK2/STAT6 pathway, which was not activated by insulin treatment. Single cell sequencing analysis 3 days after MI also showed that 3 day IGF1 treatment caused a downregulation of pro-inflammatory genes and upstream regulators in most neutrophil and many macrophage cell clusters whereas anti-inflammatory genes and upstream regulators were upregulated. Thus, IGF1 acts like an anti-inflammatory cytokine on myeloid cells in vitro and attenuates the pro-inflammatory phenotype of neutrophils and macrophages in vivo after MI. IGF1 treatment might therefore represent an effective immune modulatory therapy to improve the outcome after MI., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Nederlof, Reidel, Spychala, Gödecke, Heinen, Lautwein, Petzsch, Köhrer and Gödecke.)

Urothelial carcinoma (UC) of the urinary bladder is a prevalent cancer worldwide. Because histone deacetylases (HDACs) are important factors in cancer, targeting these epigenetic regulators is considered an attractive strategy to develop novel anticancer drugs. Whereas HDAC1 and HDAC2 promote UC, HDAC5 is often downregulated and only weakly expressed in UC cell lines, suggesting a tumor-suppressive function. We studied the effect of stable lentiviral-mediated HDAC5 overexpression in four UC cell lines with different phenotypes (RT112, VM-Cub-1, SW1710, and UM-UC-3, each with vector controls). In particular, comprehensive proteomics and RNA-seq transcriptomics analyses were performed on the four cell line pairs, which are described here. For comparison, the immortalized benign urothelial cell line HBLAK was included. These datasets will be a useful resource for researchers studying UC, and especially the influence of HDAC5 on epithelial-mesenchymal transition (EMT). Moreover, these data will inform studies on HDAC5 as a less studied member of the HDAC family in other cell types and diseases, especially fibrosis., (© 2022. The Author(s).)

Beauvericin (BEA), a mycotoxin of the enniatin family produced by various toxigenic fungi, has been attributed multiple biological activities such as anti-cancer, anti-inflammatory, and anti-microbial functions. However, effects of BEA on dendritic cells remain unknown so far. Here, we identified effects of BEA on murine granulocyte-macrophage colony-stimulating factor (GM-CSF)-cultured bone marrow derived dendritic cells (BMDCs) and the underlying molecular mechanisms. BEA potently activates BMDCs as signified by elevated IL-12 and CD86 expression. Multiplex immunoassays performed on myeloid differentiation primary response 88 (MyD88) and toll/interleukin-1 receptor (TIR) domain containing adaptor inducing interferon beta (TRIF) single or double deficient BMDCs indicate that BEA induces inflammatory cytokine and chemokine production in a MyD88/TRIF dependent manner. Furthermore, we found that BEA was not able to induce IL-12 or IFNβ production in Toll-like receptor 4 ( Tlr4 )-deficient BMDCs, whereas induction of these cytokines was not compromised in Tlr3/7/9 deficient BMDCs. This suggests that TLR4 might be the functional target of BEA on BMDCs. Consistently, in luciferase reporter assays BEA stimulation significantly promotes NF-κB activation in mTLR4/CD14/MD2 overexpressing but not control HEK-293 cells. RNA-sequencing analyses further confirmed that BEA induces transcriptional changes associated with the TLR4 signaling pathway. Together, these results identify TLR4 as a cellular BEA sensor and define BEA as a potent activator of BMDCs, implying that this compound can be exploited as a promising candidate structure for vaccine adjuvants or cancer immunotherapies., Competing Interests: Authors P-KL, UK, and JS are employed by Institute for Experimental Infection Research, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Helmholtz Centre for Infection Research and the Hannover Medical School, Hannover, Germany. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yang, Ali, Zhao, Richter, Schäfer, Schliehe-Diecks, Frank, Qi, Larsen, Skerra, Islam, Wachtmeister, Alter, Huang, Bhatia, Köhrer, Kirschning, Weighardt, Kalinke, Kalscheuer, Uhrberg and Scheu.)

Background and Objectives: Siponimod is an oral, selective sphingosine-1-phosphate receptor-1/5 modulator approved for treatment of multiple sclerosis., Methods: Mouse MRI was used to investigate remyelination in the cuprizone model. We then used a conditional demyelination Xenopus laevis model to assess the dose-response of siponimod on remyelination. In experimental autoimmune encephalomyelitis-optic neuritis (EAEON) in C57Bl/6J mice, we monitored the retinal thickness and the visual acuity using optical coherence tomography and optomotor response. Optic nerve inflammatory infiltrates, demyelination, and microglial and oligodendroglial differentiation were assessed by immunohistochemistry, quantitative real-time PCR, and bulk RNA sequencing., Results: An increased remyelination was observed in the cuprizone model. Siponimod treatment of demyelinated tadpoles improved remyelination in comparison to control in a bell-shaped dose-response curve. Siponimod in the EAEON model attenuated the clinical score, reduced the retinal degeneration, and improved the visual function after prophylactic and therapeutic treatment, also in a bell-shaped manner. Inflammatory infiltrates and demyelination of the optic nerve were reduced, the latter even after therapeutic treatment, which also shifted microglial differentiation to a promyelinating phenotype., Discussion: These results confirm the immunomodulatory effects of siponimod and suggest additional regenerative and promyelinating effects, which follow the dynamics of a bell-shaped curve with high being less efficient than low concentrations., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Testicular germ cell tumors (GCTs) are stratified into seminomas and nonseminomas. Seminomas share many histological and molecular features with primordial germ cells, whereas the nonseminoma stem cell population-embryonal carcinoma (EC)-is pluripotent and thus able to differentiate into cells of all three germ layers (teratomas). Furthermore, ECs are capable of differentiating into extra-embryonic lineages (yolk sac tumors, choriocarcinomas). In this study, we deciphered the molecular and (epi)genetic mechanisms regulating expression of CD24, a highly glycosylated signaling molecule upregulated in many cancers. CD24 is overexpressed in ECs compared with other GCT entities and can be associated with an undifferentiated pluripotent cell fate. We demonstrate that CD24 can be transactivated by the pluripotency factor SOX2, which binds in proximity to the CD24 promoter. In GCTs, CD24 expression is controlled by epigenetic mechanisms, that is, histone acetylation, since CD24 can be induced by the application histone deacetylase inhibitors. Vice versa, CD24 expression is downregulated upon inhibition of histone methyltransferases, E3 ubiquitin ligases, or bromodomain (BRD) proteins. Additionally, three-dimensional (3D) co-cultivation of EC cells with microenvironmental cells, such as fibroblasts, and endothelial or immune cells, reduced CD24 expression, suggesting that crosstalk with the somatic microenvironment influences CD24 expression. In a CRISPR/Cas9 deficiency model, we demonstrate that CD24 fulfills a bivalent role in differentiation via regulation of homeobox, and phospho- and glycoproteins; that is, it is involved in suppressing the germ cell/spermatogenesis program and mesodermal/endodermal differentiation, while poising the cells for ectodermal differentiation. Finally, blocking CD24 by a monoclonal antibody enhanced sensitivity toward cisplatin in EC cells, including cisplatin-resistant subclones, highlighting CD24 as a putative target in combination with cisplatin., (© 2021 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Background: Type II germ cell tumors (GCT) are the most common solid cancers in males of age 15 to 35 years. Treatment of these tumors includes cisplatin-based therapy achieving high cure rates, but also leading to late toxicities. As mainly young men are suffering from GCTs, late toxicities play a major role regarding life expectancy, and the development of therapy resistance emphasizes the need for alternative therapeutic options. GCTs are highly susceptible to interference with the epigenetic landscape; therefore, this study focuses on screening of drugs against epigenetic factors as a treatment option for GCTs., Results: We present seven different epigenetic inhibitors efficiently decreasing cell viability in GCT cell lines including cisplatin-resistant subclones at low concentrations by targeting epigenetic modifiers and interactors, like histone deacetylases (Quisinostat), histone demethylases (JIB-04), histone methyltransferases (Chaetocin), epigenetic readers (MZ-1, LP99) and polycomb-repressive complexes (PRT4165, GSK343). Mass spectrometry-based analyses of the histone modification landscape revealed effects beyond the expected mode-of-action of each drug, suggesting a wider spectrum of activity than initially assumed. Moreover, we characterized the effects of each drug on the transcriptome of GCT cells by RNA sequencing and found common deregulations in gene expression of ion transporters and DNA-binding factors. A kinase array revealed deregulations of signaling pathways, like cAMP, JAK-STAT and WNT., Conclusion: Our study identified seven drugs against epigenetic modifiers to treat cisplatin-resistant GCTs. Further, we extensively analyzed off-target effects and modes-of-action, which are important for risk assessment of the individual drugs., (© 2022. The Author(s).)

Background: Treatment of B-cell malignancies with CD19-directed chimeric antigen receptor (CAR) T-cells marked a new era in immunotherapy, which yet has to be successfully adopted to solid cancers. Epigenetic inhibitors of DNA methyltransferases (DNMTi) and histone deacetylases (HDACi) can induce broad changes in gene expression of malignant cells, thus making these inhibitors interesting combination partners for immunotherapeutic approaches., Methods: Urothelial carcinoma cell lines (UCC) and benign uroepithelial HBLAK cells pretreated with the DNMTi decitabine or the HDACi romidepsin were co-incubated with CAR T-cells directed against EGFR or CD44v6, and subsequent cytotoxicity assays were performed. Effects on T-cell cytotoxicity and surface antigen expression on UCC were determined by flow cytometry. We also performed next-generation mRNA sequencing of inhibitor-treated UCC and siRNA-mediated knockdown of potential regulators of CAR T-cell killing., Results: Exposure to decitabine but not romidepsin enhanced CAR T-cell cytotoxicity towards all UCC lines, but not towards the benign HBLAK cells. Increased killing could neither be attributed to enhanced target antigen expression (EGFR and CD44v6) nor fully explained by changes in the T-cell ligands PD-L1, PD-L2, ICAM-1, or CD95. Instead, gene expression analysis suggested that regulators of cell survival and apoptosis were differentially induced by the treatment. Decitabine altered the balance between survival and apoptosis factors towards an apoptosis-sensitive state associated with increased CAR T-cell killing, while romidepsin, at least partially, tilted this balance in the opposite direction. Knockdown experiments with siRNA in UCC confirmed BID and BCL2L1/BCLX as two key factors for the altered susceptibility of the UCC., Conclusion: Our data suggest that the combination of decitabine with CAR T-cell therapy is an attractive novel therapeutic approach to enhance tumor-specific killing of bladder cancer. Since BID and BCL2L1 are essential determinants for the susceptibility of a wide variety of malignant cells, their targeting might be additionally suitable for combination with immunotherapies, e.g., CAR T-cells or checkpoint inhibitors in other malignancies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Grunewald, Haist, König, Petzsch, Bister, Nößner, Wiek, Scheckenbach, Köhrer, Niegisch, Hanenberg and Hoffmann.)

Inflammation after injury of the central nervous system (CNS) is increasingly viewed as a therapeutic target. However, comparative studies in different CNS compartments are sparse. To date only few studies based on immunohistochemical data and all referring to mechanical injury have directly compared inflammation in different CNS compartments. These studies revealed that inflammation is more pronounced in spinal cord than in brain. Therefore, it is unclear whether concepts and treatments established in the cerebral cortex can be transferred to spinal cord lesions and vice versa or whether immunological treatments must be adapted to different CNS compartments. By use of transcriptomic and flow cytometry analysis of equally sized photothrombotically induced lesions in the cerebral cortex and the spinal cord, we could document an overall comparable inflammatory reaction and repair activity in brain and spinal cord between day 1 and day 7 after ischemia. However, remyelination was increased after cerebral versus spinal cord ischemia which is in line with increased remyelination in gray matter in previous analyses and was accompanied by microglia dominated inflammation opposed to monocytes/macrophages dominated inflammation after spinal cord ischemia. Interestingly remyelination could be reduced by microglia and not hematogenous macrophage depletion. Our results show that despite different cellular composition of the postischemic infiltrate the inflammatory response in cerebral cortex and spinal cord are comparable between day 1 and day 7. A striking difference was higher remyelination capacity in the cerebral cortex, which seems to be supported by microglia dominance., (© 2021 The Authors. GLIA published by Wiley Periodicals LLC.)

Background: Transcription factors (TFs) control gene expression by direct binding to regulatory regions of target genes but also by impacting chromatin landscapes and modulating DNA accessibility for other TFs. In recent years several TFs have been defined that control cell fate decisions and effector functions in the immune system. Plasmacytoid dendritic cells (pDCs) are an immune cell type with the unique capacity to produce high amounts of type I interferons quickly in response to contact with viral components. Hereby, this cell type is involved in anti-infectious immune responses but also in the development of inflammatory and autoimmune diseases. To date, the global TF reservoir in pDCs early after activation remains to be fully characterized., Results: To fill this gap, we have performed a comprehensive analysis in naïve versus TLR9-activated murine pDCs in a time course study covering early timepoints after stimulation (2 h, 6 h, 12 h) integrating gene expression (RNA-Seq) and chromatin landscape (ATAC-Seq) studies. To unravel the biological processes underlying the changes in TF expression on a global scale gene ontology (GO) analyses were performed. We found that 70% of all genes annotated as TFs in the mouse genome (1014 out of 1636) are expressed in pDCs for at least one stimulation time point and are covering a wide range of TF classes defined by their specific DNA binding mechanisms. GO analysis revealed involvement of TLR9-induced TFs in epigenetic modulation, NFκB and JAK-STAT signaling, and protein production in the endoplasmic reticulum. pDC activation predominantly "turned on" the chromatin regions associated with TF genes. Our in silico analyses pointed at the AP-1 family of TFs as less noticed but possibly important players in these cells after activation. AP-1 family members exhibit (1) increased gene expression, (2) enhanced chromatin accessibility in their promoter region, and (3) a TF DNA binding motif that is globally enriched in genomic regions that were found more accessible in pDCs after TLR9 activation., Conclusions: In this study we define the complete set of TLR9-regulated TFs in pDCs. Further, this study identifies the AP-1 family of TFs as potentially important but so far less well characterized regulators of pDC function., (© 2021. The Author(s).)

Acute myeloid leukemia (AML) is characterized by an expansion of leukemic cells and a simultaneous reduction of normal hematopoietic precursors in the bone marrow (BM) resulting in hematopoietic insufficiency, but the underlying mechanisms are poorly understood in humans. Assuming that leukemic cells functionally inhibit healthy CD34+ hematopoietic stem and progenitor cells (HSPC) via humoral factors, we exposed healthy BM-derived CD34+ HSPC to cell-free supernatants derived from AML cell lines as well as from 24 newly diagnosed AML patients. Exposure to AML-derived supernatants significantly inhibited proliferation, cell cycling, colony formation, and differentiation of healthy CD34+ HSPC. RNA sequencing of healthy CD34+ HSPC after exposure to leukemic conditions revealed a specific signature of genes related to proliferation, cell-cycle regulation, and differentiation, thereby reflecting their functional inhibition on a molecular level. Experiments with paired patient samples showed that these inhibitory effects are markedly related to the immunomagnetically enriched CD34+ leukemic cell population. Using PCR, ELISA, and RNA sequencing, we detected overexpression of TGFβ1 in leukemic cells on the transcriptional and protein level and, correspondingly, a molecular signature related to TGFβ1 signaling in healthy CD34+ HSPC. This inhibitory effect of TGFβ1 on healthy hematopoiesis was functionally corrobated and could be pharmacologically reverted by SD208, an inhibitor of TGFβ receptor 1 signaling. Overall, these data indicate that leukemic cells induce functional inhibition of healthy CD34+ HSPC, at least in part, through TGFβ1, suggesting that blockage of this pathway may improve hematopoiesis in AML., (© 2021 The Authors. STEM CELLS published by Wiley Periodicals LLC on behalf of AlphaMed Press.)

Background & Aims: Primary sclerosing cholangitis (PSC) is characterized by chronic inflammation and progressive fibrosis of the biliary tree. The bile acid receptor TGR5 (GPBAR1) is found on biliary epithelial cells (BECs), where it promotes secretion, proliferation and tight junction integrity. Thus, we speculated that changes in TGR5-expression in BECs may contribute to PSC pathogenesis., Methods: TGR5-expression and -localization were analyzed in PSC livers and liver tissue, isolated bile ducts and BECs from Abcb4 -/- , Abcb4 -/- /Tgr5 Tg and ursodeoxycholic acid (UDCA)- or 24-norursodeoxycholic acid (norUDCA)-fed Abcb4 -/- mice. The effects of IL8/IL8 homologues on TGR5 mRNA and protein levels were studied. BEC gene expression was analyzed by single-cell transcriptomics (scRNA-seq) from distinct mouse models., Results: TGR5 mRNA expression and immunofluorescence staining intensity were reduced in BECs of PSC and Abcb4 -/- livers, in Abcb4 -/- extrahepatic bile ducts, but not in intrahepatic macrophages. No changes in TGR5 BEC fluorescence intensity were detected in liver tissue of other liver diseases, including primary biliary cholangitis. Incubation of BECs with IL8/IL8 homologues, but not with other cytokines, reduced TGR5 mRNA and protein levels. BECs from Abcb4 -/- mice had lower levels of phosphorylated Erk and higher expression levels of Icam1, Vcam1 and Tgfβ2. Overexpression of Tgr5 abolished the activated inflammatory phenotype characteristic of Abcb4 -/- BECs. NorUDCA-feeding restored TGR5-expression levels in BECs in Abcb4 -/- livers., Conclusions: Reduced TGR5 levels in BECs from patients with PSC and Abcb4 -/- mice promote development of a reactive BEC phenotype, aggravate biliary injury and thus contribute to the pathogenesis of sclerosing cholangitis. Restoration of biliary TGR5-expression levels represents a previously unknown mechanism of action of norUDCA., Lay Summary: Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease-associated with progressive inflammation of the bile duct, leading to fibrosis and end-stage liver disease. Bile acid (BA) toxicity may contribute to the development and disease progression of PSC. TGR5 is a membrane-bound receptor for BAs, which is found on bile ducts and protects bile ducts from BA toxicity. In this study, we show that TGR5 levels were reduced in bile ducts from PSC livers and in bile ducts from a genetic mouse model of PSC. Our investigations indicate that lower levels of TGR5 in bile ducts may contribute to PSC development and progression. Furthermore, treatment with norUDCA, a drug currently being tested in a phase III trial for PSC, restored TGR5 levels in biliary epithelial cells., Competing Interests: Conflict of interest MR, LS, CK, KF, JS, KD, JH, EL, JV, SFG, GH, SJF, IE, DN, CDF, MGB, GG, SV, JPM, MV, KS, TL, KK, DH, TL, MH have nothing to disclose.VK has served as speaker for Falk Foundation, Albireo and Abbvie. JRH has served on the advisory board for Novartis and Orkla Health. JRH has received research support from Biogen unrelated to the current work. THK has received consulting/speaker reimbursement from Gilead and Intercept unrelated to the current work. CS has served as speaker for Falk Foundation. MT has served as speaker for Falk Foundation, Gilead, Intercept and MSD; he has advised for Albireo, BiomX, Boehringer Ingelheim, Falk Pharma GmbH, Genfit, Gilead, Intercept, Jannsen, MSD, Novartis, Phenex, Regulus and Shire. MT further received travel grants from Abbvie, Falk, Gilead and Intercept and research grants from Albireo, Cymabay, Falk, Gilead, Intercept, MSD and Takeda. MT is also co-inventor of patents on the medical use of NorUDCA filed by the Medical Universities of Graz and Vienna (WO2006119803, WO20099013334). PF received norUDCA, an unrestricted research grant, travel grants and advisory board fees from Dr. Falk Pharma GmbH. PF is listed as co-inventor of patents for the medical use of norUDCA filed by the Medical University of Graz (WO2006119803, WO20099013334). UB received grants for investigator-initiated studies by Dr Falk GmbH, Freiburg, and Intercept, San Diego., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Nine strains of a Rodentibacter -related bacterium were isolated over a period of 38 years from a laboratory mouse ( Mus musculus ), seven laboratory rats ( Rattus norvegicus ) and a Syrian hamster ( Mesocricetus auratus ) in Düsseldorf and Heidelberg, Germany. The isolates are genotypically and phenotypically distinct from all previously described Rodentibacter species. Sequence analysis of 16S rRNA and rpoB gene sequences placed the isolates as a novel lineage within the genus Rodentibacter . In addition to the single-gene analysis, the whole genome sequence of the strain 1625/19 T revealed distinct genome-to-genome distance values to the other Rodentibacter species. The genomic DNA G+C content of strain 1625/19 T was 40.8 mol% within the range of Rodentibacter . At least six phenotypic characteristics separate the new isolates from the other Rodentibacter species, with Rodentibacter heylii being the most closely related. In contrast to the latter, the new strains display β-haemolysis and are β-glucuronidase, d-mannitol and sorbitol positive, but fail to produce lysine decarboxylase and trehalose. The genotypic and phenotypic differences between the novel strains and the other closely related strains of the genus Rodentibacter indicate that they represent a novel species within the genus Rodentibacter , family Pasteurellaceae , for which the name Rodentibacter haemolyticus sp. nov. is proposed. The type strain 1625/19 T , (=DSM 111151 T =CCM 9081 T ), was isolated in 2019 from the nose of a laboratory mouse ( Mus musculus ) in Düsseldorf, Germany.

N-hydroxypipecolic acid (NHP) accumulates in the plant foliage in response to a localized microbial attack and induces systemic acquired resistance (SAR) in distant leaf tissue. Previous studies indicated that pathogen inoculation of Arabidopsis (Arabidopsis thaliana) systemically activates SAR-related transcriptional reprogramming and a primed immune status in strict dependence of FLAVIN-DEPENDENT MONOOXYGENASE 1 (FMO1), which mediates the endogenous biosynthesis of NHP. Here, we show that elevations of NHP by exogenous treatment are sufficient to induce a SAR-reminiscent transcriptional response that mobilizes key components of immune surveillance and signal transduction. Exogenous NHP primes Arabidopsis wild-type and NHP-deficient fmo1 plants for a boosted induction of pathogen-triggered defenses, such as the biosynthesis of the stress hormone salicylic acid (SA), accumulation of the phytoalexin camalexin and branched-chain amino acids, as well as expression of defense-related genes. NHP also sensitizes the foliage systemically for enhanced SA-inducible gene expression. NHP-triggered SAR, transcriptional reprogramming, and defense priming are fortified by SA accumulation, and require the function of the transcriptional coregulator NON-EXPRESSOR OF PR GENES1 (NPR1). Our results suggest that NPR1 transduces NHP-activated immune signaling modes with predominantly SA-dependent and minor SA-independent features. They further support the notion that NHP functions as a mobile immune regulator capable of moving independently of active SA signaling between leaves to systemically activate immune responses., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

In the adult heart, the epicardium becomes activated after injury, contributing to cardiac healing by secretion of paracrine factors. Here, we analyzed by single-cell RNA sequencing combined with RNA in situ hybridization and lineage tracing of Wilms tumor protein 1-positive (WT1 + ) cells, the cellular composition, location, and hierarchy of epicardial stromal cells (EpiSC) in comparison to activated myocardial fibroblasts/stromal cells in infarcted mouse hearts. We identified 11 transcriptionally distinct EpiSC populations, which can be classified into three groups, each containing a cluster of proliferating cells. Two groups expressed cardiac specification markers and sarcomeric proteins suggestive of cardiomyogenic potential. Transcripts of hypoxia-inducible factor (HIF)-1α and HIF-responsive genes were enriched in EpiSC consistent with an epicardial hypoxic niche. Expression of paracrine factors was not limited to WT1 + cells but was a general feature of activated cardiac stromal cells. Our findings provide the cellular framework by which myocardial ischemia may trigger in EpiSC the formation of cardioprotective/regenerative responses., Competing Interests: JH, CO, TL, RZ, JW, ZD, CA, AL, MG, NG, JF, GK, CD, KK, JS No competing interests declared, (© 2021, Hesse et al.)

Lymphotoxin-β-receptor deficient (LTβR -/- ) and Tumor Necrosis Factor Receptor p55 deficient (TNFRp55 -/- ) mice show defects in liver regeneration (LR) after partial hepatectomy (PHx) with significantly increased mortality. LTβR and TNFRp55 belong to the core members of the TNF/TNFR superfamily. Interestingly, combined failure of LTβR and TNFRp55 signaling after PHx leads to a complete defect in LR. Here, we first addressed the question which liver cell population crucially requires LTβR signaling for efficient LR. To this end, mice with a conditionally targeted LTβR allele (LTβR fl/fl ) were crossed to AlbuminCre and LysozymeMCre mouse lines to unravel the function of the LTβR on hepatocytes and monocytes/macrophages/Kupffer cells, respectively. Analysis of these mouse lines clearly reveals that LTβR is required on hepatocytes for efficient LR while no deficit in LR was found in LTβR fl/fl  × LysMCre mice. Second, the molecular basis for the cooperating role of LTβR and TNFRp55 signaling pathways in LR was investigated by transcriptome analysis of etanercept treated LTβR -/- (LTβR -/- /ET) mice. Bioinformatic analysis and subsequent verification by qRT-PCR identified novel target genes (Cyclin-L2, Fas-Binding factor 1, interferon-related developmental regulator 1, Leucyl-tRNA Synthetase 2, and galectin-4) that are upregulated by LTβR/TNFRp55 signaling after PHx and fail to be upregulated after PHx in LTβR -/- /ET mice., (© 2021 Ursula R. Sorg et al., published by De Gruyter, Berlin/Boston.)

The lymphotoxin β receptor (LTβR) plays an essential role in the initiation of immune responses to intracellular pathogens. In mice, the LTβR is crucial for surviving acute toxoplasmosis; however, until now, a functional analysis was largely incomplete. Here, we demonstrate that the LTβR is a key regulator required for the intricate balance of adaptive immune responses. Toxoplasma gondii -infected LTβR-deficient (LTβR -/- ) mice show globally altered interferon-γ (IFN-γ) regulation, reduced IFN-γ-controlled host effector molecule expression, impaired T cell functionality, and an absent anti-parasite-specific IgG response, resulting in a severe loss of immune control of the parasites. Reconstitution of LTβR -/- mice with toxoplasma immune serum significantly prolongs survival following T. gondii infection. Notably, analysis of RNA-seq data clearly indicates a specific effect of T. gondii infection on the B cell response and isotype switching. This study uncovers the decisive role of the LTβR in cytokine regulation and adaptive immune responses to control T. gondii ., (Copyright © 2021 Tersteegen et al.)

Leaves are asymmetric, with different functions for adaxial and abaxial tissue. The bundle sheath (BS) of C3 barley (Hordeum vulgare) is dorsoventrally differentiated into three types of cells: adaxial structural, lateral S-type, and abaxial L-type BS cells. Based on plasmodesmatal connections between S-type cells and mestome sheath (parenchymatous cell layer below bundle sheath), S-type cells likely transfer assimilates toward the phloem. Here, we used single-cell RNA sequencing to investigate BS differentiation in C4 maize (Zea mays L.) plants. Abaxial BS (abBS) cells of rank-2 intermediate veins specifically expressed three SWEET sucrose uniporters (SWEET13a, b, and c) and UmamiT amino acid efflux transporters. SWEET13a, b, c mRNAs were also detected in the phloem parenchyma (PP). We show that maize has acquired a mechanism for phloem loading in which abBS cells provide the main route for apoplasmic sucrose transfer toward the phloem. This putative route predominates in veins responsible for phloem loading (rank-2 intermediate), whereas rank-1 intermediate and major veins export sucrose from the PP adjacent to the sieve element companion cell complex, as in Arabidopsis thaliana. We surmise that abBS identity is subject to dorsoventral patterning and has components of PP identity. These observations provide insights into the unique transport-specific properties of abBS cells and support a modification to the canonical phloem loading pathway in maize., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Infection with the obligate intracellular bacterium Chlamydia trachomatis is the most common bacterial sexually transmitted disease worldwide. Since no vaccine is available to date, antimicrobial therapy is the only alternative in C. trachomatis infection. However, changes in chlamydial replicative activity and the occurrence of chlamydial persistence caused by diverse stimuli have been proven to impair treatment effectiveness. Here, we report the mechanism for C. trachomatis regulating host signaling processes and mitochondrial function, which can be used for chlamydial metabolic reprogramming during treatment with β-lactam antimicrobials. Activation of signal transducer and activator of transcription 3 (STAT3) is a well-known host response in various bacterial and viral infections. In C. trachomatis infection, inactivation of STAT3 by host protein tyrosine phosphatases increased mitochondrial respiration in both the absence and presence of β-lactam antimicrobials. However, during treatment with β-lactam antimicrobials, C. trachomatis increased the production of citrate as well as the activity of host ATP-citrate lyase involved in fatty acid synthesis. Concomitantly, chlamydial metabolism switched from the tricarboxylic acid cycle to fatty acid synthesis. This metabolic switch was a unique response in treatment with β-lactam antimicrobials and was not observed in gamma interferon (IFN-γ)-induced persistent infection. Inhibition of fatty acid synthesis was able to attenuate β-lactam-induced chlamydial persistence. Our findings highlight the importance of the mitochondrion-fatty acid interplay for the metabolic reprogramming of C. trachomatis during treatment with β-lactam antimicrobials. IMPORTANCE The mitochondrion generates most of the ATP in eukaryotic cells, and its activity is used for controlling the intracellular growth of Chlamydia trachomatis Furthermore, mitochondrial activity is tightly connected to host fatty acid synthesis that is indispensable for chlamydial membrane biogenesis. Phospholipids, which are composed of fatty acids, are the central components of the bacterial membrane and play a crucial role in the protection against antimicrobials. Chlamydial persistence that is induced by various stimuli is clinically relevant. While one of the well-recognized inducers, β-lactam antimicrobials, has been used to characterize chlamydial persistence, little is known about the role of mitochondria in persistent infection. Here, we demonstrate how C. trachomatis undergoes metabolic reprogramming to switch from the tricarboxylic acid cycle to fatty acid synthesis with promoted host mitochondrial activity in response to treatment with β-lactam antimicrobials., (Copyright © 2021 Shima et al.)

Myelinating oligodendrocytes (OLs) establish saltatory nerve conduction during white matter development. Thus, interference with oligodendrogenesis leads to an adverse outcome on brain performance in the child due to aberrant myelination. An intertwined network of hormonal, transcriptional and biosynthetic processes regulates OL development, thereby simultaneously creating various routes of interference for environmental toxicants. The flame retardant tetrabromobisphenol A (TBBPA) is debated as an endocrine disruptor, especially of the thyroid hormone (TH) system. We identified how TBBPA interferes with the establishment of a population of maturing OLs by two independent modes-of-action (MoA), dependent and independent of TH signaling. Combining the previously published oligodendrocyte maturation assay (NPC6) with large-scale transcriptomics, we describe TBBPA as a TH disruptor, impairing human OL maturation in vitro by dysregulation of oligodendrogenesis-associated genes (i.e., MBP, KLF9 and EGR1). Furthermore, TBBPA disrupts a gene expression network regulating cholesterol homeostasis, reducing OL numbers independently of TH signaling. These two MoA converge in a novel putative adverse outcome pathway (AOP) network on the key event (KE) hypomyelination. Comparative analyses of human and rat neural progenitor cells (NPCs) revealed that human oligodendrogenesis is more sensitive to endocrine disruption by TBBPA. Therefore, ethical, cost-efficient and species-overarching in vitro assays are needed for developmental neurotoxicity hazard assessment. By incorporation of large-scale transcriptomic analyses, we brought the NPC6 assay to a higher readiness level for future applications in a regulatory context. The combination of phenotypic and transcriptomic analyses helps to study MoA to eventually build AOPs for a better understanding of neurodevelopmental toxicity.

Human genomes contain about 100,000 LINE-1 (L1) retroelements, of which more than 100 are intact. L1s are normally tightly controlled by epigenetic mechanisms, which often fail in cancer. In bladder urothelial carcinoma (UC), particularly, L1s become DNA-hypomethylated, expressed and contribute to genomic instability and tumor growth. It is, however, unknown which individual L1s are activated. Following RNA-immunoprecipitation with a L1-specific antibody, third generation nanopore sequencing detected transcripts of 90 individual elements in the VM-Cub-1 UC line with high overall L1 expression. In total, 10 L1s accounted for >60% of the reads. Analysis of five specific L1s by RT-qPCR revealed generally increased expression in UC tissues and cell lines over normal controls, but variable expression among tumor cell lines from bladder, prostate and testicular cancer. Chromatin immunoprecipitation demonstrated active histone marks at L1 sequences with increased expression in VM-Cub-1, but not in a different UC cell line with low L1 expression. We conclude that many L1 elements are epigenetically activated in bladder cancer in a varied pattern. Our findings indicate that expression of individual L1s is highly heterogeneous between and among cancer types.

After myocardial infarction (MI), epicardial cells reactivate their embryonic program, proliferate and migrate into the damaged tissue to differentiate into fibroblasts, endothelial cells and, if adequately stimulated, to cardiomyocytes. Targeting epicardium-derived stromal cells (EpiSC) by specific ligands might enable the direct imaging of EpiSCs after MI to better understand their biology, but also may permit the cell-specific delivery of small molecules to improve the post-MI healing process. Therefore, the aim of this study was to identify specific peptides by phage display screening to enable EpiSC specific cargo delivery by active targeting. To this end, we utilized a sequential panning of a phage library on cultured rat EpiSCs and then subtracted phage that nonspecifically bound blood immune cells. EpiSC specific phage were analyzed by deep sequencing and bioinformatics analysis to identify a total of 78 300 ± 31 900 different, EpiSC-specific, peptide insertion sequences. Flow cytometry of the five most highly abundant peptides (EP1, -2, -3, -7 or EP9) showed strong binding to EpiSCs but not to blood immune cells. The best binding properties were found for EP9 which was further studied by surface plasmon resonance (SPR). SPR revealed rapid and stable association of EpiSCs with EP9. As a negative control, THP-1 monocytes did not associate with EP9. Coupling of EP9 to perfluorocarbon nanoemulsions (PFCs) resulted in the efficient delivery of 19 F cargo to EpiSCs and enabled their visualization by 19 F MRI. Moreover, active targeting of EpiSCs by EP9-labelled PFCs was able to outcompete the strong phagocytic uptake of PFCs by circulating monocytes. In summary, we have identified a 7-mer peptide, (EP9) that binds to EpiSCs with high affinity and specificity. This peptide can be used to deliver small molecule cargos such as contrast agents to permit future in vivo tracking of EpiSCs by molecular imaging and to transfer small pharmaceutical molecules to modulate the biological activity of EpiSCs.

The internal transcribed spacer (ITS) regions of Rodentibacter pneumotropicus , R. heylii , R. rarus , R. ratti , and R. heidelbergensis and of a Rodentibacter- related β-hemolytic Pasteurellaceae taxon isolated from laboratory rodents were studied for their feasibility to discriminate among these species. The 6 species analyzed showed species-specific ITS patterns that were shared by the type strains and clinical isolates and that allowed their identification. Nevertheless, differentiating between the ITS band patterns of R. pneumotropicus and R. ratti is visually challenging. In all species tested, sequence analysis of the ITS fragments revealed a larger ITS ile+ala , which contained the genes for tRNA Ile(GAU) and tRNA Ala(UGC) , and a smaller ITS glu with the tRNA Glu(UUC) gene. The ITS sequences varied among the 6 species evaluated, displaying identity levels ranging from 62% to 86% for ITS ile+ala and 68% to 90% for ITS glu . Overall, ITS amplification proved to be a reliable method to differentiate among these important Pasteurellaceae species of laboratory rodents. Moreover, the ITS sequence variations recorded here might facilitate the design of probes for specific identification of these species. The ability to diagnose these organisms to the species level could increase our understanding of their clinical significance.

Background: Mobile genetic elements are found in genomes throughout the microbial world, mediating genome plasticity and important prokaryotic phenotypes. Even the cell wall-less mycoplasmas, which are known to harbour a minimal set of genes, seem to accumulate mobile genetic elements. In Mycoplasma hominis, a facultative pathogen of the human urogenital tract and an inherently very heterogeneous species, four different MGE-classes had been detected until now: insertion sequence ISMhom-1, prophage MHoV-1, a tetracycline resistance mediating transposon, and ICEHo, a species-specific variant of a mycoplasma integrative and conjugative element encoding a T4SS secretion system (termed MICE)., Results: To characterize the prevalence of these MGEs, genomes of 23 M. hominis isolates were assembled using whole genome sequencing and bioinformatically analysed for the presence of mobile genetic elements. In addition to the previously described MGEs, a new ICEHo variant was found, which we designate ICEHo-II. Of 15 ICEHo-II genes, five are common MICE genes; eight are unique to ICEHo-II; and two represent a duplication of a gene also present in ICEHo-I. In 150 M. hominis isolates and based on a screening PCR, prevalence of ICEHo-I was 40.7%; of ICEHo-II, 28.7%; and of both elements, 15.3%. Activity of ICEHo-I and -II was demonstrated by detection of circularized extrachromosomal forms of the elements through PCR and subsequent Sanger sequencing., Conclusions: Nanopore sequencing enabled the identification of mobile genetic elements and of ICEHo-II, a novel MICE element of M. hominis, whose phenotypic impact and potential impact on pathogenicity can now be elucidated.

The majority of childhood leukemias are precursor B-cell acute lymphoblastic leukemias (pB-ALLs) caused by a combination of prenatal genetic predispositions and oncogenic events occurring after birth. Although genetic predispositions are frequent in children (>1% to 5%), fewer than 1% of genetically predisposed carriers will develop pB-ALL. Although infectious stimuli are believed to play a major role in leukemogenesis, the critical determinants are not well defined. Here, by using murine models of pB-ALL, we show that microbiome disturbances incurred by antibiotic treatment early in life were sufficient to induce leukemia in genetically predisposed mice, even in the absence of infectious stimuli and independent of T cells. By using V4 and full-length 16S ribosomal RNA sequencing of a series of fecal samples, we found that genetic predisposition to pB-ALL (Pax5 heterozygosity or ETV6-RUNX1 fusion) shaped a distinct gut microbiome. Machine learning accurately (96.8%) predicted genetic predisposition using 40 of 3983 amplicon sequence variants as proxies for bacterial species. Transplantation of either wild-type (WT) or Pax5+/- hematopoietic bone marrow cells into WT recipient mice revealed that the microbiome is shaped and determined in a donor genotype-specific manner. Gas chromatography-mass spectrometry (GC-MS) analyses of sera from WT and Pax5+/- mice demonstrated the presence of a genotype-specific distinct metabolomic profile. Taken together, our data indicate that it is a lack of commensal microbiota rather than the presence of specific bacteria that promotes leukemia in genetically predisposed mice. Future large-scale longitudinal studies are required to determine whether targeted microbiome modification in children predisposed to pB-ALL could become a successful prevention strategy., (© 2020 by The American Society of Hematology.)

Background: Germ cell tumours (GCTs) are the most common solid malignancies in young men. Although high cure rates can be achieved, metastases, resistance to cisplatin-based therapy and late toxicities still represent a lethal threat, arguing for the need of new therapeutic options. In this study, we analysed the potential of cyclin-dependent kinase 4/6 (CDK4/6) inhibitors palbociclib and ribociclib (PaRi) as molecular drugs to treat cisplatin-resistant and -sensitive paediatric and adult GCTs., Methods: Ten GCT cell lines, including cisplatin-resistant subclones and non-malignant controls, were treated with PaRi and screened for changes in viability (triphenyl tetrazolium chloride (XTT) assay), apoptosis rates (flow cytometry, caspase assay), the cell cycle (flow cytometry), the transcriptome (RNA-sequencing, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and on protein level (western blot). Expression profiling was performed on paediatric and adult GCT tissues (expression microarrays, qRT-PCR, immunohistochemistry, 'The Cancer Genome Atlas' database)., Results: We demonstrate that adult GCTs highly express CDK4, while paediatric GCTs strongly express CDK6 instead. Thus, both GCT types are potentially treatable by PaRi. GCTs presented as highly sensitive towards PaRi, which caused a decrease in viability, cell cycle arrest and apoptosis. Although GCTs mainly arrested in the G1/G0 phase, some embryonal carcinoma cell lines were able to bypass the G1/S checkpoint and progressed to the G2/M phase. We found that upregulation of CDK3 and downregulation of many mitosis regulation factors, like the HAUS genes, might be responsible for bypassing the G1/S checkpoint and termination of mitosis, respectively. We postulate that GCT cells do not tolerate these alterations in the cell cycle and eventually induce apoptosis., Conclusion: Our study highlights PaRi as therapeutic options for cisplatin-resistant and -sensitive paediatric and adult GCTs.

Neurotoxicity is mediated by a variety of modes-of-actions leading to disturbance of neuronal function. In order to screen larger numbers of compounds for their neurotoxic potential, in vitro functional neuronal networks (NN) might be helpful tools. We established and characterized human NN (hNN) from hiPSC-derived neural progenitor cells by comparing hNN formation with two different differentiation media: in presence (CINDA) and absence (neural differentiation medium (NDM)) of maturation-supporting factors. As a NN control we included differentiating rat NN (rNN) in the study. Gene/protein expression and electrical activity from in vitro developing NN were assessed at multiple time points. Transcriptomes of 5, 14 and 28 days in vitro CINDA-grown hNN were compared to gene expression profiles of in vivo human developing brains. Molecular expression analyses as well as measures of electrical activity indicate that NN mature into neurons of different subtypes and astrocytes over time. In contrast to rNN, hNN are less electrically active within the same period of differentiation time, yet hNN grown in CINDA medium develop higher firing rates than hNN without supplements. Challenge of NN with neuronal receptor stimulators and inhibitors demonstrate presence of inhibitory, GABAergic neurons, whereas glutamatergic responses are limited. hiPSC-derived GABAergic hNN grown in CINDA medium might be a useful tool as part of an in vitro battery for assessing neurotoxicity., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

The initial phases of neuronal differentiation are key to neuronal function. A particularly informative model to study these initial phases are retinoic acid-stimulated SH-SY5Y cells. Although these progressions are associated with redox-sensitive processes, it is largely undefined how the cellular proteome underpins redox dynamics and the management of reactive oxygen species. Here, we map the global cysteine-based redox landscape of SH-SY5Y cells using quantitative redox proteomics. We find evidence that redox alterations occurred early in differentiation and affect the expression of neuronal marker proteins and the extension of neurites. The spatiotemporal analysis of reactive oxygen species suggests a NOX2-dependent peak in cytoplasmic superoxide anions/hydrogen peroxide generation 2 h after retinoic acid stimulation. At the same time point, 241 out of 275 proteins with an altered cysteine redox state are reversibly oxidized in response to retinoic acid. Our analyses pinpoint redox alterations of proteins involved in the retinoic acid homeostasis and cytoskeletal dynamics.

The histone demethylase UTX (gene: KDM6A ) directs cell and tissue differentiation during development. Deleterious mutations in KDM6A occur in many human cancers, most frequently in urothelial carcinoma. The consequences of these mutations are poorly understood; plausibly, they may disturb urothelial differentiation. We therefore investigated the effects of UTX siRNA-mediated knockdown in two in vitro models of urothelial differentiation; namely, primary cultures of urothelial epithelial cells treated with troglitazone and PD153035 and the immortalized urothelial cell line HBLAK treated with high calcium and serum. In both models, efficient UTX knockdown did not block morphological and biochemical differentiation. An apparent delay was due to a cytotoxic effect on the cell cultures before the initiation of differentiation, which induced apoptosis partly in a p53-dependent manner. As a consequence, slowly cycling, smaller, KRT14 high precursor cells in the HBLAK cell line were enriched at the expense of more differentiated, larger, proliferating KRT14 low cells. UTX knockdown induced apoptosis and enriched KRT14 high cells in the BFTC-905 papillary urothelial carcinoma cell line as well. Our findings suggest an explanation for the frequent occurrence of KDM6A mutations across all stages and molecular subtypes of urothelial carcinoma, whereby loss of UTX function does not primarily impede later stages of urothelial differentiation, but favors the expansion of precursor populations to provide a reservoir of potential tumor-initiating cells.

Aims: Myocardial infarction (MI) leads to activation of cardiac fibroblasts (aCFs) and at the same time induces the formation of epicardium-derived cells at the heart surface. To discriminate between the two cell populations, we elaborated a fast and efficient protocol for the simultaneous isolation and characterization of aCFs and epicardial stromal cells (EpiSCs) from the infarcted mouse heart., Methods and Results: For the isolation of aCFs and EpiSCs, infarcted hearts (50 min ischaemia/reperfusion) were digested by perfusion with a collagenase-containing medium for only 8 min, while EpiSCs were enzymatically removed from the outside by applying mild shear forces via a motor driven device. Cardiac fibroblasts (CFs) isolated from unstressed hearts served as control. Viability of isolated cells was >90%. Purity of EpiSCs was confirmed by immunofluorescence staining and qPCR of various mesenchymal markers including Wilms-tumor-protein-1. Microarray analysis of CFs, aCFs, and EpiSCs on day 5 post-MI revealed a unique gene expression pattern in the EpiSC fraction, which was enriched for epithelial markers and epithelial to mesenchymal transition-related genes. Compared to aCFs, 336 significantly altered gene entities were identified in the EpiSC fraction. qPCR analysis showed high expression of Serpinb2, Cxcl13, Adora2b, and Il10 in EpiSCs relative to CFs and aCFs. Furthermore, microarray data identified Ddah1 and Cemip to be highly up-regulated in aCFs compared to CFs. Immunostaining of the infarcted heart revealed a unique distribution of Dermokine, Aquaporin-1, Cytokeratin, Lipocalin2, and Periostin within the epicardial cell layer., Conclusions: We describe the simultaneous isolation of viable, purified fractions of aCFs and EpiSCs from the infarcted mouse heart. In this study, several differentially expressed markers for aCFs and EpiSCs were identified, underlining the importance of cell separation to study heterogeneity of stromal cells in the healing process after MI., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

Background Aryl hydrocarbon receptor (AHR)-deficient mice do not support the expansion of dendritic epidermal T cells (DETC), a resident immune cell population in the murine epidermis, which immigrates from the fetal thymus to the skin around birth. Material and Methods In order to identify the gene expression changes underlying the DETC disappearance in AHR-deficient mice, we analyzed microarray RNA-profiles of DETC, sorted from the skin of two-week-old AHR-deficient mice and their heterozygous littermates. In vitro studies were done for verification, and IL-10, AHR repressor (AHRR), and c-Kit deficient mice analyzed for DETC frequency. Results We identified 434 annotated differentially expressed genes. Gene set enrichment analysis demonstrated that the expression of genes related to proliferation, ion homeostasis and morphology differed between the two mouse genotypes. Importantly, with 1767 pathways the cluster-group "inflammation" contained the majority of AHR-dependently regulated pathways. The most abundant cluster of differentially expressed genes was "inflammation." DETC of AHR-deficient mice were inflammatory active and had altered calcium and F-actin levels. Extending the study to the AHRR, an enigmatic modulator of AHR-activity, we found approximately 50% less DETC in AHRR-deficient mice than in wild-type-littermates. Conclusion AHR-signaling in DETC dampens their inflammatory default potential and supports their homeostasis in the skin.

Macrophages have important protective functions during infection with herpes simplex virus type 1 (HSV-1). However, molecular mechanisms that restrict viral propagation and protect from severe disease are unclear. Here we show that macrophages take up HSV-1 via endocytosis and transport the virions into multivesicular bodies (MVBs). In MVBs, acid ceramidase (aCDase) converts ceramide into sphingosine and increases the formation of sphingosine-rich intraluminal vesicles (ILVs). Once HSV-1 particles reach MVBs, sphingosine-rich ILVs bind to HSV-1 particles, which restricts fusion with the limiting endosomal membrane and prevents cellular infection. Lack of aCDase in macrophage cultures or in Asah1 -/- mice results in replication of HSV-1 and Asah1 -/- mice die soon after systemic or intravaginal inoculation. The treatment of macrophages with sphingosine enhancing compounds blocks HSV-1 propagation, suggesting a therapeutic potential of this pathway. In conclusion, aCDase loads ILVs with sphingosine, which prevents HSV-1 capsids from penetrating into the cytosol.

During invasion of host cells, Chlamydia pneumoniae secretes the effector protein CPn0678, which facilitates internalization of the pathogen by remodeling the target cell's plasma membrane and recruiting sorting nexin 9 (SNX9), a central multifunctional endocytic scaffold protein. We show here that the strongly amphipathic N-terminal helix of CPn0678 mediates binding to phospholipids in both the plasma membrane and synthetic membranes, and is sufficient to induce extensive membrane tubulations. CPn0678 interacts via its conserved C-terminal polyproline sequence with the Src homology 3 domain of SNX9. Thus, SNX9 is found at bacterial entry sites, where C. pneumoniae is internalized via EGFR-mediated endocytosis. Moreover, depletion of human SNX9 significantly reduces internalization, whereas ectopic overexpression of CPn0678-GFP results in a dominant-negative effect on endocytotic processes in general, leading to the uptake of fewer chlamydial elementary bodies and diminished turnover of EGFR. Thus, CPn0678 is an early effector involved in regulating the endocytosis of C. pneumoniae in an EGFR- and SNX9-dependent manner., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Occurrence of cisplatin-resistance in bladder cancer is frequent and results in disease progression. Thus, novel therapeutic approaches are a high medical need for patients suffering from chemotherapy failure. The purpose of this study was to test the combination of the DNA methyltransferase inhibitor decitabine (DAC) with the histone deacetylase inhibitor entinostat (ENT) in bladder cancer cells with different platinum sensitivities: J82, cisplatin-resistant J82CisR, and RT-112. Intermittent treatment of J82 cells with cisplatin resulted in the six-fold more cisplatin-resistant cell line J82CisR. Combinations of DAC and/or ENT plus cisplatin could not reverse chemoresistance. However, the combination of DAC and ENT acted cytotoxic in a highly synergistic manner as shown by Chou-Talalay analysis via induction of apoptosis and cell cycle arrest. Importantly, this effect was cancer cell-selective as no synergism was found for the combination in the non-cancerous urothelial cell line HBLAK. Expression analysis indicated that epigenetic treatment led to up-regulation of forkhead box class O1 (FoxO1) and further activated proapoptotic Bim and the cell cycle regulator p21 and reduced expression of survivin in J82CisR. In conclusion, the combination of DAC and ENT is highly synergistic and has a promising potential for therapy of bladder cancer, particularly in cases with platinum resistance.

TGR5 (Gpbar1) is a G protein-coupled receptor responsive to bile acids (BAs), which is expressed in different non-parenchymal cells of the liver, including biliary epithelial cells, liver-resident macrophages, sinusoidal endothelial cells (LSECs), and activated hepatic stellate cells (HSCs). Mice with targeted deletion of TGR5 are more susceptible towards cholestatic liver injury induced by cholic acid-feeding and bile duct ligation, resulting in a reduced proliferative response and increased liver injury. Conjugated lithocholic acid (LCA) represents the most potent TGR5 BA ligand and LCA-feeding has been used as a model to rapidly induce severe cholestatic liver injury in mice. Thus, TGR5 knockout (KO) mice and wildtype (WT) littermates were fed a diet supplemented with 1% LCA for 84 h. Liver injury and gene expression changes induced by the LCA diet revealed an enrichment of pathways associated with inflammation, proliferation, and matrix remodeling. Knockout of TGR5 in mice caused upregulation of endothelin-1 (ET-1) expression in the livers. Analysis of TGR5-dependent ET-1 signaling in isolated LSECs and HSCs demonstrated that TGR5 activation reduces ET-1 expression and secretion from LSECs and triggers internalization of the ET-1 receptor in HSCs, dampening ET-1 responsiveness. Thus, we identified two independent mechanisms by which TGR5 inhibits ET-1 signaling and modulates portal pressure.

The DNA-damaging compound cisplatin is broadly employed for cancer chemotherapy. The mutagenic effects of cisplatin on cancer cell genomes are poorly studied and might even contribute to drug resistance. We have therefore analyzed mutations and chromosomal alterations in four cisplatin-resistant bladder cancer cell lines (LTTs) by whole-exome-sequencing and array-CGH. 720-7479 genes in the LTTs contained point mutations, with a characteristic mutational signature. Only 53 genes were mutated in all LTTs, including the presumed cisplatin exporter ATP7B. Chromosomal alterations were characterized by segmented deletions and gains leading to severely altered karyotypes. The few chromosomal changes shared among LTTs included gains involving the anti-apoptotic BCL2L1 gene and losses involving the NRF2 regulator KEAP1. Overall, the extent of genomic changes paralleled cisplatin treatment concentrations. In conclusion, bladder cancer cell lines selected for cisplatin-resistance contain abundant and characteristic drug-induced genomic changes. Cisplatin treatment may therefore generate novel tumor genomes during patient treatment.

The extra-hospital epidemiology of Acinetobacter infections is a subject of debate. In recent years, the prevalence of animal multidrug-resistant Acinetobacter infections has increased considerably. The goal of the present study was to specify Acinetobacter species isolated from laboratory mice and to test them for their antimicrobial susceptibility. During routine microbiological monitoring of laboratory mice, 12 Acinetobacter spp. were isolated. By means of 16S rRNA and rpoB gene sequencing, seven of the isolates were identified as Acinetobacter radioresistens , three isolates belonged to Acinetobacter genomospecies 14BJ, one isolate was classified as Acinetobacter pitii and one as Acinetobacter sp. ANC 4051. The distribution of the minimal inhibitory concentration (MIC) values was uniform for 21 of the 23 antimicrobial agents tested, whereas a broad MIC distribution was recorded for tulathromycin and streptomycin. The MIC values recorded were low for the majority of the antibiotics tested. Nevertheless, very high MIC values, which will probably render a therapeutic approach using these substances unsuccessful, were recorded for florfenicol, tiamulin, tilmicosin and cephalothin in most of the isolates. In conclusion, we document colonization of laboratory mice with different Acinetobacter species, displaying similar antibiotic susceptibility profiles, with possible implications in the Acinetobacter epidemiology as well as in the husbandry and experimentation of the colonized animals.

Introduction: Recent evidence shows that numerous microRNAs (miRNAs) regulate pain-related genes in chronic pain. The aim of the present study was to further explore the regulation of miRNAs and their effect on the expression of pain-associated target genes in experimental neuropathic pain., Methods: Male Wistar rats underwent chronic constriction injury (CCI) of the sciatic nerve or Sham procedure. After assessment of mechanical allodynia, the ipsilateral dorsal root ganglia (DRG) were harvested. MiRNA expression levels were analysed with Agilent microRNA microarrays and real time quantitative PCR. An interaction between miRNAs and pain-relevant genes was confirmed by luciferase assays. Western Blot analysis and ELISA were performed to evaluate protein expression, respectively., Results: Mechanical allodynia developed within 6 days after CCI. MiRNA-arrays revealed the differential expression of 49 miRNAs after 4 h, of 3 miRNAs after 1 d, of 26 miRNAs after 6 d and of 28 miRNAs after 12 d in the CCI group versus Sham. Time-dependent down regulation of miR-34a was verified by qPCR. Bioinformatic prediction revealed an interaction with several pain-relevant targets including voltage-gated sodium channel β2 subunit (SCN2B) and vesicle-associated membrane protein 2 (VAMP-2), both of which were subsequently confirmed by luciferase assay. VAMP-2 expression was statistically significantly increased 12 d after CCI. A non-significant upregulation of SCN2B in the DRG after CCI was confirmed by ELISA., Discussion: Peripheral mononeuropathic pain in rats was associated with distinct alterations of miRNA expression in the ipsilateral DRG. Notably, miR-34a was time-dependently down regulated. We validated SCN2B and VAMP-2 as new targets of miR-34a. While SCN2B expression was only marginally altered, VAMP-2 expression was increased. The present study underlines that the induction and maintenance of neuropathic pain is accompanied by expression changes of miRNAs in the peripheral nervous system, adding several previously unreported miRNAs, including miR-34a., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

The p38 MAPK downstream targets MAPKAP kinases (MK) 2 and 3 are critical for the regulation of the macrophage response to LPS. The extents to which these two kinases act cooperatively and distinctly in regulating LPS-induced inflammatory cytokine expression are still unclear. To address this uncertainty, whole transcriptome analyses were performed using bone marrow-derived macrophages (BMDM) generated from MK2 -/- or MK2/3 -/- animals and their wild-type littermates. The results suggest that in BMDM, MK2 and MK3 not only cooperatively regulate the transcript expression of signaling intermediates, including IL-10, IL-19, CXCL2 and the IL-4 receptor (IL-4R)α subunit, they also exert distinct regulatory effects on the expression of specific transcripts. Based on the differential regulation of gene expression by MK2 and MK3, at least six regulatory patterns were identified. Importantly, we confirmed our previous finding, which showed that in the absence of MK2, MK3 negatively regulates IFN-β. Moreover, this genome-wide analysis identified the regulation of Cr1A, NOD1 and Serpina3f as similar to that of IFN-β. In the absence of MK2, MK3 also delayed the nuclear translocation of NFκB by delaying the ubiquitination and subsequent degradation of IκBβ, reflecting the substantial plasticity of the response of BMDM to LPS.

Since 1967, it is known that the loss of GLI3 causes very severe defects in murine eye development. GLI3 is able to act as a transcriptional activator (GLI3-A) or as a transcriptional repressor (GLI3-R). Soon after the discovery of these GLI3 isoforms, the question arose which of the different isoforms is involved in eye formation - GLI3-A, GLI3-R or even both. For several years, this question remained elusive. By analysing the eye morphogenesis of Gli3 XtJ/XtJ mouse embryos that lack GLI3-A and GLI3-R and of Gli3 Δ699/Δ699 mouse embryos in which only GLI3-A is missing, we revealed that GLI3-A is dispensable in vertebrate eye formation. Remarkably, our study shows that GLI3-R is sufficient for the creation of morphologically normal eyes although the molecular setup deviates substantially from normality. In depth-investigations elucidated that GLI3-R controls numerous key players in eye development and governs lens and retina development at least partially via regulating WNT/β-CATENIN signalling., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Currently, the clinical diagnosis of schizophrenia relies solely on self-reporting and clinical interview, and likely comprises heterogeneous biological subsets. Such subsets may be defined by an underlying biology leading to solid biomarkers. A transgenic rat model modestly overexpressing the full-length, non-mutant Disrupted-in-Schizophrenia 1 (DISC1) protein (tgDISC1 rat) was generated that defines such a subset, inspired by our previous identification of insoluble DISC1 protein in post mortem brains from patients with chronic mental illness. Besides specific phenotypes such as DISC1 protein pathology, abnormal dopamine homeostasis, and changes in neuroanatomy and behavior, this animal model also shows subtle disturbances in overarching signaling pathways relevant for schizophrenia. In a reverse-translational approach, assuming that both the animal model and a patient subset share common disturbed signaling pathways, we identified differentially expressed transcripts from peripheral blood mononuclear cells of tgDISC1 rats that revealed an interconnected set of dysregulated genes, led by decreased expression of regulator of G-protein signaling 1 (RGS1), chemokine (C-C) ligand 4 (CCL4), and other immune-related transcripts enriched in T-cell and macrophage signaling and converging in one module after weighted gene correlation network analysis. Testing expression of this gene network in two independent cohorts of patients with schizophrenia versus healthy controls (n = 16/50 and n = 54/45) demonstrated similar expression changes. The two top markers RGS1 and CCL4 defined a subset of 27% of patients with 97% specificity. Thus, analogous aberrant signaling pathways can be identified by a blood test in an animal model and a corresponding schizophrenia patient subset, suggesting that in this animal model tailored pharmacotherapies for this patient subset could be achieved.

Class I histone deacetylases (HDACs) generally promote cell proliferation and tumorigenesis, whereas class IIA HDACs like HDAC4 and HDAC5 may promote or impede cancer development in a tissue-dependent manner. In urothelial carcinoma (UC), HDAC5 is often downregulated. Accordingly, HDAC5 was weakly expressed in UC cell lines suggesting a possible tumor-suppressive function. We therefore characterized the effects of stable HDAC5 expression in four UC cell lines (RT112, VM-Cub-1, SW1710 and UM-UC-3) with different phenotypes reflecting the heterogeneity of UC, by assessing proliferation, clonogenicity and migration ability. Further, we detailed changes in the proteome and transcriptome by immunoblotting, mass spectrometry and RNA sequencing analysis. We observed that HDAC5 overexpression in general decreased cell proliferation, but in one cell line (VM-Cub-1) induced a dramatic change from an epitheloid to a mesenchymal phenotype, i.e., epithelial-mesenchymal transition (EMT). These phenotypical changes were confirmed by comprehensive proteomics and transcriptomics analyses. In contrast to HDAC5, overexpression of HDAC4 exerted only weak effects on cell proliferation and phenotypes. We conclude that overexpression of HDAC5 may generally decrease proliferation in UC, but, intriguingly, may induce EMT on its own in certain circumstances.

The histone demethylase Ubiquitously Transcribed Tetratricopeptide Repeat Protein X-Linked (UTX/KDM6A) demethylates H3K27me2/3 at genes and enhancers and is often inactivated by mutations in urothelial carcinoma (UC). The consequences of its inactivation are however poorly understood. We have investigated the consequences of moderate UTX overexpression across a range of UC cell lines with or without mutations in KDM6A or its interaction partners and in a normal control cell line. Effects on cell proliferation, especially long-term, varied dramatically between the cell lines, ranging from deleterious to beneficial. Similarly, effects on global gene expression determined by RNA-Seq were variable with few overlapping up- or downregulated genes between the cell lines. Our data indicate that UTX does not act in a uniform fashion in UC. Rather, its effect depends on several contingencies including, prominently, the status of KMT2C and KMT2D which interact with UTX in the COMPASS complex. In particular, we provide evidence that these factors determine the amount of nuclear UTX., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Insulin-like growth factor 1 (IGF1) is an anabolic hormone that controls the growth and metabolism of many cell types. However, IGF1 also mediates cardio-protective effects after acute myocardial infarction (AMI), but the underlying mechanisms and cellular targets are not fully understood. Here we demonstrate that short-term IGF1 treatment for 3 days after AMI improved cardiac function after 1 and 4 weeks. Regional wall motion was improved in ischemic segments, scar size was reduced, and capillary density increased in the infarcted area and the border zone. Unexpectedly, inducible inactivation of the IGF1 receptor (IGF1R) in cardiomyocytes did not attenuate the protective effect of IGF1. Sequential cardiac transcriptomic analysis indicated an altered myeloid cell response in the acute phase after AMI, and, notably, myeloid-cell Igf1r -/- mice lost the protective IGF1 function after I/R. In addition, IGF1 induced an M2-like anti-inflammatory phenotype in bone marrow-derived macrophages and enhanced the number of anti-inflammatory macrophages in heart tissue on day 3 after AMI in vivo. In summary, modulation of the acute inflammatory phase after AMI by IGF1 represents an effective mechanism to preserve cardiac function after I/R., (Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Enterobacter cloacae complex is a common cause of hospital outbreaks. A retrospective and prospective molecular analysis of carbapenem-resistant clinical isolates in a tertiary care center demonstrated an outbreak of a German-imipenemase-1 (GIM-1) metallo-beta-lactamase-producing Enterobacter hormaechei ssp. steigerwaltii affecting 23 patients between 2009 and 2016. Thirty-three isolates were sequence type 89 by conventional multilocus sequence typing (MLST) and displayed a maximum difference of 49 out of 3,643 targets in the ad-hoc core-genome MLST (cgMLST) scheme (SeqSphere+ software; Ridom, Münster, Germany). The relatedness of all isolates was confirmed by further maximum-likelihood phylogeny. One clonal complex of highly related isolates (≤15 allele difference in cgMLST) contained 17 patients, but epidemiological data only suggested five transmission events. The bla GIM-1 -gene was embedded in a class-1-integron (In770) and the Tn21-subgroup transposon Tn6216 (KC511628) on a 25-kb plasmid. Environmental screening detected one colonized sink trap in a service room. The outbreak was self-limited as no further bla GIM-1 -positive E. hormaechei has been isolated since 2016. Routine molecular screening of carbapenem-nonsusceptible gram-negative isolates detected a long-term, low-frequency outbreak of a GIM-1-producing E. hormaechei ssp. steigerwaltii clone. This highlights the necessity of molecular surveillance.

It is widely accepted that human brain development has unique features that cannot be represented by rodents. Obvious reasons are the evolutionary distance and divergent physiology. This might lead to false predictions when rodents are used for safety or pharmacological efficacy studies. For a better translation of animal-based research to the human situation, human in vitro systems might be useful. In this study, we characterize developing neural progenitor cells from prenatal human and time-matched rat and mouse brains by analyzing the changes in their transcriptome profile during neural differentiation. Moreover, we identify hub molecules that regulate neurodevelopmental processes like migration and differentiation. Consequences of modulation of three of those hubs on these processes were studied in a species-specific context. We found that although the gene expression profiles of the three species largely differ qualitatively and quantitatively, they cluster in similar GO terms like cell migration, gliogenesis, neurogenesis or development of multicellular organism. Pharmacological modulation of the identified hub molecules triggered species-specific cellular responses. This study underlines the importance of understanding species differences on the molecular level and advocates the use of human based in vitro models for pharmacological and toxicological research., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The albumin D-box binding protein (DBP) is a member of the PAR bZip (proline and acidic amino acid-rich basic leucine zipper) transcription factor family and functions as important regulator of circadian core and output gene expression. Gene expression of DBP itself is under the control of E-box-dependent binding by the Bmal1-Clock heterodimer and CRE-dependent binding by the cAMP responsive element binding protein (CREB). However, the signaling mechanism mediating CREB-dependent regulation of DBP expression in the peripheral clock remains elusive. In this study, we examined the role of the GPCR (G-protein-coupled receptor)/Gα i3 (Galphai3) controlled cAMP-CREB signaling pathway in the regulation of hepatic expression of core clock and clock-regulated genes, including Dbp . Analysis of circadian gene expression revealed that rhythmicity of hepatic transcript levels of the majority of core clock (including Per1) and clock-regulated genes were not affected by Gα i3 deficiency. Consistently, the period length of primary Gα i3 deficient tail fibroblasts expressing a Bmal1-Luciferase reporter was not affected. Interestingly, however, Gα i3 deficient female but not male mice showed a tendentiously increased activation of CREB (nuclear pSer133-CREB) accompanied by an advanced peak in Dbp gene expression and elevated mRNA levels of the cytochrome P 450 family member Cyp3a11, a target gene of DBP. Accordingly, selective inhibition of CREB led to a strongly decreased expression of DBP and CYP3A4 (human Cyp3a11 homologue) in HepG2 liver cells. In summary, our data suggest that the Gα i3 -pCREB signalling pathway functions as a regulator of sexual-dimorphic expression of DBP and its xenobiotic target enzymes Cyp3a11/CYP3A4., Competing Interests: CONFLICTS OF INTEREST The authors state no conflicts of interest.