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Intestinal microbiota regulates multiple host metabolic and immunological processes. Consequently, any difference in its qualitative and quantitative composition is susceptible to exert significant effects, in particular along the gut-liver axis. Indeed, recent findings suggest that such changes modulate the severity and the evolution of a wide spectrum of hepatobiliary disorders. However, the mechanisms linking intestinal microbiota and the pathogenesis of liver disease remain largely unknown. In this work, we investigated how a distinct composition of the intestinal microbiota, in comparison with germ-free conditions, may lead to different outcomes in an experimental model of acute cholestasis. Acute cholestasis was induced in germ-free (GF) and altered Schaedler’s flora (ASF) colonized mice by common bile duct ligation (BDL). Studies were performed 5 days after BDL and hepatic histology, gene expression, inflammation, lipids metabolism, and mitochondrial functioning were evaluated in normal and cholestatic mice. Differences in plasma concentration of bile acids (BA) were evaluated by UHPLC-HRMS. The absence of intestinal microbiota was associated with significant aggravation of hepatic bile infarcts after BDL. At baseline, we found the absence of gut microbiota induced altered expression of genes involved in the metabolism of fatty and amino acids. In contrast, acute cholestasis induced altered expression of genes associated with extracellular matrix, cell cycle, autophagy, activation of MAPK, inflammation, metabolism of lipids, and mitochondrial functioning pathways. Ductular reactions, cell proliferation, deposition of collagen 1 and autophagy were increased in the presence of microbiota after BDL whereas GF mice were more susceptible to hepatic inflammation as evidenced by increased gene expression levels of osteopontin, interleukin (IL)-1β and activation of the ERK/MAPK pathway as compared to ASF colonized mice. Additonally, we found that the presence of microbiota provided partial protection to the mitochondrial functioning and impairment in the fatty acid metabolism after BDL. The concentration of the majority of BA markedly increased after BDL in both groups without remarkable differences according to the hygiene status of the mice. In conclusion, acute cholestasis induced more severe liver injury in GF mice compared to mice with limited intestinal bacterial colonization. This protective effect was associated with different hepatic gene expression profiles mostly related to tissue repair, metabolic and immune functions. Our findings suggest that microbial-induced differences may impact the course of cholestasis and modulate liver injury, offering a background for novel therapies based on the modulation of the intestinal microbiota.

The exopolysaccharide capsule of Streptococcus pneumoniae is an important virulence factor, but the mechanisms that regulate capsule thickness are not fully understood. Here, we investigated the effects of various exogenously supplied carbohydrates on capsule production and gene expression in several pneumococcal serotypes. Microscopy analyses indicated a near absence of the capsular polysaccharide (CPS) when S. pneumoniae was grown on fructose. Moreover, serotype 7F pneumococci produced much less CPS than strains of other serotypes (6B, 6C, 9V, 15, and 23F) when grown on glucose or sucrose. RNA-sequencing revealed carbon source-dependent regulation of distinct genes of WT strains and capsule-switch mutants of serotypes 6B and 7F, but could not explain the mechanism of capsule thickness regulation. In contrast, 31P NMR of whole-cell extract from capsule-knockout strains (Δcps) clearly revealed the accumulation or absence of capsule precursor metabolites when cells were grown on glucose or fructose, respectively. This finding suggests that fructose uptake mainly results in intracellular fructose 1-phosphate, which is not converted to CPS precursors. In addition, serotype 7F strains accumulated more precursors than did 6B strains, indicating less efficient conversion of precursor metabolites into the CPS in 7F, in line with its thinner capsule. Finally, isotopologue sucrose labeling and NMR analyses revealed that the uptake of the labeled fructose subunit into the capsule is

INTRODUCTION Preterm birth is a major cause of neonatal morbidity and mortality. There is an urgent need to accurately predict imminent delivery to enable necessary interventions such as tocolytic, glucocorticoid, and magnesium sulfate administration. We aimed to evaluate placental α-macroglobulin-1 as a new diagnostic marker in the prediction of preterm birth. MATERIAL AND METHODS We performed a prospective observational trial in women with intact membranes between 24+0 and 36+6 weeks of gestation. We included both women with and without threatened preterm labor symptoms. We evaluated the test performance of placental α-macroglobulin-1 measurements in cervicovaginal fluid regarding three different presentation-to-delivery intervals: ≤2, ≤7, ≤14 days. In addition, we calculated placental α-macroglobulin-1 performance in combination with other prognostic factors such as ultrasonographic cervical length measurements. RESULTS We included 126 women in the study. We detected high specificity (97%-98%) and negative predictive value (89%-97%) for placental α-macroglobulin-1 at all time intervals. We assessed placental α-macroglobulin-1 in combination with cervical length measurements (≤15 mm) in the sub-group of women presenting with threatened preterm labor symptoms (n = 63) and detected high positive predictive values (100%) for 7- and 14-day presentation-to-delivery intervals. CONCLUSIONS Our study provides evidence that placental α-macroglobulin-1 testing in cervicovaginal fluid, in combination with cervical length measurements, accurately predicts preterm birth in women with preterm labor symptoms. This novel test combination may be used clinically to triage women presenting with threatened preterm labor, avoiding overtreatment and unnecessary hospitalizations.

Constitutive JAK2 signaling is central to myeloproliferative neoplasm (MPN) pathogenesis and results in activation of STAT, PI3K/AKT, and MEK/ERK signaling. However, the therapeutic efficacy of current JAK2 inhibitors is limited. We investigated the role of MEK/ERK signaling in MPN cell survival in the setting of JAK inhibition. Type I and II JAK2 inhibition suppressed MEK/ERK activation in MPN cell lines in vitro, but not in Jak2V617F and MPLW515L mouse models in vivo. JAK2 inhibition ex vivo inhibited MEK/ERK signaling, suggesting that cell-extrinsic factors maintain ERK activation in vivo. We identified PDGFRα as an activated kinase that remains activated upon JAK2 inhibition in vivo, and PDGF-AA/PDGF-BB production persisted in the setting of JAK inhibition. PDGF-BB maintained ERK activation in the presence of ruxolitinib, consistent with its function as a ligand-induced bypass for ERK activation. Combined JAK/MEK inhibition suppressed MEK/ERK activation in Jak2V617F and MPLW515L mice with increased efficacy and reversal of fibrosis to an extent not seen with JAK inhibitors. This demonstrates that compensatory ERK activation limits the efficacy of JAK2 inhibition and dual JAK/MEK inhibition provides an opportunity for improved therapeutic efficacy in MPNs and in other malignancies driven by aberrant JAK-STAT signaling.

OBJECTIVES Stem cells secrete significant amounts of bioactive factors in their secretome that can be immunosuppressive. We studied the effect of the secretome obtained from bone marrow-derived mesenchymal stem cells (BMSC-sec) in combination with cyclosporine A following acute rejection of lung allografts in the rat. METHODS Lung allotransplants were performed from male Brown Norway donor rats to recipient male Fisher 344 rats. Rat BMSC-sec was introduced intratracheally in the recipient every day after the transplant until the day the animal was sacrificed. Group A (n = 5) received control medium and cyclosporine A (2.5 mg/kg body weight intraperitoneally) for 5 days post-transplant and group B (n = 5) received BMSC-sec and cyclosporine A. Blood gas analysis was performed to assess graft function at day 5 only from the graft, and the tissue was sampled for measurement of the wet/dry ratio and histological grading of rejection. RESULTS All control animals (group A) showed severe signs of rejection. At day 5 grafts in group B showed improved gas exchange (i.e. mean PaO2 mmHg 237.9 ± 130 mmHg vs 24.9 ± 7.8 mmHg in group A). Histological examination according to the International Society of Heart and Lung Transplantation (ISHLT) revealed moderate to severe rejection in all animals in group A (III B) and a significant improvement in group B (I-IIA). The wet/dry ratio was also reduced in group B to 6.19 ± 0.6 compared to 9.36 ± 2 in group A. Furthermore, in vitro T-cell proliferation was reduced after treatment with BMSC-sec for CD 3 cells (69.55 ± 07 vs 73 ± 0.84), for CD 4 (24.95 ± 1.2 vs 27.75 ± 0.21) and for CD 8 cells (3.75 ± 0.2 vs 5.68 ± 0.02). CONCLUSIONS The BMSC-sec is a promising novel cell-based therapeutic option for acute rejection in a rat lung allograft model.

Simple Summary Macrophages play essential role in repair, regeneration and tissue remodeling. Role of macrophages in progression of lung fibrosis is established. Secretome of Induced pluripotent stem cells (iPSC-CM) has shown to reduce lung fibrosis and regulate macrophage phenotype, however exact mechanism is not known. Using advanced bioinformatics analysis by gene network analysis in this study we identified two components AAP and ELAVL-1 present in the iPSC-CM playing important role in regulation of macrophage phenotype. In this invitro study we confirmed experimentally that AAP and ELAVL1 play essential role by changing the profibrotic phenotype of the macrophages to pro resolution macrophages. We demonstrate reduction in gene expression and cytokine secretion of profibrotic macrophages after iPSC-CM treatment. Our study confirms antifibrotic and regenerative potential of iPSC-CM. Abstract Induced pluripotent stem cell secretome (iPSC-CM) mitigate organ injury and help in repair. Macrophages play a critical role in tissue repair and regeneration and can be directed to promote tissue repair by iPSC-CM, although the exact mechanisms are not known. In the current investigative study, we evaluated the possible mechanism by which iPSC-CM regulates the phenotype and secretory pattern of macrophages in vitro. Macrophages were obtained from human peripheral blood mononuclear cells and differentiated to various subpopulations and treated with either iPSC-CM or control media in vitro. Macrophage phenotype was assessed by flow cytometry, gene expression changes by qRT PCR and secretory pattern by multiplex protein analysis. The protein and gene interaction network revealed the involvement of Amyloid precursor protein (APP) and ELAV-like protein 1 (ELAVL-1) both present in the iPSC-CM to play an important role in regulating the macrophage phenotype and their secretory pattern. This exploratory study reveals, in part, the possible mechanism and identifies two potential targets by which iPSC-CM regulate macrophages and help in repair and regeneration.

Lung injury in mice induces mobilization of discrete subsets of epithelial progenitor cells to promote new airway and alveolar structures. However, whether similar cell types exist in human lung remains unresolved. Using flow cytometry, we identified a distinct cluster of cells expressing the epithelial cell adhesion molecule (EpCAM), a cell surface marker expressed on epithelial progenitor cells, enriched in the ecto-5′-nucleotidase CD73 in unaffected postnatal human lungs resected from pediatric patients with congenital lung lesions. Within the EpCAM+CD73+ population, a small subset coexpresses integrin β4 and HTII-280. This population remained stable with age. Spatially, EpCAM+CD73+ cells were positioned along the basal membrane of respiratory epithelium and alveolus next to CD73+ cells lacking EpCAM. Expanded EpCAM+CD73+ cells give rise to a pseudostratified epithelium in a two-dimensional air–liquid interface or a clonal three-dimensional organoid assay. Organoids generated under alveolar differentiation conditions were cystic-like and lacked robust alveolar mature cell types. Compared with unaffected postnatal lung, congenital lung lesions were marked by clusters of EpCAM+CD73+ cells in airway and cystic distal lung structures lined by simple epithelium composed of EpCAM+SCGB1A1+ cells and hyperplastic EpCAM+proSPC+ cells. In non-small-cell lung cancer (NSCLC), there was a marked increase in EpCAM+CD73+ tumor cells enriched in inhibitory immune checkpoint molecules CD47 and programmed death-ligand 1 (PD-L1), which was associated with poor survival in lung adenocarcinoma (LUAD). In conclusion, EpCAM+CD73+ cells are rare novel epithelial progenitor cells in the human lung. Importantly, reemergence of CD73 in lung adenocarcinoma enriched in negative immune checkpoint molecules may serve as a novel therapeutic target.

Background Idiopathic pulmonary fibrosis (IPF) is a chronic fatal lung disease without a cure and new drug strategies are urgently needed. Differences in behavior between diseased and healthy cells are well known and drug response can be different between cells isolated from IPF patients and controls. The macrolide Azithromycin (AZT) has anti-inflammatory and immunomodulatory properties. Recently anti-fibrotic effects have been described. However, the anti-fibrotic effects on primary IPF-fibroblasts (FB) directly compared to control-FB are unknown. We hypothesized that IPF-FB react differently to AZT in terms of anti-fibrotic effects. Methods Primary normal human lung and IPF-FB were exposed to TGF-β (5 ng/ml), Azithromycin (50 μM) alone or in combination prior to gene expression analysis. Pro-collagen Iα1 secretion was assessed by ELISA and protein expression by western blot (αSMA, Fibronectin, ATP6V1B2, LC3 AB (II/I), p62, Bcl-xL). Microarray analysis was performed to screen involved genes and pathways after Azithromycin treatment in control-FB. Apoptosis and intraluminal lysosomal pH were analyzed by flow cytometry. Results AZT significantly reduced collagen secretion in TGF-β treated IPF-FB compared to TGF-β treatment alone, but not in control-FB. Pro-fibrotic gene expression was similarly reduced after AZT treatment in IPF and control-FB. P62 and LC3II/I western blot revealed impaired autophagic flux after AZT in both control and IPF-FB with significant increase of LC3II/I after AZT in control and IPF-FB, indicating enhanced autophagy inhibition. Early apoptosis was significantly higher in TGF-β treated IPF-FB compared to controls after AZT. Microarray analysis of control-FB treated with AZT revealed impaired lysosomal pathways. The ATPase and lysosomal pH regulator ATP6V0D2 was significantly less increased after additional AZT in IPF-FB compared to controls. Lysosomal function was impaired in both IPF and control FB, but pH was significantly more increased in TGF-β treated IPF-FB. Conclusion We report different treatment responses after AZT with enhanced anti-fibrotic and pro-apoptotic effects in IPF compared to control-FB. Possibly impaired lysosomal function contributes towards these effects. In summary, different baseline cell phenotype and behavior of IPF and control cells contribute to enhanced anti-fibrotic and pro-apoptotic effects in IPF-FB after AZT treatment and strengthen its role as a new potential anti-fibrotic compound, that should further be evaluated in clinical studies.

Idiopathic pulmonary fibrosis (IPF) is a complex disease involving various cell types. Macrophages are essential in maintenance of physiological homeostasis, wound repair and fibrosis in the lung. Macrophages play a crucial role in repair and remodeling by altering their phenotype and secretory pattern in response to injury. The secretome of induced pluripotent stem cells (iPSC-cm) attenuates injury and fibrosis in bleomycin injured rat lungs. In the current study, we evaluate the effect of iPSC-cm on gene expression and phenotype of interstitial macrophage in bleomycin injured rat lungs in vivo. iPSC-cm was intratracheally instilled 7 days after bleomycin induced lung injury and assessed 7 days later and single cell isolation was performed. Macrophages were FACS sorted and microarray analysis was performed. We characterized changes in the rat lung interstitial macrophages using transcriptional profiling. iPSC-cm reduced the total collagen content of the lung and reduced different macrophage populations. Gene set enrichment analysis revealed involvement of three essential pathways (a) immune modulation, (b) branching morphogenesis and (c) canonical Wnt signaling. This study demonstrates that iPSC-cm reduces fibrosis in bleomycin injured rat lung by partially altering the macrophages and regulating their gene expression. Electronic supplementary material The online version of this article (10.1007/s12015-017-9790-9) contains supplementary material, which is available to authorized users.

Triggering receptor expressed on myeloid cells-1 (TREM-1) is a potent amplifier of pro-inflammatory innate immune responses. Increasing evidence suggests a role for TREM-1 not only in acute pathogen-induced reactions but also in chronic and non-infectious inflammatory disorders, including various types of cancer. Here, we demonstrate that genetic deficiency in Trem1 protects from colorectal cancer. In particular, Trem1−/− mice exhibited reduced tumor numbers and load in an experimental model of inflammation-driven tumorigenesis. Gene expression analysis of Trem1−/− versus Trem1+/+ tumor tissue demonstrated distinct immune signatures. Whereas Trem1−/− tumors showed an increased abundance of transcripts linked to adaptive immunity, Trem1+/+ tumors were characterized by overexpression of innate pro-inflammatory genes associated with tumorigenesis. Compared to adjacent tumor-free colonic mucosa, expression of Trem1 was increased in murine and human colorectal tumors. Unexpectedly, TREM-1 was not detected on tumor-associated Ly6C− MHC class II+ macrophages. In contrast, TREM-1 was highly expressed by tumor-infiltrating neutrophils which represented the predominant myeloid population in Trem1+/+ but not in Trem1−/− tumors. Collectively, our findings demonstrate a clear role of TREM-1 for intestinal tumorigenesis and indicate TREM-1-expressing neutrophils as critical players in colorectal tumor development.

Diagnostic markers are needed for accidental or deliberate radiation exposure that could cause acute and chronic radiation toxicity. Biomarkers of temporal, dose-dependent, aging-attenuated and multiple radiation exposures have been previously described by others. However, the physiological origin and biochemical networks that generate these biomarkers and their association at the molecular level have yet to be explored. Hence, the discovery and identification of total-body-irradiation-induced tissue specific biomarkers remains an enormous challenge within radiation biodosimetry research. To determine the tissue level response of total-body exposure (6 Gy), metabolomics analysis was carried out on radiosensitive tissues bone marrow, ileum, liver, muscle and lung as well as serum and on urine within 12 h postirradiation. Differences in the metabolic signatures between the sham and gamma-irradiated groups were analyzed by hydrophilic interaction liquid chromatography (HILIC)-based ultra-performance liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS). A panel of 67 biomarkers identified in radiosensitive tissues and biofluids (serum and urine) at a 6 Gy dose. Among the identified biomarkers, 3-methylglutarylcarnitine (3-MGC) was found to be a novel metabolite in liver, serum and urine that could potentially be an early radiation response marker. The degree of metabolic changes among different tissues showed perturbations in pathways including DNA methylation, energy, nucleic acid, amino acid, glutathione and bile acid metabolism. These results highlight metabolomics as a potential novel approach to understand functional alterations in the metabolome that could be adapted for use in the rapid assessment of radiation exposure and triage protocols in the case of nuclear incidents.

In childhood acute lymphoblastic leukemia, treatment failure is associated with resistance to glucocorticoid agents. Resistance to this class of drugs represents one of the strongest indicators of poor clinical outcome. We show that leukemic cells, which are resistant to the glucocorticoid drug methylprednisolone, display a higher demand of glucose associated with a deregulation of metabolic pathways, in comparison to sensitive cells. Interestingly, a combinatorial treatment of glucocorticoid and the glucose analog 2-deoxy-D-glucose displayed a synergistic effect in methylprednisolone-resistant cells, in an oxygen tension-independent manner. Unlike solid tumors, where 2-deoxy-D-glucose promotes inhibition of glycolysis by hexokinase II exclusively under hypoxic conditions, we were able to show that the antileukemic effects of 2-deoxy-D-glucose are far more complex in leukemia. We demonstrate a hexokinase II-independent cell viability decrease and apoptosis induction of the glucose analog in leukemia. Additionally, due to the structural similarity of 2-deoxy-D-glucose with mannose, we could confirm that the mechanism by which 2-deoxy-D-glucose predominantly acts in leukemia is via modification in N-linked glycosylation, leading to endoplasmic reticulum stress and consequently induction of the unfolded protein response., Oxidative Medicine and Cellular Longevity, 2017, ISSN:1942-0994, ISSN:1942-0900

The liver has an intrinsic capacity to regenerate in response to injury or surgical resection. Nevertheless, circumstances in which hepatocytes are unresponsive to proliferative signals result in impaired regeneration and hepatic failure. As the Hippo pathway has a canonical role in the maintenance of liver size, we investigated whether it could serve as a therapeutic target to support regeneration. Using a standard two‐thirds partial hepatectomy (PH) model in young and aged mice, we demonstrate that the Hippo pathway is modulated across the phases of liver regeneration. The activity of the core kinases MST1 and LATS1 increased during the early hypertrophic phase and returned to steady state levels in the proliferative phase, coinciding with activation of YAP1 target genes and hepatocyte proliferation. Moreover, following PH in aged mice, we demonstrate that Hippo signaling is anomalous in non‐regenerating livers. We provide pre‐clinical evidence that silencing the Hippo core kinases MST1 and MST2 with siRNA provokes hepatocyte proliferation in quiescent livers and rescues liver regeneration in aged mice following PH. Our data suggest that targeting the Hippo core kinases MST1/2 has therapeutic potential to improve regeneration in non‐regenerative disorders.

CD8+ T cell immunosurveillance is crucial in solid tumors and T cell dysfunction leads to tumor progression. In contrast, the role of CD8+ T cells in the control of leukemia is less clear. We characterized the molecular signature of leukemia stem/progenitor cells (LSPCs) and paired CD8+ T cells in patients with acute myeloid leukemia (AML). Epigenetic alterations via histone deacetylation reduced the expression of immune-related genes in bone marrow (BM)-infiltrating CD8+ T cells. Surprisingly, a silenced gene expression pattern in CD8+ T cells significantly correlated with an improved prognosis. To define interactions between CD8+ T cells and LSPCs, we performed comprehensive correlative network modeling. This analysis indicated that CD8+ T cells contribute to the maintenance/expansion of LSPCs, particularly in favorable risk AML. Functionally, CD8+ T cells in favorable AML induced the expansion of LSPCs by stimulating the autocrine production of important hematopoietic cytokines such as interleukin (IL)-3. In contrast, LSPCs in aggressive AML were characterized by a higher activation of stemness/proliferation-related pathways and develop independent of BM CD8+ T cells. Overall, our study indicates that CD8+ T cells support and expand LSPCs in favorable risk AML whereas intermediate and adverse risk AML possess the intrinsic molecular abnormalities to develop independently.

The composition of immune infiltrates strongly affects the prognosis of patients with colorectal cancer (CRC). Interleukin (IL)-33 and regulatory T cells (Tregs) in the tumor microenvironment have been separately implicated in CRC; however their contribution to intestinal carcinogenesis is still controversial. Here, we reveal that IL-33 signaling promotes CRC by changing the phenotype of Tregs. In mice with CRC, tumor-infiltrating Tregs preferentially upregulate IL-33 receptor (ST2), and IL-33/ST2 signaling positively correlates with tumor number and size. Transcriptomic and flow cytometry analyses demonstrate that ST2 expression induces a more activated and migratory phenotype in FOXP3+ Tregs, which favors their accumulation in the tumor environment. Consequently, genetic ablation of St2 reduces Treg infiltration and concomitantly enhances the frequencies of effector CD8+ T cells, thereby restraining CRC. Mechanistically, IL-33 curtails IL-17 production by FOXP3+ Tregs and inhibits Th17 differentiation. In humans, numbers of activated ST2-expressing Tregs are increased in blood and tumor lesions of CRC patients, suggesting a similar mode of regulation. Together, these data indicate a central role of IL-33/ST2 signaling in shaping an immunosuppressive environment during intestinal tumorigenesis. Blockade of this pathway may provide a strategy to modulate the composition of CRC immune infiltrates.

Although TH1, TH2, and TH17 cells are well-defined TH cell lineages in humans, it remains debated whether IL-9-producing TH cells represent a bona fide "TH9" lineage. Our understanding of the cellular characteristics and functions of IL-9-producing TH cells in humans is still nascent. Here, we report that human IL-9-producing TH cells express the chemokine receptors CCR4 and CCR8, produce high levels of IL-5 and IL-13, and express TH2 lineage-associated transcription factors. In these cells, IL-9 production is activation dependent, transient, and accompanied by down-regulation of TH2 cytokines, leading to an apparent "TH9" phenotype. IL-9+ TH2 cells can be distinguished from "conventional" TH2 cells based on their expression of the transcription factor PPAR-γ. Accordingly, PPAR-γ is induced in naive TH cells by priming with IL-4 and TGF-β ("TH9" priming) and is required for IL-9 production. In line with their identity as early activated TH2 cells, IL-9+ TH2 cells are found in acute allergic skin inflammation in humans. We propose that IL-9-producing TH cells are a phenotypically and functionally distinct subpopulation of TH2 cells that depend on PPAR-γ for full effector functions.

Emerging evidence suggests an immunosuppressive role of altered tumor glycosylation due to downregulation of innate immune responses via immunoregulatory Siglecs. In contrast, human T cells, a major anticancer effector cell, only rarely express Siglecs. However, here, we report that the majority of intratumoral, but not peripheral blood, cytotoxic CD8+ T cells expressed Siglec-9 in melanoma. We identified Siglec-9+ CD8+ T cells as a subset of effector memory cells with high functional capacity and signatures of clonal expansion. This cytotoxic T-cell subset was functionally inhibited in the presence of Siglec-9 ligands or by Siglec-9 engagement by specific antibodies. TCR signaling pathways and key effector functions (cytotoxicity, cytokine production) of CD8+ T cells were suppressed by Siglec-9 engagement, which was associated with the phosphorylation of the inhibitory protein tyrosine phosphatase SHP-1, but not SHP-2. Expression of cognate Siglec-9 ligands was observed on the majority of tumor cells in primary and metastatic melanoma specimens. Targeting the tumor-restricted, glycosylation-dependent Siglec-9 axis may unleash this intratumoral T-cell subset, while confining T-cell activation to the tumor microenvironment.

Interferon-γ (IFN-γ) plays an important role in innate and adaptive immunity against intracellular infections and is used clinically for the prevention and control of infections in chronic granulomatous disease (CGD) and inborn defects in the IFN-γ/interleukin (IL)-12 axis. Using transcriptome profiling (RNA-seq), we sought to identify differentially expressed genes, transcripts and exons in Epstein-Barr virus-transformed B lymphocytes (B-EBV) cells from CGD patients, IFN-γ receptor deficiency patients, and normal controls, treated in vitro with IFN-γ for 48 hours. Our results show that IFN-γ increased the expression of a diverse array of genes related to different cellular programs. In cells from normal controls and CGD patients, IFN-γ-induced expression of genes relevant to oxidative killing, nitric oxide synthase pathway, proteasome-mediated degradation, antigen presentation, chemoattraction, and cell adhesion. IFN-γ also upregulated genes involved in diverse stages of messenger RNA (mRNA) processing including pre-mRNA splicing, as well as others implicated in the folding, transport, and assembly of proteins. In particular, differential exon expression of WARS (encoding tryptophanyl-transfer RNA synthetase, which has an essential function in protein synthesis) induced by IFN-γ in normal and CGD cells suggests that this gene may have an important contribution to the benefits of IFN-γ treatment for CGD. Upregulation of mRNA and protein processing related genes in CGD and IFNRD cells could mediate some of the effects of IFN-γ treatment. These data support the concept that IFN-γ treatment may contribute to increased immune responses against pathogens through regulation of genes important for mRNA and protein processing.

// Reut Shainer 1 , Osnat Almogi-Hazan 1 , Arye Berger 1 , Liad Hinden 1 , Martin Mueller 2, 3 , Chaya Brodie 4 , Cedric Simillion 5 , Michael Paidas 2 , Eytan R. Barnea 6, 7, * , Reuven Or 1, * 1 Department of Bone Marrow Transplantation and Cancer Immunotherapy, Hadassah-Hebrew University Medical Center, Jerusalem, 91120, Israel 2 Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale University School of Medicine, New Haven, CT 06510, USA 3 Department of Obstetrics and Gynecology, University Hospital Bern, Bern, 3003, Switzerland 4 Henry Ford Hospital, Detroit, MI 48202, USA 5 Department of Clinical Research, University of Bern, Bern, 3003, Switzerland 6 The Society for The Investigation of Early Pregnancy (SIEP), Cherry Hill, NJ 08003, USA 7 BioIncept, LLC (PreImplantation Factor* Proprietary), Cherry Hill, NJ 08003, USA * These authors contributed equally to this work Correspondence to: Eytan R. Barnea, email: barnea@earlypregnancy.org Keywords: ARS, PreImplantation Factor (PIF), oxidative stress, local & systemic protection Received: May 03, 2016 Accepted: June 30, 2016 Published: July 16, 2016 ABSTRACT Acute Radiation Syndrome (ARS) may lead to cancer and death and has few effective countermeasures. Efficacy of synthetic PIF treatment was demonstrated in preclinical autoimmune and transplantation models. PIF protected against inflammation and mortality following lethal irradiation in allogeneic bone marrow transplant (BMT) model. Herein, we demonstrate that PIF imparts comprehensive local and systemic protection against lethal and sub-lethal ARS in murine models. PIF treatment 2 h after lethal irradiation led to 100% survival and global hematopoietic recovery at 2 weeks after therapy. At 24 h after irradiation PIF restored hematopoiesis in a semi-allogeneic BMT model. PIF-preconditioning provided improved long-term engraftment. The direct effect of PIF on bone marrow cells was also demonstrated in vitro : PIF promoted pre-B cell differentiation and increased immunoregulatory properties of BM-derived mesenchymal stromal cells. PIF treatment also improved hematopoietic recovery and reduced systemic inflammatory cytokine production after sub-lethal radiation exposure. Here, PIF also prevented colonic crypt and basal membrane damage coupled with reduced nitric oxide synthetase ( iNOS ) and increased (B7h1) expression. Global upper GI gene pathway analysis revealed PIF’s involvement in protein-RNA interactions, mitochondrial oxidative pathways, and responses to cellular stress. Some effects may be attributed to PIF’s influence on macrophage differentiation and function. PIF demonstrated a regulatory effect on irradiated macrophages and on classically activated M1 macrophages, reducing inflammatory gene expression ( iNOS, Cox2 ), promoting protective ( Arg1 ) gene expression and inducing pro-tolerance cytokine secretion. Notably, synthetic PIF is stable for long-term field use. Overall, clinical investigation of PIF for comprehensive ARS protection is warranted.

After publication of our article [1], we have been notified that an extra alpha symbol (α) was mistakenly added at the beginning of the title.

Regular physical exercise has many beneficial effects, including antitumor properties, and is associated with a reduced risk of developing hepatocellular carcinoma (HCC). Less is known about the impact of exercise on HCC growth and progression. Here, we investigated the effects of exercise on HCC progression and assessed whether any beneficial effects would be evident under sorafenib treatment and could be mimicked by metformin. American Cancer Institute rats with orthotopic syngeneic HCC derived from Morris Hepatoma-3924A cells were randomly assigned to exercise (Exe) and sedentary groups, or sorafenib±Exe groups or sorafenib±metformin groups. The Exe groups ran on a motorized treadmill for 60 minutes/day, 5 days/week for 4 weeks. Tumor viable area was decreased by exercise, while cell proliferation and vascular density were reduced. Exercise increased the expression of phosphatase and tensin homolog deleted from chromosome 10 and increased the phosphorylation of adenosine monophosphate-activated protein kinase, while the phosphorylation of protein kinase B, S6 ribosomal protein, and signal transducer and activator of transcription 3 were decreased. Transcriptomic analysis suggested major effects of exercise were on nontumoral liver rather than tumor tissue. Exercise demonstrated similar effects when combined with sorafenib. Moreover, similar effects were observed in the group treated with sorafenib+metformin, revealing an exercise-mimicking effect of metformin. Conclusion: Exercise attenuates HCC progression associated with alterations in key signaling pathways, cellular proliferation, tumor vascularization, and necrosis. These beneficial effects are maintained when combined with sorafenib and can be mimicked by metformin. (Hepatology Communications 2018;2:607-620).

Altered surface glycosylation is a key feature of cancers, including gynecologic malignancies. Hypersialylation, the overexpression of sialic acid, is known to promote tumor progression and to dampen antitumor responses by mechanisms that also involve sialic acid binding immunoglobulin-like lectins (Siglecs), inhibitory immune receptors. Here, we discuss the expression patterns of Siglecs and sialyltransferases (STs) in gynecologic cancers, including breast, ovarian, and uterine malignancies, based on evidence from The Cancer Genome Atlas. The balance between sialosides generated by specific STs within the tumor microenvironment and Siglecs on leukocytes may play a decisive role for antitumor immunity. An interdisciplinary effort is required to decipher the characteristics and biological impact of the altered tumor sialome in gynecologic cancers and to exploit this knowledge to the clinical benefit of patients.

Aberrant alternative pre-mRNA splicing (AS) events have been associated with several disorders. However, it is unclear whether deregulated AS directly contributes to disease. Here, we reveal a critical role of the AS regulator epithelial splicing regulator protein 1 (ESRP1) for intestinal homeostasis and pathogenesis. In mice, reduced ESRP1 function leads to impaired intestinal barrier integrity, increased susceptibility to colitis and altered colorectal cancer (CRC) development. Mechanistically, these defects are produced in part by modified expression of ESRP1-specific Gpr137 isoforms differently activating the Wnt pathway. In humans, ESRP1 is downregulated in inflamed biopsies from inflammatory bowel disease patients. ESRP1 loss is an adverse prognostic factor in CRC. Furthermore, generation of ESRP1-dependent GPR137 isoforms is altered in CRC and expression of a specific GPR137 isoform predicts CRC patient survival. These findings indicate a central role of ESRP1-regulated AS for intestinal barrier integrity. Alterations in ESRP1 function or expression contribute to intestinal pathology., eLife digest The lining of the intestine is just one cell thick, and yet it can act as an effective barrier between the inside of the body and the contents of the digestive system. This lining is often disturbed during bowel cancer, inflammatory bowel disease and other intestinal diseases, causing the barrier to fail and the gut to become leaky. These diseases often reduce patient life expectancy and quality of life. Intestinal epithelial cells make up the lining of the intestine and the normal activities of these cells are often disturbed during intestinal disease. In the intestine, a protein called ESRP1 is only found in epithelial cells, but its role in maintaining a healthy intestinal lining was not clear. Here, Mager et al. studied the intestines of mice that had been genetically engineered to produce a form of ESRP1 that is less active than normal. The experiments show that lower levels of ESRP1 activity leads to a broken intestinal barrier. The genetically engineered mice were more likely to develop inflammatory bowel disease and more aggressive forms of cancer. ESRP1 controls a gene that encodes another protein called GPR137, which helps to relay signals to the epithelial cells. Lower levels of ESRP1 resulted in a longer form of the GPR137 protein being produced. This in turn affected the protein’s signaling role and disturbed the activities of intestinal epithelial cells. Further experiments on biopsies taken from patients with inflammatory bowel disease or colorectal cancer revealed that these patients had lower levels of ESRP1 compared to healthy individuals. Furthermore, low levels of ESRP1 and increased levels of the long version of GPR137 were associated with poorer outcomes for cancer patients. Together, these findings may help us to better understand how the intestinal barrier fails in mice and humans and could lead to new ways to monitor and treat intestinal disease.

AIMS OF THE STUDY Induction of labour after previous caesarean section (CS) is a challenge for obstetricians due to the increased risk of uterine rupture. Common methods for labour induction are balloon catheters and oxytocin as they are considered safe. However, the effectiveness remains unclear as currently available data are limited. Therefore, we aimed to determine safety and effectiveness of balloon catheter or oxytocin for labour induction after CS. METHODS We included 179 consecutive women with a previous CS and labour induction in this retrospective study. We performed labour induction using a balloon catheter in case of a Bishop score of 6 and/or premature rupture of membranes. The primary outcome was the rate of successful vaginal deliveries. We adjusted for multiple factors that may have impacted on the rate of vaginal delivery as well. The secondary outcomes were the rate of maternal and neonatal morbidities. RESULTS We detected a vaginal delivery success rate of 45.8% in the catheter and of 63.9% in the oxytocin group. We identified previous vaginal birth as an independent predictive factor for successful vaginal delivery in both groups. Induction using oxytocin was a negative predictive factor for neonatal admissions. Multivariate analysis showed that post-term pregnancy decreased the likelihood of vaginal delivery. We did not detect any factors predicting uterine rupture or uterine dehiscence, which occurred with similar frequency in both groups. Finally, the neonatal admission rate was less likely with higher gestational age and oxytocin as an induction method, whereas previous vaginal birth increased the risk. CONCLUSIONS Our study indicates that induction of labour with balloon catheter or oxytocin seems to be safe in women with previous CS. Labour induction using a balloon catheter in women with previous CS and unfavourable cervix has a disappointingly low success rate. We identified factors influencing vaginal delivery success rates. Women with previous CS and indications for labour induction should be informed about vaginal birth success rates and the alternative of elective repeat CS needs to be discussed.

Background aims Wharton's jelly mesenchymal stromal cells (WJ-MSCs) might be ideal candidates to treat perinatal brain damage. Their secretome has been shown to have beneficial effects on neuroregeneration, in part through interaction with neural progenitor cells (NPCs). However, it remains unclear whether cell-to-cell contact decisively contributes to this positive effect. The objective of this study was to elucidate the mechanism through which differentiation in NPCs is triggered after exposure to WJ-MSCs. Furthermore, given that WJ-MSCs can be derived from term (tWJ-MSCs) or preterm (ptWJ-MSCs) deliveries and that WJ-MSCs might be used for transplantations independent of gestational age, the influence of tWJ-MSCs versus ptWJ-MSCs on the differentiation capacities of NPCs was studied. Methods The effect of tWJ-MSCs and ptWJ-MSCs on the expression of neuroglial markers in NPCs was assessed in co-culture (CC), conditioned medium (CM) or transwell CC experiments by immunocytochemistry, real-time polymerase chain reaction and Western blot. Additionally, mass spectrometry was used to study their secretomes. Results NPCs showed an increased expression of glial markers after CC with WJ-MSCs or exposure to WJ-MSC-CMs. CC had a more prominent effect on the expression of glial markers compared with CM or transwell CCs. tWJ-MSCs more strongly induced the expression of mature oligodendroglial markers compared with ptWJ-MSCs. A possible role in enhancing this maturation could be attributed to the laminin α2-subunit. Conclusions Cell-to-cell contact between WJ-MSCs and NPCs induces oligodendrogenesis on NPCs, whereas trophic factor secretion is sufficient to promote astrogenesis. Thus, transplanting WJ-MSCs may promote endogenous neuroregeneration in perinatal brain damage.

Triggering receptor expressed on myeloid cells-1 (TREM-1) is a potent amplifier of pro-inflammatory innate immune responses, but its significance in non-infectious diseases remains unclear. Here, we demonstrate that TREM-1 promotes cardiovascular disease by exacerbating atherosclerosis. TREM-1 is expressed in advanced human atheromas and is highly upregulated under dyslipidemic conditions on circulating and on lesion-infiltrating myeloid cells in the Apoe−/− mouse model. TREM-1 strongly contributes to high-fat, high-cholesterol diet (HFCD)-induced monocytosis and synergizes with HFCD serum-derived factors to promote pro-inflammatory cytokine responses and foam cell formation of human monocyte/macrophages. Trem1−/−Apoe−/− mice exhibit substantially attenuated diet-induced atherogenesis. In particular, our results identify skewed monocyte differentiation and enhanced lipid accumulation as novel mechanisms through which TREM-1 can promote atherosclerosis. Collectively, our findings illustrate that dyslipidemia induces TREM-1 surface expression on myeloid cells and subsequently synergizes with TREM-1 to enhance monopoiesis, pro-atherogenic cytokine production and foam cell formation., TREM-1 is a receptor that amplifies acute pro-inflammatory responses in infection. Here the authors show that TREM-1 plays an important role in atherosclerosis, a chronic and non-infectious disease, by critically skewing myelopoiesis towards preferential monocyte differentiation and by contributing to CD36-driven cellular lipid accumulation.

Pulmonary fibrosis is a progressive lung disease, resulting in death of the patient. Macrophages play a critical role in tissue remodeling in this disease. Recently, we have shown that secretome of induced pluripotent stem cells (iPSC-cm) attenuates fibrosis in bleomycin injured rat lungs. We hypothesize that iPSC-cm exerts its antifibrotic effect by altering the lung macrophage transcription profile due to multiple factors present in the secretome. Adult male rats were instilled with bleomycin, and were treated by iPSC-cm or control media. Macrophages were sorted out and cDNA was synthesized. Differential genes expression was determined using Agilent microarrays. The protein composition of the iPSC-cm was examined using LC/MS2 shotgun proteomics. Both datasets were integrated using the STRING protein interaction database. Percentage of total macrophages increased in bleomycin injured lungs compared to normal control (26.2±1.4% vs 14.4±1.0%). iPS-cm treatment reduced total percentage of macrophages to 17.4±1.2%; moreover, the original macrophage phenotype was partially restored (M1: 18.0%±8.3% vs 32.3±2.0%; M2: 1.3%±0.7 vs. 5.7%±0.3%). Pathway analysis of the microarray data indicated that treatment with iPSC-cm specifically regulates genes involved in the organization of the basal membrane. Subsequent network analysis identified several factors in the secretome that could be responsible for the observed antifibrotic effect.iPSC-cm is rich in many antifibrotic molecules that plays critical role in changing macrophage phenotype thus helping in tissue remodeling and reducing fibrosis. This abstract has been presented previously at the European Respiratory Society9s Lung Science Conference in March 2016.

Cellular senescence might be a tumour suppressing mechanism as well as a contributor to age-related loss of tissue function. It has been characterised classically as the result of the loss of DNA sequences called telomeres at the end of chromosomes. However, recent studies have revealed that senescence is in fact an intricate process, involving the sequential activation of multiple cellular processes, which have proven necessary for the establishment and maintenance of the phenotype. Here, we review some of these processes, namely, the role of mitochondrial function and reactive oxygen species, senescence-associated secreted proteins and chromatin remodelling. Finally, we illustrate the use of systems biology to address the mechanistic, functional and biochemical complexity of senescence.

BACKGROUND AIMS: Unhealthy lifestyles predispose people to non alcoholic steatohepatitis (NASH) which may further result in the development of hepatocellular carcinoma (HCC). Although NASH patients benefit from physical activity it is unknown whether regular exercise reduces the risk of developing HCC. Therefore we studied the effect of regular exercise on the development of HCC in male hepatocyte specific PTEN deficient mice (AlbCrePten(flox/flox)) which develop steatohepatitis and HCC spontaneously. METHODS: Mice were fed a standardized 10 fat diet and were randomly divided into exercise or sedentary groups. The exercise group ran on a motorized treadmill for 60 min/day 5 days/week during 32 weeks. RESULTS: After 32 weeks of regular exercise 71 of exercised mice developed nodules larger than 15 mm(3)vs. 100 of mice in the sedentary group. The mean number of tumors per liver was reduced by exercise as well as the total tumoral volume per liver. Exercise did not affect steatosis and had no effect on the non alcoholic fatty liver disease (NAFLD) Activity Score (NAS). Exercise decreased tumor cell proliferation. Mechanistically exercise stimulated the phosphorylation of AMPK and its substrate raptor which decreased the kinase activity of mTOR. CONCLUSIONS: These data show a beneficial effect of regular exercise on the development of HCC in an experimental model of NASH and offer a rationale for encouraging predisposed patients to increase their physical activity for the prevention of HCC. Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

We describe the genome sequence of the protist Trichomonas vaginalis , a sexually transmitted human pathogen. Repeats and transposable elements comprise about two-thirds of the ∼160-megabase genome, reflecting a recent massive expansion of genetic material. This expansion, in conjunction with the shaping of metabolic pathways that likely transpired through lateral gene transfer from bacteria, and amplification of specific gene families implicated in pathogenesis and phagocytosis of host proteins may exemplify adaptations of the parasite during its transition to a urogenital environment. The genome sequence predicts previously unknown functions for the hydrogenosome, which support a common evolutionary origin of this unusual organelle with mitochondria.

The complete genomic analysis of Arabidopsis thaliana has shown that a major fraction of the genome consists of paralogous genes that probably originated through one or more ancient large-scale gene or genome duplication events. However, the number and timing of these duplications still remains unclear, and several different hypotheses have been put forward recently. Here, we reanalyzed duplicated blocks found in the Arabidopsis genome described previously and determined their date of divergence based on silent substitution estimations between the paralogous genes and, where possible, by phylogenetic reconstruction. We show that methods based on averaging protein distances of heterogeneous classes of duplicated genes lead to unreliable conclusions and that a large fraction of blocks duplicated much more recently than assumed previously. We found clear evidence for one large-scale gene or even complete genome duplication event somewhere between 70 to 90 million years ago. Traces pointing to a much older (probably more than 200 million years) large-scale gene duplication event could be detected. However, for now it is impossible to conclude whether these old duplicates are the result of one or more large-scale gene duplication events.

Plasma cell malignancies, such as multiple myeloma, are characterized by an extensively developed endoplasmic reticulum (ER) to accommodate the high secretion rate of immunoglobulins. An adaptive stress response mechanism, termed the unfolded protein response (UPR), is markedly elevated in multiple myeloma cells to ensure a homeostatic balance between ER burden and ER capacity. One branch of the UPR consists of the activation of PERK under chronic ER stress: PERK phosphorylates and impairs the function of the translation initiation factor, eIF2α, thereby downregulating global protein synthesis, and reducing the secretory burden on the ER. To test the therapeutic potential of PERK inhibitors in multiple myeloma, we identified two chemically diverse PERK inhibitors: both are sub-nM inhibitors of PERK, and have a >100-fold window against other kinases (including other eIF2α kinases). These compounds inhibit phosphorylation of eIF2α at 10-20nM (IC50) in HEK293 cells, incubated with the ER stressor tunicamycin. Both PERK inhibitors were selectively anti-proliferative in an ER-stressed epithelial cancer model (A549 cells with tunicamycin) at nM concentrations, but not in the absence of ER stress. Furthermore, in the absence of an exogenous ER stressor, both PERK inhibitors induced ER stress (eg, as evidenced by induction of the pro-apoptotic CHOP gene) selectively in multiple myeloma cell lines (and certain B-cell lymphoma lines) at low nM concentrations but not in normal or malignant epithelial cells (induction of CHOP at 10 μM). The magnitude of this induction was comparable to well-established ER stressors, such as bortezomib or tunicamycin, and correlated closely with reduced proliferation in malignant B-cell lines. The potent induction of CHOP occurred both in vitro and upon oral dosing of mice, with a xenografted multiple myeloma tumour (JIM-1); however, in both instances, the induction of ER stress was maximal at a dose corresponding to approximately 50-75% inhibition of PERK, whereas, remarkably, at dose levels corresponding to more complete PERK inhibition (as evidenced by inhibition of P-eIF2α), CHOP induction (and ER-stress induction in general as determined by a genome-wide expression profile) was reduced, eventually down to baseline levels. Consistently, both PERK inhibitors resulted in a biphasic concentration-dependent reduction of proliferation of certain myeloma and B-cell lymphoma lines. Hence, PERK inhibition triggers a biphasic induction of ER stress and inhibition of cell proliferation, selectively in B-cell malignancies. The possible underlying mechanism as well as the therapeutic implications of these findings will be discussed. Citation Format: Matthias Versele, Tamara Geerts, Jeroen Van De Ven, Ilse Van den Wyngaert, Peter Vermeulen, Inge Beerden, Danielle Peeters, Johnny Liebregts, Kurt Van Baelen, Cedric Simillion, Boudewijn Janssen, Tinne Verhulst, Norbert Esser, Christophe Meyer, Ian Stansfield, James Bischoff. Selective PERK kinase inhibition triggers a biphasic concentration-dependent induction of ER stress in multiple myeloma and B-cell lymphoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2926. doi:10.1158/1538-7445.AM2013-2926

Androgens drive the onset and progression of prostate cancer (PCa) by modulating androgen receptor (AR) transcriptional activity. Although several microarray-based studies have identified androgen-regulated genes, here we identify in-parallel global androgen-dependent changes in both gene and alternative mRNA isoform expression by exon-level analyses of the LNCaP transcriptome. While genome-wide gene expression changes correlated well with previously-published studies, we additionally uncovered a subset of 226 novel androgen-regulated genes. Gene expression pathway analysis of this subset revealed gene clusters associated with, and including the tyrosine kinase LYN, as well as components of the mTOR (mammalian target of rapamycin) pathway, which is commonly dysregulated in cancer. We also identified 1279 putative androgen-regulated alternative events, of which 325 (∼25%) mapped to known alternative splicing events or alternative first/last exons. We selected 30 androgen-dependent alternative events for RT-PCR validation, including mRNAs derived from genes encoding tumour suppressors and cell cycle regulators. Of seven positively-validating events (∼23%), five events involved transcripts derived from alternative promoters of known AR gene targets. In particular, we found a novel androgen-dependent mRNA isoform derived from an alternative internal promoter within the TSC2 tumour suppressor gene, which is predicted to encode a protein lacking an interaction domain required for mTOR inhibition. We confirmed that expression of this alternative TSC2 mRNA isoform was directly regulated by androgens, and chromatin immunoprecipitation indicated recruitment of AR to the alternative promoter region at early timepoints following androgen stimulation, which correlated with expression of alternative transcripts. Together, our data suggest that alternative mRNA isoform expression might mediate the cellular response to androgens, and may have roles in clinical PCa.

Positive-sense RNA viruses hijack intracellular membranes that provide niches for viral RNA synthesis and a platform for interactions with host proteins. However, little is known about host factors at the interface between replicase complexes and the host cytoplasm. We engineered a biotin ligase into a coronaviral replication/transcription complex (RTC) and identified >500 host proteins constituting the RTC microenvironment. siRNA-silencing of each RTC-proximal host factor demonstrated importance of vesicular trafficking pathways, ubiquitin-dependent and autophagy-related processes, and translation initiation factors. Notably, detection of translation initiation factors at the RTC was instrumental to visualize and demonstrate active translation proximal to replication complexes of several coronaviruses. Collectively, we establish a spatial link between viral RNA synthesis and diverse host factors of unprecedented breadth. Our data may serve as a paradigm for other positive-strand RNA viruses and provide a starting point for a comprehensive analysis of critical virus-host interactions that represent targets for therapeutic intervention., eLife digest Coronaviruses can infect the nose and throat and are a main cause of the common cold. Infections are usually mild and short-lived, but sometimes they can turn nasty. In 2002 and 2012, two dangerous new coronaviruses emerged and caused diseases known as SARS and MERS. These viruses caused much more serious symptoms and in some cases proved deadly. The question is, why are some coronaviruses more dangerous than others? Scientists know that the body's response to virus infection can make a difference to whether someone had mild or severe disease. So, to understand why some coronaviruses cause a cold and others kill, they also need to learn how people react to virus infection. Coronaviruses hijack membranes inside cells and turn them into virus factories. Within these factories, the viruses build molecular machinery called replicase complexes to copy their genetic code, which is needed for the next generation of virus particles. The viruses steal and repurpose proteins from their host cell that will assist in the copying process. However, scientists do not yet know which host proteins are essential for the virus to multiply. So, to find out, V’kovski et al. developed a way to tag any host protein that came near the virus factories. The new technique involved attaching an enzyme called a biotin ligase to the replicase complex. This enzyme acts as a molecular label gun, attaching a chemical tag to any protein that comes within ten nanometres. The label gun revealed that more than 500 different proteins come into contact with the replicase complex. To find out what these proteins were doing, the next step was to switch off their genes one by one. This revealed the key cell machinery that coronaviruses hijack when they are replicating. It included the cell's cargo transport system, the waste disposal system, and the protein production system. Using these systems allows the viruses to copy their genetic code next to machines that can turn it straight into viral proteins. These new results provide clues about which proteins viruses actually need from their host cells. They also do not just apply to coronaviruses. Other viruses use similar strategies to complete their infection cycle. These findings could help researchers to understand more generally about how viruses multiply. In the future, this knowledge could lead to new ways to combat virus infections.