Caspase 5 depletion is linked to hyper-inflammatory response and progeroid syndrome.

A progeroid family was found to harbor a pathogenic variant in the CASP5 gene that encodes inflammatory caspase 5. Caspase 5-depleted fibroblasts exhibited hyper-activation of inflammatory cytokines in response to pro-inflammatory stimuli. Long-term intermittent hyper-inflammatory response is likely the cause of the accelerated aging phenotype comprised of earlier onset of common aging diseases, supporting inflammaging as a potential common disease mechanism of progeroid syndromes and possibly normative aging.

Serum and Urine Metabolomic Profiling of Newly Diagnosed Treatment-Naïve Inflammatory Bowel Disease Patients.

BACKGROUND AND AIMS: Inflammatory bowel disease (IBD) is a prevalent chronic noncurable disease associated with profound metabolic changes. The discovery of novel molecular indicators for unraveling IBD etiopathogenesis and the diagnosis and prognosis of IBD is therefore pivotal. We sought to determine the distinctive metabolic signatures from the different IBD subgroups before treatment initiation. METHODS: Serum and urine samples from newly diagnosed treatment-naïve IBD patients and age and sex-matched healthy control (HC) individuals were investigated using proton nuclear magnetic resonance spectroscopy. Metabolic differences were identified based on univariate and multivariate statistical analyses. RESULTS: A total of 137 Crohn's disease patients, 202 ulcerative colitis patients, and 338 HC individuals were included. In the IBD cohort, several distinguishable metabolites were detected within each subgroup comparison. Most of the differences revealed alterations in energy and amino acid metabolism in IBD patients, with an increased demand of the body for energy mainly through the ketone bodies. As compared with HC individuals, differences in metabolites were more marked and numerous in Crohn's disease than in ulcerative colitis patients, and in serum than in urine. In addition, clustering analysis revealed 3 distinct patient profiles with notable differences among them based on the analysis of their clinical, anthropometric, and metabolomic variables. However, relevant phenotypical differences were not found among these 3 clusters. CONCLUSIONS: This study highlights the molecular alterations present within the different subgroups of newly diagnosed treatment-naïve IBD patients. The metabolomic profile of these patients may provide further understanding of pathogenic mechanisms of IBD subgroups. Serum metabotype seemed to be especially sensitive to the onset of IBD.

Inflammatory macrophages reprogram to immunosuppression by reducing mitochondrial translation.

Acute inflammation can either resolve through immunosuppression or persist, leading to chronic inflammation. These transitions are driven by distinct molecular and metabolic reprogramming of immune cells. The anti-diabetic drug Metformin inhibits acute and chronic inflammation through mechanisms still not fully understood. Here, we report that the anti-inflammatory and reactive-oxygen-species-inhibiting effects of Metformin depend on the expression of the plasticity factor ZEB1 in macrophages. Using mice lacking Zeb1 in their myeloid cells and human patient samples, we show that ZEB1 plays a dual role, being essential in both initiating and resolving inflammation by inducing macrophages to transition into an immunosuppressed state. ZEB1 mediates these diverging effects in inflammation and immunosuppression by modulating mitochondrial content through activation of autophagy and inhibition of mitochondrial protein translation. During the transition from inflammation to immunosuppression, Metformin mimics the metabolic reprogramming of myeloid cells induced by ZEB1. Mechanistically, in immunosuppression, ZEB1 inhibits amino acid uptake, leading to downregulation of mTORC1 signalling and a decrease in mitochondrial translation in macrophages. These results identify ZEB1 as a driver of myeloid cell metabolic plasticity, suggesting that targeting its expression and function could serve as a strategy to modulate dysregulated inflammation and immunosuppression.

Transcriptomics and translatomics identify a robust inflammatory gene signature in brain endothelial cells after ischemic stroke.

Vascular endothelial function is challenged during cerebral ischemia and reperfusion. The endothelial responses are involved in inflammatory leukocyte attraction, adhesion and infiltration, blood-brain barrier leakage, and angiogenesis. This study investigated gene expression changes in brain endothelial cells after acute ischemic stroke using transcriptomics and translatomics. We isolated brain endothelial mRNA by: (i) translating ribosome affinity purification, enabling immunoprecipitation of brain endothelial ribosome-attached mRNA for translatome sequencing and (ii) isolating CD31+ endothelial cells by fluorescence-activating cell sorting for classical transcriptomic analysis. Both techniques revealed similar pathways regulated by ischemia but they showed specific differences in some transcripts derived from non-endothelial cells. We defined a gene set characterizing the endothelial response to acute stroke (24h) by selecting the differentially expressed genes common to both techniques, thus corresponding with the translatome and minimizing non-endothelial mRNA contamination. Enriched pathways were related to inflammation and immunoregulation, angiogenesis, extracellular matrix, oxidative stress, and lipid trafficking and storage. We validated, by flow cytometry and immunofluorescence, the protein expression of several genes encoding cell surface proteins. The inflammatory response was associated with the endothelial upregulation of genes related to lipid storage functions and we identified lipid droplet biogenesis in the endothelial cells after ischemia. The study reports a robust translatomic signature of brain endothelial cells after acute stroke and identifies enrichment in novel pathways involved in membrane signaling and lipid storage. Altogether these results highlight the endothelial contribution to the inflammatory response, and identify novel molecules that could be targets to improve vascular function after ischemic stroke.

Macrophage CD5L is a target for cancer immunotherapy.

BACKGROUND: Reprogramming of immunosuppressive tumor-associated macrophages (TAMs) presents an attractive therapeutic strategy in cancer. The aim of this study was to explore the role of macrophage CD5L protein in TAM activity and assess its potential as a therapeutic target. METHODS: Monoclonal antibodies (mAbs) against recombinant CD5L were raised by subcutaneous immunization of BALB/c mice. Peripheral blood monocytes were isolated from healthy donors and stimulated with IFN/LPS, IL4, IL10, and conditioned medium (CM) from different cancer cell lines in the presence of anti-CD5L mAb or controls. Subsequently, phenotypic markers, including CD5L, were quantified by flow cytometry, IF and RT-qPCR. Macrophage CD5L protein expression was studied in 55 human papillary lung adenocarcinoma (PAC) samples by IHC and IF. Anti-CD5L mAb and isotype control were administered intraperitoneally into a syngeneic Lewis Lung Carcinoma mouse model and tumor growth was measured. Tumor microenvironment (TME) changes were determined by flow cytometry, IHC, IF, Luminex, RNAseq and RT-qPCR. FINDINGS: Cancer cell lines CM induced an immunosuppressive phenotype (increase in CD163, CD206, MERTK, VEGF and CD5L) in cultured macrophages. Accordingly, high TAM expression of CD5L in PAC was associated with poor patient outcome (Log-rank (Mantel-Cox) test p = 0.02). We raised a new anti-CD5L mAb that blocked the immunosuppressive phenotype of macrophages in vitro. Its administration in vivo inhibited tumor progression of lung cancer by altering the intratumoral myeloid cell population profile and CD4+ T-cell exhaustion phenotype, thereby significantly modifying the TME and increasing the inflammatory milieu. INTERPRETATION: CD5L protein plays a key function in modulating the activity of macrophages and their interactions within the TME, which supports its role as a therapeutic target in cancer immunotherapy. FUNDING: For a full list of funding bodies, please see the Acknowledgements.

Metabolomics as a tool to predict the risk of decompensation or liver-related death in patients with compensated cirrhosis.

BACKGROUND AND AIMS: Patients with compensated cirrhosis with clinically significant portal hypertension (CSPH: HVPG > 10 mm Hg) have a high risk of decompensation. HVPG is, however, an invasive procedure not available in all centers. The present study aims to assess whether metabolomics can improve the capacity of clinical models in predicting clinical outcomes in these compensated patients. APPROACH AND RESULTS: This is a nested study from the PREDESCI cohort (an RCT of nonselective beta-blockers vs. placebo in 201 patients with compensated cirrhosis and CSPH), including 167 patients for whom a blood sample was collected. A targeted metabolomic serum analysis, using ultra-high-performance liquid chromatography-mass spectrometry, was performed. Metabolites underwent univariate time-to-event cox regression analysis. Top-ranked metabolites were selected using Log-Rank p -value to generate a stepwise cox model. Comparison between models was done using DeLong test. Eighty-two patients with CSPH were randomized to nonselective beta-blockers and 85 to placebo. Thirty-three patients developed the main endpoint (decompensation/liver-related death). The model, including HVPG, Child-Pugh, and treatment received ( HVPG/Clinical model ), had a C-index of 0.748 (CI95% 0.664-0.827). The addition of 2 metabolites, ceramide (d18:1/22:0) and methionine (HVPG/Clinical/Metabolite model), significantly improved the model's performance [C-index of 0.808 (CI95% 0.735-0.882); p =0.032]. The combination of these 2 metabolites together with Child-Pugh and the type of treatment received (Clinical/Metabolite model) had a C-index of 0.785 (CI95% 0.710-0.860), not significantly different from the HVPG-based models including or not metabolites. CONCLUSIONS: In patients with compensated cirrhosis and CSPH, metabolomics improves the capacity of clinical models and achieves similar predictive capacity than models including HVPG.

When Heart Beats Differently in Depression: Review of Nonlinear Heart Rate Variability Measures.

BACKGROUND: Disturbed heart dynamics in depression seriously increases mortality risk. Heart rate variability (HRV) is a rich source of information for studying this dynamics. This paper is a meta-analytic review with methodological commentary of the application of nonlinear analysis of HRV and its possibility to address cardiovascular diseases in depression. OBJECTIVE: This paper aimed to appeal for the introduction of cardiological screening to patients with depression, because it is still far from established practice. The other (main) objective of the paper was to show that nonlinear methods in HRV analysis give better results than standard ones. METHODS: We systematically searched on the web for papers on nonlinear analyses of HRV in depression, in line with PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) 2020 framework recommendations. We scrutinized the chosen publications and performed random-effects meta-analysis, using the esci module in jamovi software where standardized effect sizes (ESs) are corrected to yield the proof of the practical utility of their results. RESULTS: In all, 26 publications on the connection of nonlinear HRV measures and depression meeting our inclusion criteria were selected, examining a total of 1537 patients diagnosed with depression and 1041 healthy controls (N=2578). The overall ES (unbiased) was 1.03 (95% CI 0.703-1.35; diamond ratio 3.60). We performed 3 more meta-analytic comparisons, demonstrating the overall effectiveness of 3 groups of nonlinear analysis: detrended fluctuation analysis (overall ES 0.364, 95% CI 0.237-0.491), entropy-based measures (overall ES 1.05, 95% CI 0.572-1.52), and all other nonlinear measures (overall ES 0.702, 95% CI 0.422-0.982). The effectiveness of the applied methods of electrocardiogram analysis was compared and discussed in the light of detection and prevention of depression-related cardiovascular risk. CONCLUSIONS: We compared the ESs of nonlinear and conventional time and spectral methods (found in the literature) and demonstrated that those of the former are larger, which recommends their use for the early screening of cardiovascular abnormalities in patients with depression to prevent possible deleterious events.

Genome-wide survey of D/E repeats in human proteins uncovers their instability and aids in identifying their role in the chromatin regulator ATAD2.

D/E repeats are stretches of aspartic and/or glutamic acid residues found in over 150 human proteins. We examined genomic stability of D/E repeats and functional characteristics of D/E repeat-containing proteins vis-à-vis the proteins with poly-Q or poly-A repeats, which are known to undergo pathologic expansions. Mining of tumor sequencing data revealed that D/E repeat-coding regions are similar to those coding poly-Qs and poly-As in increased incidence of trinucleotide insertions/deletions but differ in types and incidence of substitutions. D/E repeat-containing proteins preferentially function in chromatin metabolism and are the more likely to be nuclear and interact with core histones, the longer their repeats are. One of the longest D/E repeats of unknown function is in ATAD2, a bromodomain family ATPase frequently overexpressed in tumors. We demonstrate that D/E repeat deletion in ATAD2 suppresses its binding to nascent and mature chromatin and to the constitutive pericentromeric heterochromatin, where ATAD2 represses satellite transcription.

Computational Literature-based Discovery for Natural Products Research: Current State and Future Prospects.

Literature-based discovery (LBD) mines existing literature in order to generate new hypotheses by finding links between previously disconnected pieces of knowledge. Although automated LBD systems are becoming widespread and indispensable in a wide variety of knowledge domains, little has been done to introduce LBD to the field of natural products research. Despite growing knowledge in the natural product domain, most of the accumulated information is found in detached data pools. LBD can facilitate better contextualization and exploitation of this wealth of data, for example by formulating new hypotheses for natural product research, especially in the context of drug discovery and development. Moreover, automated LBD systems promise to accelerate the currently tedious and expensive process of lead identification, optimization, and development. Focusing on natural product research, we briefly reflect the development of automated LBD and summarize its methods and principal data sources. In a thorough review of published use cases of LBD in the biomedical domain, we highlight the immense potential of this data mining approach for natural product research, especially in context with drug discovery or repurposing, mode of action, as well as drug or substance interactions. Most of the 91 natural product-related discoveries in our sample of reported use cases of LBD were addressed at a computer science audience. Therefore, it is the wider goal of this review to introduce automated LBD to researchers who work with natural products and to facilitate the dialogue between this community and the developers of automated LBD systems.

Discovery of host-directed modulators of virus infection by probing the SARS-CoV-2-host protein-protein interaction network.

The ongoing coronavirus disease 2019 (COVID-19) pandemic has highlighted the need to better understand virus-host interactions. We developed a network-based method that expands the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-host protein interaction network and identifies host targets that modulate viral infection. To disrupt the SARS-CoV-2 interactome, we systematically probed for potent compounds that selectively target the identified host proteins with high expression in cells relevant to COVID-19. We experimentally tested seven chemical inhibitors of the identified host proteins for modulation of SARS-CoV-2 infection in human cells that express ACE2 and TMPRSS2. Inhibition of the epigenetic regulators bromodomain-containing protein 4 (BRD4) and histone deacetylase 2 (HDAC2), along with ubiquitin-specific peptidase (USP10), enhanced SARS-CoV-2 infection. Such proviral effect was observed upon treatment with compounds JQ1, vorinostat, romidepsin and spautin-1, when measured by cytopathic effect and validated by viral RNA assays, suggesting that the host proteins HDAC2, BRD4 and USP10 have antiviral functions. We observed marked differences in antiviral effects across cell lines, which may have consequences for identification of selective modulators of viral infection or potential antiviral therapeutics. While network-based approaches enable systematic identification of host targets and selective compounds that may modulate the SARS-CoV-2 interactome, further developments are warranted to increase their accuracy and cell-context specificity.

Epigenome-Wide DNA Methylation Profiling of Normal Mucosa Reveals HLA-F Hypermethylation as a Biomarker Candidate for Serrated Polyposis Syndrome.

Serrated polyposis syndrome (SPS) is associated with a high risk for colorectal cancer. Intense promoter hypermethylation is a frequent molecular finding in the serrated pathway and may be present in normal mucosa, predisposing to the formation of serrated lesions. To identify novel biomarkers for SPS, fresh-frozen samples of normal mucosa from 50 patients with SPS and 19 healthy individuals were analyzed by using the 850K BeadChip Technology (Infinium). Aberrant methylation levels were correlated with gene expression using a next-generation transcriptome profiling tool. Two validation steps were performed on independent cohorts: first, on formalin-fixed, paraffin-embedded tissue of the normal mucosa; and second, on 24 serrated lesions. The most frequently hypermethylated genes were HLA-F, SLFN12, HLA-DMA, and RARRES3; and the most frequently hypomethylated genes were PIWIL1 and ANK3 (Δß = 10%; P < 0.05). Expression levels of HLA-F, SLFN12, and HLA-DMA were significantly different between SPS patients and healthy individuals and correlated well with the methylation status of the corresponding differentially methylated region (fold change, >20%; r > 0.55; P < 0.001). Significant hypermethylation of CpGs in the gene body of HLA-F was also found in serrated lesions (Δß = 23%; false discovery rate = 0.01). Epigenome-wide methylation profiling has revealed numerous differentially methylated CpGs in normal mucosa from SPS patients. Significant hypermethylation of HLA-F is a novel biomarker candidate for SPS.

Differentially Deregulated MicroRNAs as Novel Biomarkers for Neoplastic Progression in Ulcerative Colitis.

INTRODUCTION: Colorectal cancer (CRC) is a potentially life-threatening complication of long-standing ulcerative colitis (UC). MicroRNAs (miRNA) are epigenetic regulators that have been involved in the development of UC-associated CRC. However, their role as potential mucosal biomarkers of neoplastic progression has not been adequately studied. METHODS: In this study, we analyzed the expression of 96 preselected miRNAs in human formalin-fixed and paraffin-embedded tissue of 52 case biopsies (20 normal mucosa, 20 dysplasia, and 12 UC-associated CRCs) and 50 control biopsies (10 normal mucosa, 21 sporadic adenomas, and 19 sporadic CRCs) by using Custom TaqMan Array Cards. For validation of deregulated miRNAs, we performed individual quantitative real-time polymerase chain reaction in an independent cohort of 50 cases (13 normal mucosa, 25 dysplasia, and 12 UC-associated CRCs) and 46 controls (7 normal mucosa, 19 sporadic adenomas, and 20 sporadic CRCs). RESULTS: Sixty-four miRNAs were found to be differentially deregulated in the UC-associated CRC sequence. Eight of these miRNAs were chosen for further validation. We confirmed miR-31, -106a, and -135b to be significantly deregulated between normal mucosa and dysplasia, as well as across the UC-associated CRC sequence (all P < 0.01). Notably, these miRNAs also confirmed to have a significant differential expression compared with sporadic CRC (all P < 0.05). DISCUSSION: UC-associated and sporadic CRCs have distinct miRNA expression patterns, and some miRNAs indicate early neoplastic progression.

Impact of Diabetes Mellitus on Voice: A Methodological Commentary.

Recent research describes the effect of Type 2 diabetes (T2D) on voice, suggesting that it can be diagnosed based on vocal clues. Although these studies have similar experimental designs with respect to the voice data and the analysis methods, the conclusions regarding the voice changes differ substantially and are at times contradictory. This is unexpected, since the mechanism of pathological deterioration behind the observed changes is the same. This year in an article published in J. of Voice it was suggested that vocal changes may be different among ethnicities. Before this hypothesis can be accepted, the study protocols should be improved and unified, to ensure that the empirical evidence is reliable. Additionally, given the recently published data about the temporal voice changes as a result of glucose swings, we propose that the persons in hypo- and hyperglycemic conditions should be excluded from the experiment. Since no study succeeded in diabetes detection, it is timely to mention that there is an alternative methodology for disease detection from voice, which is far more sensitive than the state of the art procedure. We propose a script that is available from the first author on request.

Blood Glucose Estimation From Voice: First Review of Successes and Challenges.

The possibility to estimate glucose value from voice would make a breakthrough in diabetes treatment: namely, remove the delay in the nonintrusive instantaneous blood glucose estimation, relieve medical budgets and significantly improve wellbeing of diabetics. In this review, different approaches have been described and systematized, in order to provide an objective snapshot of the state of the art. Since nonintrusive glucose estimation is notoriously difficult, we included a Transparence and Reproducibility Score aimed at revealing the biases in the primary research articles. The review is completed with the discussion on future research pathways.

Treatment With Simvastatin and Rifaximin Restores the Plasma Metabolomic Profile in Patients With Decompensated Cirrhosis.

Patients with decompensated cirrhosis, particularly those with acute-on-chronic liver failure (ACLF), show profound alterations in plasma metabolomics. The aim of this study was to investigate the effect of treatment with simvastatin and rifaximin on plasma metabolites of patients with decompensated cirrhosis, specifically on compounds characteristic of the ACLF plasma metabolomic profile. Two cohorts of patients were investigated. The first was a descriptive cohort of patients with decompensated cirrhosis (n = 42), with and without ACLF. The second was an intervention cohort from the LIVERHOPE-SAFETY randomized, double-blind, placebo-controlled trial treated with simvastatin 20 mg/day plus rifaximin 1,200 mg/day (n = 12) or matching placebo (n = 13) for 3 months. Plasma samples were analyzed using ultrahigh performance liquid chromatography-tandem mass spectroscopy for plasma metabolomics characterization. ACLF was characterized by intense proteolysis and lipid alterations, specifically in pathways associated with inflammation and mitochondrial dysfunction, such as the tryptophan-kynurenine and carnitine beta-oxidation pathways. An ACLF-specific signature was identified. Treatment with simvastatin and rifaximin was associated with changes in 161 of 985 metabolites in comparison to treatment with placebo. A remarkable reduction in levels of metabolites from the tryptophan-kynurenine and carnitine pathways was found. Notably, 18 of the 32 metabolites of the ACLF signature were affected by the treatment. Conclusion: Treatment with simvastatin and rifaximin modulates some of the pathways that appear to be key in ACLF development. This study unveils some of the mechanisms involved in the effects of treatment with simvastatin and rifaximin in decompensated cirrhosis and sets the stage for the use of metabolomics to investigate new targeted therapies in cirrhosis to prevent ACLF development.

Innate immunity mediator STING modulates nascent DNA metabolism at stalled forks in human cells.

Background: The cGAS/STING pathway, part of the innate immune response to foreign DNA, can be activated by cell's own DNA arising from the processing of the genome, including the degradation of nascent DNA at arrested replication forks, which can be upregulated in cancer cells. Recent evidence raises a possibility that the cGAS/STING pathway may also modulate the very processes that trigger it, e.g., DNA damage repair or processing of stalled forks. Methods: We manipulated STING levels in human cells by depleting or re-expressing it, and assessed the effects of STING on replication using microfluidics-assisted replication track analysis, or maRTA, a DNA fiber assay, as well as immuno-precipitation of nascent DNA, or iPOND. We also assessed STING subcellular distribution and its ability to activate. Results: Depletion of STING suppressed and its re-expression in STING-deficient cancer cells upregulated the degradation of nascent DNA at arrested replication forks. Replication fork arrest was accompanied by the STING pathway activation, and a STING mutant that does not activate the pathway failed to upregulate nascent DNA degradation. cGAS was required for STING's effect on degradation, but this requirement could be bypassed by treating cells with a STING agonist. Cells expressing inactive STING had a reduced level of RPA on parental and nascent DNA of arrested forks and a reduced CHK1 activation compared to cells with the wild type STING. STING also affected unperturbed fork progression in a subset of cell lines. STING fractionated to the nuclear fractions enriched for structural components of chromatin and nuclear envelope, and furthermore, it associated with the chromatin of arrested replication forks as well as post-replicative chromatin. Conclusion: Our data highlight STING as a determinant of stalled replication fork integrity, thus revealing a novel connection between the replication stress and innate immune responses.

Non-invasive alloimmune risk stratification of long-term liver transplant recipients.

BACKGROUND & AIMS: Management of long-term immunosuppression following liver transplantation (LT) remains empirical. Surveillance liver biopsies in combination with transcriptional profiling could overcome this challenge by identifying recipients with active alloimmune-mediated liver damage despite normal liver tests, but this approach lacks applicability. Our aim was to investigate the utility of non-invasive tools for the stratification of stable long-term survivors of LT, according to their immunological risk and need for immunosuppression. METHODS: We conducted a cross-sectional multicentre study of 190 adult LT recipients assessed to determine their eligibility to participate in an immunosuppression withdrawal trial. Patients had stable liver allograft function and had been transplanted for non-autoimmune non-replicative viral liver disease >3 years before inclusion. We performed histological, immunogenetic and serological studies and measured the intrahepatic transcript levels of an 11-gene classifier highly specific for T cell-mediated rejection (TCMR). RESULTS: In this cohort, 35.8% of patients harboured clinically silent fibro-inflammatory liver lesions (13.7% had mild damage and 22.1% had moderate-to-severe damage). The severity of liver allograft damage was positively associated with TCMR-related transcripts, class II donor-specific antibodies (DSAs), ALT, AST, and liver stiffness measurement (LSM), and negatively correlated with serum creatinine and tacrolimus trough levels. Liver biopsies were stratified according to their TCMR transcript levels using a cut-off derived from biopsies with clinically significant TCMR. Two multivariable prediction models, integrating ALT+LSM or ALT+class II DSAs, had a high discriminative capacity for classifying patients with or without alloimmune damage. The latter model performed well in an independent cohort of 156 liver biopsies obtained from paediatric liver recipients with similar inclusion/exclusion criteria. CONCLUSION: ALT, class II DSAs and LSM are valuable tools to non-invasively identify stable LT recipients without significant underlying alloimmunity who could benefit from minimisation of immunosuppression. LAY SUMMARY: A large proportion of liver transplant patients with normal liver tests have inflammatory liver lesions, which in 17% of cases are molecularly indistinguishable from those seen at the time of rejection. ALT, class II donor-specific antibodies and liver stiffness are useful in identifying patients with this form of subclinical rejection. We propose these markers as a useful tool to help clinicians determine if the immunosuppression administered is adequate.

Overexpression of oncogenic H-Ras in hTERT-immortalized and SV40-transformed human cells targets replicative and specialized DNA polymerases for depletion.

DNA polymerases play essential functions in replication fork progression and genome maintenance. DNA lesions and drug-induced replication stress result in up-regulation and re-localization of specialized DNA polymerases η and κ. Although oncogene activation significantly alters DNA replication dynamics, causing replication stress and genome instability, little is known about DNA polymerase expression and regulation in response to oncogene activation. Here, we investigated the consequences of mutant H-RAS G12V overexpression on the regulation of DNA polymerases in h-TERT immortalized and SV40-transformed human cells. Focusing on DNA polymerases associated with the replication fork, we demonstrate that DNA polymerases are depleted in a temporal manner in response to H-RAS G12V overexpression. The polymerases targeted for depletion, as cells display markers of senescence, include the Pol α catalytic subunit (POLA1), Pol δ catalytic and p68 subunits (POLD1 and POLD3), Pol η, and Pol κ. Both transcriptional and post-transcriptional mechanisms mediate this response. Pol η (POLH) depletion is sufficient to induce a senescence-like growth arrest in human foreskin fibroblast BJ5a cells, and is associated with decreased Pol α expression. Using an SV-40 transformed cell model, we observed cell cycle checkpoint signaling differences in cells with H-RasG12V-induced polymerase depletion, as compared to Pol η-deficient cells. Our findings contribute to our understanding of cellular events following oncogene activation and cellular transformation.

Co-expression gene network analysis reveals novel regulatory pathways involved in porto-sinusoidal vascular disease.

BACKGROUND & AIMS: Porto-sinusoidal vascular disease (PSVD) is a rare vascular liver disease of unknown etiology that causes portal hypertension. It usually affects young individuals and shortens live expectancy. The deregulated pathways involved in PSVD development are unknown and therefore we lack curative treatments. The purpose of this study was to integrate transcriptomic and clinical data by comprehensive network-based modeling in order to uncover altered biological processes in patients with PSVD. METHODS: We obtained liver tissue samples from 20 consecutive patients with PSVD and 21 sex- and age-matched patients with cirrhosis and 13 histologically normal livers (HNL) (initial cohort) and performed transcriptomic analysis. Microarray data were analyzed using weighted gene correlation network analysis to identify clusters of highly correlated genes differently expressed in patients with PSVD. We next evaluated the molecular pathways enriched in patients with PSVD and the core-related genes from the most significantly enriched pathways in patients with PSVD. Our main findings were validated using RNA sequencing in a different cohort of PSVD, cirrhosis and HNL (n = 8 for each group). RESULTS: Patients with PSVD have a distinctive genetic profile enriched mainly in canonical pathways involving hemostasis and coagulation but also lipid metabolism and oxidative phosphorylation. Serpin family (SERPINC1), the apolipoproteins (APOA, APOB, APOC), ATP synthases (ATP5G1, ATP5B), fibrinogen genes (FGB, FGA) and alpha-2-macroglobulin were identified as highly connective genes that may have an important role in PSVD pathogenesis. CONCLUSION: PSVD has a unique transcriptomic profile and we have identified deregulation of pathways involved in vascular homeostasis as the main pathogenic event of disease development. LAY SUMMARY: Porto-sinusoidal vascular disease is a rare but life-shortening disease that affects mainly young people. Knowledge of the disrupted pathways involved in its development will help to identify novel therapeutic targets and new treatments. Using a systems biology approach, we identify that pathways regulating endothelial function and tone may act as drivers of porto-sinusoidal vascular disease.

SARS-CoV-2-specific serological and functional T cell immune responses during acute and early COVID-19 convalescence in solid organ transplant patients.

The description of protective humoral and T cell immune responses specific against SARS-CoV-2 has been reported among immunocompetent (IC) individuals developing COVID-19 infection. However, its characterization and determinants of poorer outcomes among the at-risk solid organ transplant (SOT) patient population have not been thoroughly investigated. Cytokine-producing T cell responses, such as IFN-γ, IL-2, IFN-γ/IL-2, IL-6, IL-21, and IL-5, against main immunogenic SARS-CoV-2 antigens and IgM/IgG serological immunity were tracked in SOT (n = 28) during acute infection and at two consecutive time points over the following 40 days of convalescence and were compared to matched IC (n = 16) patients admitted with similar moderate/severe COVID-19. We describe the development of a robust serological and functional T cell immune responses against SARS-CoV-2 among SOT patients, similar to IC patients during early convalescence. However, at the infection onset, SOT displayed lower IgG seroconversion rates (77% vs. 100%; p = .044), despite no differences on IgG titers, and a trend toward decreased SARS-CoV-2-reactive T cell frequencies, especially against the membrane protein (7 [0-34] vs. 113 [15-245], p = .011, 2 [0-9] vs. 45 [5-74], p = .009, and 0 [0-2] vs. 13 [1-24], p = .020, IFN-γ, IL-2, and IFN-γ/IL-2 spots, respectively). In summary, our data suggest that despite a certain initial delay, SOT population achieve comparable functional immune responses than the general population after moderate/severe COVID-19.