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Abstract The ongoing evolution of SARS-CoV-2 to evade vaccines and therapeutics underlines the need for innovative therapies with high genetic barriers to resistance. Therefore, there is pronounced interest in identifying new pharmacological targets in the SARS-CoV-2 viral life cycle. The small molecule PAV-104, identified through a cell-free protein synthesis and assembly screen, was recently shown to target host protein assembly machinery in a manner specific to viral assembly. In this study, we investigate the capacity of PAV-104 to inhibit SARS-CoV-2 replication in human airway epithelial cells (AECs). We show that PAV-104 inhibits >99% of infection with diverse SARS-CoV-2 variants in immortalized AECs, and in primary human AECs cultured at the air-liquid interface (ALI) to represent the lung microenvironment in vivo. Our data demonstrate that PAV-104 inhibits SARS-CoV-2 production without affecting viral entry, mRNA transcription, or protein synthesis. PAV-104 interacts with SARS-CoV-2 nucleocapsid (N) and interferes with its oligomerization, blocking particle assembly. Transcriptomic analysis reveals that PAV-104 reverses SARS-CoV-2 induction of the type-I interferon response and the maturation of nucleoprotein signaling pathway known to support coronavirus replication. Our findings suggest that PAV-104 is a promising therapeutic candidate for COVID-19 with a mechanism of action that is distinct from existing clinical management approaches.

Summary: Small intestine (SI) maturation during early life is pivotal in preventing the onset of gut diseases. In this study we interrogated the milestones of SI development by gene expression profiling and ingenuity pathway analyses. We identified a set of cytokines as main regulators of changes observed across different developmental stages. Upon cytokines stimulation, with IFNγ as the most contributing factor, human fetal organoids (HFOs) increase brush border gene expression and enzyme activity as well as trans-epithelial electrical resistance. Electron microscopy revealed developed brush border and loss of fetal cell characteristics in HFOs upon cytokine stimulation. We identified T cells as major source of IFNγ production in the fetal SI lamina propria. Co-culture of HFOs with T cells recapitulated the major effects of cytokine stimulation. Our findings underline pro-inflammatory cytokines derived from T cells as pivotal factors inducing functional SI maturation in vivo and capable of modulating the barrier maturation of HFOs in vitro.

BackgroundDegradation of the endothelial protective glycocalyx layer during COVID-19 infection leads to shedding of major glycocalyx components. These circulating proteins and their degradation products may feedback on immune and endothelial cells and activate molecular signaling cascades in COVID-19 associated microvascular injury. To test this hypothesis, we measured plasma glycocalyx components in patients with SARS-CoV-2 infection of variable disease severity and identified molecular signaling networks activated by glycocalyx components in immune and endothelial cells.MethodsWe studied patients with RT-PCR confirmed COVID-19 pneumonia, patients with COVID-19 Acute Respiratory Distress Syndrome (ARDS) and healthy controls (wildtype, n=20 in each group) and measured syndecan-1, heparan sulfate and hyaluronic acid. The in-silico construction of signaling networks was based on RNA sequencing (RNAseq) of mRNA transcripts derived from blood cells and of miRNAs isolated from extracellular vesicles from the identical cohort. Differentially regulated RNAs between groups were identified by gene expression analysis. Both RNAseq data sets were used for network construction of circulating glycosaminoglycans focusing on immune and endothelial cells.ResultsPlasma concentrations of glycocalyx components were highest in COVID-19 ARDS. Hyaluronic acid plasma levels in patients admitted with COVID-19 pneumonia who later developed ARDS during hospital treatment (n=8) were significantly higher at hospital admission than in patients with an early recovery. RNAseq identified hyaluronic acid as an upregulator of TLR4 in pneumonia and ARDS. In COVID-19 ARDS, syndecan-1 increased IL-6, which was significantly higher than in pneumonia. In ARDS, hyaluronic acid activated NRP1, a co-receptor of activated VEGFA, which is associated with pulmonary vascular hyperpermeability and interacted with VCAN (upregulated), a proteoglycan important for chemokine communication.ConclusionsCirculating glycocalyx components in COVID-19 have distinct biologic feedback effects on immune and endothelial cells and result in upregulation of key regulatory transcripts leading to further immune activation and more severe systemic inflammation. These consequences are most pronounced during the early hospital phase of COVID-19 before pulmonary failure develops. Elevated levels of circulating glycocalyx components may early identify patients at risk for microvascular injury and ARDS. The timely inhibition of glycocalyx degradation could provide a novel therapeutic approach to prevent the development of ARDS in COVID-19.

Abstract Background Progranulin is a widely expressed pleiotropic growth factor with a central regulatory effect during the early immune response in sepsis. Progranulin signaling has not been systematically studied and compared between sepsis, community-acquired pneumonia (CAP), COVID-19 pneumonia and a sterile systemic inflammatory response (SIRS). We delineated molecular networks of progranulin signaling by next-generation sequencing (NGS), determined progranulin plasma concentrations and quantified the diagnostic performance of progranulin to differentiate between the above-mentioned disorders using the established biomarkers procalcitonin (PCT), interleukin-6 (IL-6) and C-reactive protein (CRP) for comparison. Methods The diagnostic performance of progranulin was operationalized by calculating AUC and ROC statistics for progranulin and established biomarkers in 241 patients with sepsis, 182 patients with SIRS, 53 patients with CAP, 22 patients with COVID-19 pneumonia and 53 healthy volunteers. miRNAs and mRNAs in blood cells from sepsis patients (n = 7) were characterized by NGS and validated by RT-qPCR in an independent cohort (n = 39) to identify canonical gene networks associated with upregulated progranulin at sepsis onset. Results Plasma concentrations of progranulin (ELISA) in patients with sepsis were 57.5 (42.8–84.9, Q25–Q75) ng/ml and significantly higher than in CAP (38.0, 33.5–41.0 ng/ml, p

Abstract Background Leveraging previously identified viral interactions with human host proteins, we apply a machine learning-based approach to connect SARS-CoV-2 viral proteins to relevant host biological functions, diseases, and pathways in a large-scale knowledge graph derived from the biomedical literature. Our goal is to explore how SARS-CoV-2 could interfere with various host cell functions, and to identify drug targets amongst the host genes that could potentially be modulated against COVID-19 by repurposing existing drugs. The machine learning model employed here involves gene embeddings that leverage causal gene expression signatures curated from literature. In contrast to other network-based approaches for drug repurposing, our approach explicitly takes the direction of effects into account, distinguishing between activation and inhibition. Results We have constructed 70 networks connecting SARS-CoV-2 viral proteins to various biological functions, diseases, and pathways reflecting viral biology, clinical observations, and co-morbidities in the context of COVID-19. Results are presented in the form of interactive network visualizations through a web interface, the Coronavirus Network Explorer (CNE), that allows exploration of underlying experimental evidence. We find that existing drugs targeting genes in those networks are strongly enriched in the set of drugs that are already in clinical trials against COVID-19. Conclusions The approach presented here can identify biologically plausible hypotheses for COVID-19 pathogenesis, explicitly connected to the immunological, virological and pathological observations seen in SARS-CoV-2 infected patients. The discovery of repurposable drugs is driven by prior knowledge of relevant functional endpoints that reflect known viral biology or clinical observations, therefore suggesting potential mechanisms of action. We believe that the CNE offers relevant insights that go beyond more conventional network approaches, and can be a valuable tool for drug repurposing. The CNE is available at https://digitalinsights.qiagen.com/coronavirus-network-explorer .

BackgroundExtracellular vesicles (EVs) are mediators of cell-to-cell communication in inflammatory lung diseases. They function as carriers for miRNAs which regulate mRNA transcripts and signaling pathways after uptake into recipient cells. We investigated whether miRNAs associated with circulating EVs regulate immunologic processes in COVID-19.MethodsWe prospectively studied 20 symptomatic patients with COVID-19 pneumonia, 20 mechanically ventilated patients with severe COVID-19 (severe acute respiratory corona virus-2 syndrome, ARDS) and 20 healthy controls. EVs were isolated by precipitation, total RNA was extracted, profiled by small RNA sequencing and evaluated by differential gene expression analysis (DGE). Differentially regulated miRNAs between groups were bioinformatically analyzed, mRNA target transcripts identified and signaling networks constructed, thereby comparing COVID-19 pneumonia to the healthy state and pneumonia to severe COVID-19 ARDS.ResultsDGE revealed 43 significantly and differentially expressed miRNAs (25 downregulated) in COVID-19 pneumonia when compared to controls, and 20 miRNAs (15 downregulated) in COVID-19 ARDS patients in comparison to those with COVID-19 pneumonia. Network analysis for comparison of COVID-19 pneumonia to healthy controls showed upregulated miR-3168 (log2FC=2.28, padjusted

Exposure to stressful environments weakens immunity evidenced by a detectable reactivation of dormant viruses. The mechanism behind this observation remains unclear. We performed next generation sequencing from RNA extracted from blood samples of 8 male subjects collected before, during and after a 12-month stay at the Antarctic station Concordia. RNA-seq data analysis was done using QIAGEN Ingenuity Pathway Analysis (IPA) software. Data revealed the inactivation of key immune functions such as chemotaxis and leukocyte recruitment which persisted after return. Next to the activation of the stress response eIF2 pathway, interferon signaling was predicted inactivated due to a downregulation of 14 downstream genes involved in antiviral immunity. Among them, the interferon stimulated genes (ISGs) IFITM2 and 3 as well as IFIT3 exhibited the strongest fold changes and IFIT3 remained downregulated even after return. Impairment of antiviral immunity in winter-over crew can be explained by the downregulation of a battery of ISGs.

The most common scoring system for critically ill patients is the Sequential Organ Failure Assessment (SOFA) score. Little is known about specific molecular signaling networks underlying the SOFA criteria. We characterized these networks and identified specific key regulatory molecules. We prospectively studied seven patients with sepsis and six controls with high-throughput RNA sequencing (RNAseq). Quantitative reverse transcription PCR (RT-qPCR) confirmation was performed in a second independent cohort. Differentially and significantly expressed miRNAs and their target mRNA transcripts were filtered for admission SOFA criteria and marker RNAs for the respective criteria identified. We bioinformatically constructed molecular signaling networks specifically reflecting these criteria followed by RT-qPCR confirmation of RNAs with important regulatory functions in the networks in the second cohort. RNAseq identified 82 miRNAs (45% upregulated) and 3254 mRNAs (50% upregulated) differentially expressed between sepsis patients and controls. Bioinformatic analysis characterized 6 miRNAs and 76 mRNA target transcripts specific for the SOFA criteria. RT-qPCR validated miRNA and mRNAs included IGFBP2 (respiratory system); MMP9 and PDE4B (nervous system); PPARG (cardiovascular system); AKR1B1, ANXA1, and LNC2/NGAL (acute kidney injury); GFER/ALR (liver); and miR-30c-3p (coagulopathy). There are specific canonical networks underlying the SOFA score. Key regulatory miRNA and mRNA transcripts support its biologic validity.

Background: Metastatic prostate cancer (CaP) treatments are evolving rapidly but without evidence-based biomarkers to predict responses, and to maximize remissions and survival. Objective: To determine the activity of androgen receptor (AR), the target for default first-line systemic treatment, in localized treatment-naïve CaP and its association with clinical risk factors, molecular markers, CaP subtypes, and predictors of treatment response. Design, setting, and participants: We examined 452 bona fide AR target genes in clinical-grade expression profiles from 6532 such CaPs collected between 2013 and 2017 by US physicians ordering the Decipher RP test. Results were validated in three independent smaller cohorts (n = 73, 90, and 127) and clinical CaP AR ChIP-Seq data. Association with CaP differentiation and progression was analyzed in independent datasets. Outcome measurements and statistical analysis: Unsupervised clustering of CaPs based on AR target gene expression was aligned with clinical variables, differentiation scores, molecular subtypes, and predictors of response to hormonal therapy, radiotherapy, and chemotherapy. AR target gene sets were analyzed via Gene Set Enrichment Analysis for differentiation and treatment resistance, Ingenuity Pathway Analysis for associated biology, and Cistrome for genomic AR binding site (ARBS) composition. Results and limitations: Expression of eight AR target gene subsignatures gave rise to five CaP clusters, which were preferentially associated with CaP molecular subtypes, differentiation, and predictors of treatment response rather than with clinical variables. Subsignatures differed in contribution to CaP progression, luminal/basal differentiation, CaP biology, and ARBS composition. Validation in prospective trials and optimized quantitation are needed for clinical implementation. Conclusions: Measurement of AR activity patterns in treatment-naïve CaP may serve as a first branch of an evidence-based decision tree to optimize personalized treatment plans. Patient summary: Treatment options for metastatic prostate cancer are increasing without information needed to choose the right treatment for the right patient. We found variation in the behavior of the target for the default first-line therapy before treatment, which may help optimize treatment plans.

The circadian clock component NR1D1 (REVERBα) is considered a dominant regulator of lipid metabolism, with global Nr1d1 deletion driving dysregulation of white adipose tissue (WAT) lipogenesis and obesity. However, a similar phenotype is not observed under adipocyte-selective deletion (Nr1d1Flox2-6:AdipoqCre), and transcriptional profiling demonstrates that, under basal conditions, direct targets of NR1D1 regulation are limited, and include the circadian clock and collagen dynamics. Under high-fat diet (HFD) feeding, Nr1d1Flox2-6:AdipoqCre mice do manifest profound obesity, yet without the accompanying WAT inflammation and fibrosis exhibited by controls. Integration of the WAT NR1D1 cistrome with differential gene expression reveals broad control of metabolic processes by NR1D1 which is unmasked in the obese state. Adipocyte NR1D1 does not drive an anticipatory daily rhythm in WAT lipogenesis, but rather modulates WAT activity in response to alterations in metabolic state. Importantly, NR1D1 action in adipocytes is critical to the development of obesity-related WAT pathology and insulin resistance.

Standard treatment for metastatic prostate cancer (CaP) prevents ligand-activation of androgen receptor (AR). Despite initial remission, CaP progresses while relying on AR. AR transcriptional output controls CaP behavior and is an alternative therapeutic target, but its molecular regulation is poorly understood. Here, we show that action of activated AR partitions into fractions that are controlled preferentially by different coregulators. In a 452-AR-target gene panel, each of 18 clinically relevant coregulators mediates androgen-responsiveness of 0–57% genes and acts as a coactivator or corepressor in a gene-specific manner. Selectivity in coregulator-dependent AR action is reflected in differential AR binding site composition and involvement with CaP biology and progression. Isolation of a novel transcriptional mechanism in which WDR77 unites the actions of AR and p53, the major genomic drivers of lethal CaP, to control cell cycle progression provides proof-of-principle for treatment via selective interference with AR action by exploiting AR dependence on coregulators.

ABSTRACT Lyme disease is a tick-borne illness caused by the bacterium Borrelia burgdorferi, and approximately 10 to 20% of patients report persistent symptoms lasting months to years despite appropriate treatment with antibiotics. To gain insights into the molecular basis of acute Lyme disease and the ensuing development of post-treatment symptoms, we conducted a longitudinal transcriptome study of 29 Lyme disease patients (and 13 matched controls) enrolled at the time of diagnosis and followed for up to 6 months. The differential gene expression signature of Lyme disease following the acute phase of infection persisted for at least 3 weeks and had fewer than 44% differentially expressed genes (DEGs) in common with other infectious or noninfectious syndromes. Early Lyme disease prior to antibiotic therapy was characterized by marked upregulation of Toll-like receptor signaling but lack of activation of the inflammatory T-cell apoptotic and B-cell developmental pathways seen in other acute infectious syndromes. Six months after completion of therapy, Lyme disease patients were found to have 31 to 60% of their pathways in common with three different immune-mediated chronic diseases. No differential gene expression signature was observed between Lyme disease patients with resolved illness to those with persistent symptoms at 6 months post-treatment. The identification of a sustained differential gene expression signature in Lyme disease suggests that a panel of selected human host-based biomarkers may address the need for sensitive clinical diagnostics during the “window period” of infection prior to the appearance of a detectable antibody response and may also inform the development of new therapeutic targets. IMPORTANCE Lyme disease is the most common tick-borne infection in the United States, and some patients report lingering symptoms lasting months to years despite antibiotic treatment. To better understand the role of the human host response in acute Lyme disease and the development of post-treatment symptoms, we conducted the first longitudinal gene expression (transcriptome) study of patients enrolled at the time of diagnosis and followed up for up to 6 months after treatment. Importantly, we found that the gene expression signature of early Lyme disease is distinct from that of other acute infectious diseases and persists for at least 3 weeks following infection. This study also uncovered multiple previously undescribed pathways and genes that may be useful in the future as human host biomarkers for diagnosis and that constitute potential targets for the development of new therapies.

BACKGROUND:MicroRNAs (miRNAs) regulate gene expression by interfering translation or stability of target transcripts. This interplay between miRNA and their mRNA has been proposed as an important process in cancer development and progression. We have investigated molecular networks impacted by predicted mRNA targets of differentially expressed miRNAs in patients with clear cell renal cell carcinoma (ccRCC) diagnosed with or without metastasis. MATERIAL AND METHODS:miRNA and mRNA microarray expression profiles derived from primary ccRCC from patients with (16 samples) or without diagnosed metastasis (22 samples) were used to identify anti-correlated miRNA-mRNA interaction in ccRCC. For this purpose, Ingenuity pathway analysis microRNA Target Filter, which enables prioritization of experimentally validated and predicted mRNA targets was used. By applying an expression pairing tool, the analysis was focused on targets exhibiting altered expression in our analysis, finding miRNAs and their target genes with opposite or same expression. The resulting identified interactions were revalidated by RT-qPCR in another cohort of ccRCC patients. A selection of the predicted miRNA-mRNA interactions was tested by functional analyses using miRNA knockdown and overexpression experiments in renal cancer cell lines. RESULTS:Among the significantly differentially expressed miRNAs, we have identified three miRNAs (miR-146a-5p, miR-128a-3p, and miR-17-5p) that were upregulated in primary tumors from patients without metastasis and downregulated in primary tumors from patients with metastasis. We have further identified mRNA targets, which expression were inversely correlated to these 3 miRNAs, and have been previously experimentally demonstrated in cancer setting in humans. Specifically, we showed that CXCL8/IL8, UHRF1, MCM10, and CDKN3 were downregulated and targeted by miR-146a-5p. The interaction between miR-146a-5p and their targets CXCL8 and UHRF1 was validated in cell culture experiments. CONCLUSIONS:We identified novel target genes of dysregulated miRNAs, which are involved in the transition from primary RCC without metastases into tumors generating distant metastasis.