Time-dependent recruitment of GAF, ISGF3 and IRF1 complexes shapes IFNα and IFNγ-activated transcriptional responses and explains mechanistic and functional overlap.

To understand in detail the transcriptional and functional overlap of IFN-I- and IFN-II-activated responses, we used an integrative RNAseq-ChIPseq approach in Huh7.5 cells and characterized the genome-wide role of pSTAT1, pSTAT2, IRF9 and IRF1 in time-dependent ISG expression. For the first time, our results provide detailed insight in the timely steps of IFNα- and IFNγ-induced transcription, in which pSTAT1- and pSTAT2-containing ISGF3 and GAF-like complexes and IRF1 are recruited to individual or combined ISRE and GAS composite sites in a phosphorylation- and time-dependent manner. Interestingly, composite genes displayed a more heterogeneous expression pattern, as compared to GAS (early) and ISRE genes (late), with the time- and phosphorylation-dependent recruitment of GAF, ISGF3 and IRF1 after IFNα stimulation and GAF and IRF1 after IFNγ. Moreover, functional composite genes shared features of GAS and ISRE genes through transcription factor co-binding to closely located sites, and were able to sustain IFN responsiveness in STAT1-, STAT2-, IRF9-, IRF1- and IRF9/IRF1-mutant Huh7.5 cells compared to Wt cells. Thus, the ISRE + GAS composite site acted as a molecular switch, depending on the timely available components and transcription factor complexes. Consequently, STAT1, STAT2 and IRF9 were identified as functional composite genes that are part of a positive feedback loop controlling long-term IFNα and IFNγ responses. More important, in the absence of any one of the components, the positive feedback regulation of the ISGF3 and GAF components appeared to be preserved. Together, these findings provide further insight in the existence of a novel ISRE + GAS composite-dependent intracellular amplifier circuit prolonging ISG expression and controlling cellular responsiveness to different types of IFNs and subsequent antiviral activity. It also offers an explanation for the existing molecular and functional overlap between IFN-I- and IFN-II-activated ISG expression.

ISGF3 and STAT2/IRF9 Control Basal and IFN-Induced Transcription through Genome-Wide Binding of Phosphorylated and Unphosphorylated Complexes to Common ISRE-Containing ISGs.

In addition to the canonical ISGF3 and non-canonical STAT2/IRF9 complexes, evidence is emerging of the role of their unphosphorylated counterparts in IFN-dependent and -independent ISG transcription. To better understand the relation between ISGF3 and U-ISGF3 and STAT2/IRF9 and U-STAT2/IRF9 in IFN-I-stimulated transcriptional responses, we performed RNA-Seq and ChIP-Seq, in combination with phosphorylation inhibition and antiviral experiments. First, we identified a group of ISRE-containing ISGs that were commonly regulated in IFNα-treated WT and STAT1-KO cells. Thus, in 2fTGH and Huh7.5 WT cells, early and long-term IFNα-inducible transcription and antiviral activity relied on the DNA recruitment of the ISGF3 components STAT1, STAT2 and IRF9 in a phosphorylation- and time-dependent manner. Likewise, in ST2-U3C and Huh-STAT1KO cells lacking STAT1, delayed IFN responses correlated with DNA binding of phosphorylated STAT2/IRF9 but not U-STAT2/IRF9. In addition, comparative experiments in U3C (STAT1-KO) cells overexpressing all the ISGF3 components (ST1-ST2-IRF9-U3C) revealed U-ISGF3 (and possibly U-STAT2/IRF9) chromatin interactions to correlate with phosphorylation-independent ISG transcription and antiviral activity. Together, our data point to the dominant role of the canonical ISGF3 and non-canonical STAT2/IRF9, without a shift to U-ISGF3 or U-STAT2/IRF9, in the regulation of early and prolonged ISG expression and viral protection. At the same time, they suggest the threshold-dependent role of U-ISFG3, and potentially U-STAT2/IRF9, in the regulation of constitutive and possibly long-term IFNα-dependent responses.

Preoperative cell-free DNA concentration in plasma as a diagnostic and prognostic biomarker of clear cell renal cell carcinoma.

Introduction: Assessment of renal tumour masses is based on conventional imaging studies (computer tomography or magnetic resonance), which does not allow characterisation of the histopathological type. Moreover, the prediction of prognosis in localised and metastatic renal cell carcinoma requires improvement as well. Analysis of circulating free DNA (cfDNA) in blood is one of the variants of liquid biopsy that may improve diagnostics and prognosis issues of patients with renal tumour masses suspected to be renal cell carcinoma. The aim of the study was to assess the diagnostic and prognostic role of preoperative cfDNA concentration in the plasma samples of clear cell renal cell carcinoma (ccRCC) patients. Material and methods: The preoperative plasma cfDNA concentration was assessed in ccRCC patients (n = 46) and healthy individuals (control group) (n = 17). The circulating free DNA concentration was reflected by the 90 bp DNA fragments determined by real-time polymerase chain reaction. Results: The median cfDNA concentration was significantly higher in ccRCC patients (n = 46) compared to the control g roup (n = 17) (2588 ±2554 copies/ml vs. 960 ±490 copies/ml, p < 0.01). In multivariate analysis, the preoperative plasma cfDNA concentration was the significant factor increasing the probability of ccRCC detection (OR: 1.003; 95% CI: 1.001-1.005). The median cfDNA concentration depended on the stage of ccRCC; it was higher in metastatic ccRCC patients (n = 11) compared to non-metastatic ccRCC patients (n = 35) (3619 ±4059 copies/ml vs. 2473 ±1378 copies/ml, p < 0.03). Kaplan-Meier survival analysis demon-strated that patients with high cfDNA values (above 2913 copies/ml) had significantly worse cancer-specific survival (HR: 4.5; 95% CI: 1.3-16.9, log-rank Mantel-Cox test p = 0.015). Conclusions: Preoperative plasma cfDNA concentration has diagnostic and prognostic potential in ccRCC pa-tients.

Novel Demodex detection method involving non-invasive sebum collection and next-generation sequencing.

Introduction: Demodex mites are common human ectoparasites found across a broad geographical range. They reside in pilosebaceous units of the skin and feed on sebum, epithelial and glandular cells. D. folliculorum is the more common mite, inhabiting the upper end of the pilosebaceous unit while D. brevis resides deeper in the skin and meibomian glands. Until now, Demodex mites have been obtained by various techniques such as skin scraping, cellophane tape, plucking eyelashes, and also by invasive biopsies. Aim: To assess whether non-invasively collected sebum samples of patients suspected of rosacea or demodicosis are suitable for NGS DNA Demodex analysis. Material and methods: Suspicion of seborrheic dermatitis or rosacea was the inclusion criterion. The study group consisted of 20 males, 1 female, age: 33-83, median: 58. Nasal dorsum was moisturized and an adhesive strip was applied. DNA was isolated from the sebum and sequenced with the use of MiSeq® Reagent Kit v2 and MiSeq® System. Results: Out of 7 patients who were positive by microscopy, 6 were found positive by NGS. Additional 4 patients were found positive only by NGS, adding to a total of ten. The NGS approach showed superior sensitivity compared to light microscopy (63% and 44%, respectively). In 3 patients, both Demodex species were identified by NGS. Conclusions: We believe to have proven that it is possible to study Demodex mites by NGS with sebum as the input sample. Furthermore, it is possible to identify and distinguish Demodex folliculorum from D. brevis in individual patients.

Identification of candidate genes and regulatory factors related to growth rate through hypothalamus transcriptome analyses in broiler chickens.

BACKGROUND: Intensive selection for growth rate (GR) in broiler chickens carries negative after-effects, such as aberrations in skeletal development and the immune system, heart failure, and deterioration of meat quality. In Poland, fast-growing chicken populations are highly non-uniform in term of growth rate, which is highly unprofitable for poultry producers. Therefore, the identification of genetic markers for boiler GR that could support the selection process is needed. The hypothalamus is strongly associated with growth regulation by inducing important pituitary hormones. Therefore, the present study used this tissue to pinpoint genes involved in chicken growth control. RESULTS: The experiment included male broilers of Ross 308 strain in two developmental stages, after 3rd and 6th week of age, which were maintained in the same housing and feeding conditions. The obtained results show for the overexpression of genes related to orexigenic molecules, such as neuropeptide Y (NPY), aldehyde dehydrogenase 1 family, member A1 (ALDH1A1), galanin (GAL), and pro-melanin concentrating hormone (PMCH) in low GR cockerels. CONCLUSION: The results reveal strong associations between satiety centre and the growth process. The present study delivers new insights into hypothalamic regulation in broiler chickens and narrows the area for the searching of genetic markers for GR.

Massive parallel sequencing in forensics: advantages, issues, technicalities, and prospects.

In the last decade, next-generation sequencing (NGS) technology, alternatively massive parallel sequencing (MPS), was applied to all fields of biological research. Its introduction to the field of forensics was slower, mainly due to lack of accredited sequencers, kits, and relatively higher sequencing error rates as compared with standardized Sanger sequencing. Currently, a majority of the problematic issues have been solved, which is proven by the body of reports in the literature. Here, we discuss the utility of NGS sequencing in forensics, emphasizing the advantages, issues, the technical aspects of the experiments, commercial solutions, and the potentially interesting applications of MPS.

Lipid Stores and Lipid Metabolism Associated Gene Expression in Porcine and Bovine Parthenogenetic Embryos Revealed by Fluorescent Staining and RNA-seq.

Compared to other mammalian species, porcine oocytes and embryos are characterized by large amounts of lipids stored mainly in the form of droplets in the cytoplasm. The amount and the morphology of lipid droplets (LD) change throughout the preimplantation development, however, relatively little is known about expression of genes involved in lipid metabolism of early embryos. We compared porcine and bovine blastocyst stage embryos as well as dissected inner cell mass (ICM) and trophoblast (TE) cell populations with regard to lipid droplet storage and expression of genes functionally annotated to selected lipid gene ontology terms using RNA-seq. Comparing the number and the volume occupied by LD between bovine and porcine blastocysts, we have found significant differences both at the level of single embryo and a single blastomere. Aside from different lipid content, we found that embryos regulate the lipid metabolism differentially at the gene expression level. Out of 125 genes, we found 73 to be differentially expressed between entire porcine and bovine blastocyst, and 36 and 51 to be divergent between ICM and TE cell lines. We noticed significant involvement of cholesterol and ganglioside metabolism in preimplantation embryos, as well as a possible shift towards glucose, rather than pyruvate dependence in bovine embryos. A number of genes like DGAT1, CD36 or NR1H3 may serve as lipid associated markers indicating distinct regulatory mechanisms, while upregulated PLIN2, APOA1, SOAT1 indicate significant function during blastocyst formation and cell differentiation in both models.

Thyroid cancers of follicular origin in a genomic light: in-depth overview of common and unique molecular marker candidates.

In recent years, thyroid malignances have become more prevalent, especially among women. The most common sporadic types of thyroid tumors of follicular origin include papillary, follicular and anaplastic thyroid carcinomas. Although modern diagnosis methods enable the identification of tumors of small diameter, tumor subtype differentiation, which is imperative for the correct choice of treatment, is still troublesome. This review discusses the recent advances in the field of molecular marker identification via next-generation sequencing and microarrays. The potential use of these biomarkers to distinguish among the most commonly occurring sporadic thyroid cancers is presented and compared. Geographical heterogeneity might be a differentiator, although not necessarily a limiting factor, in biomarker selection. The available data advocate for a subset of mutations common for the three subtypes as well as mutations that are unique for a particular tumor subtype. Tumor heterogeneity, a known issue occurring within solid malignancies, is also discussed where applicable. Public databases with datasets derived from high-throughput experiments are a valuable source of information that aid biomarker research in general, including the identification of molecular hallmarks of thyroid cancer.

Examining the Genetic Background of Porcine Muscle Growth and Development Based on Transcriptome and miRNAome Data.

Recently, selection in pigs has been focused on improving the lean meat content in carcasses; this focus has been most evident in breeds constituting a paternal component in breeding. Such sire-breeds are used to improve the meat quantity of cross-breed pig lines. However, even in one breed, a significant variation in the meatiness level can be observed. In the present study, the comprehensive analysis of genes and microRNA expression profiles in porcine muscle tissue was applied to identify the genetic background of meat content. The comparison was performed between whole gene expression and miRNA profiles of muscle tissue collected from two sire-line pig breeds (Pietrain, Hampshire). The RNA-seq approach allowed the identification of 627 and 416 differentially expressed genes (DEGs) between pig groups differing in terms of loin weight between Pietrain and Hampshire breeds, respectively. The comparison of miRNA profiles showed differential expression of 57 microRNAs for Hampshire and 34 miRNAs for Pietrain pigs. Next, 43 genes and 18 miRNAs were selected as differentially expressed in both breeds and potentially related to muscle development. According to Gene Ontology analysis, identified DEGs and microRNAs were involved in the regulation of the cell cycle, fatty acid biosynthesis and regulation of the actin cytoskeleton. The most deregulated pathways dependent on muscle mass were the Hippo signalling pathway connected with the TGF-ß signalling pathway and controlling organ size via the regulation of ubiquitin-mediated proteolysis, cell proliferation and apoptosis. The identified target genes were also involved in pathways such as the FoxO signalling pathway, signalling pathways regulating pluripotency of stem cells and the PI3K-Akt signalling pathway. The obtained results indicate molecular mechanisms controlling porcine muscle growth and development. Identified genes (SOX2, SIRT1, KLF4, PAX6 and genes belonging to the transforming growth factor beta superfamily) could be considered candidate genes for determining muscle mass in pigs.

Potential protective role of Grainyhead-like genes in the development of clear cell renal cell carcinoma.

The involvement of Grainyhead-like (GRHL) transcription factors in various cancers is well documented. However, little is known about their role in clear cell renal cell carcinoma (ccRCC). We discovered that the expression of two of these factors-GRHL1 and GRHL2-are downregulated in ccRCC samples, and their expression is correlated with the expression of VHL gene. This suggests a functional link between the GRHL transcription factors and one of the best known tumor suppressors. Although the GRHL genes are not mutated in ccRCC, some of the single nucleotide polymorphisms in these genes may indicate an increased risk of ccRCC development and/or may allow to assess patients' prognoses and predict their responses to various forms of therapy. Silencing of GRHL2 expression in non-tumorigenic kidney cell line results in increased cell proliferation, increased resistance to apoptosis, as well as changes in the levels of selected proteins involved in the pathogenesis of ccRCC. These changes support the potential role for GRHL2 as a suppressor of ccRCC.

STAT2/IRF9 directs a prolonged ISGF3-like transcriptional response and antiviral activity in the absence of STAT1.

Evidence is accumulating for the existence of a signal transducer and activator of transcription 2 (STAT2)/interferon regulatory factor 9 (IRF9)-dependent, STAT1-independent interferon alpha (IFNα) signalling pathway. However, no detailed insight exists into the genome-wide transcriptional regulation and the biological implications of STAT2/IRF9-dependent IFNα signalling as compared with interferon-stimulated gene factor 3 (ISGF3). In STAT1-defeicient U3C cells stably overexpressing human STAT2 (hST2-U3C) and STAT1-deficient murine embryonic fibroblast cells stably overexpressing mouse STAT2 (mST2-MS1KO) we observed that the IFNα-induced expression of 2'-5'-oligoadenylate synthase 2 (OAS2) and interferon-induced protein with tetratricopeptide repeats 1 (Ifit1) correlated with the kinetics of STAT2 phosphorylation, and the presence of a STAT2/IRF9 complex requiring STAT2 phosphorylation and the STAT2 transactivation domain. Subsequent microarray analysis of IFNα-treated wild-type (WT) and STAT1 KO cells overexpressing STAT2 extended our observations and identified ∼120 known antiviral ISRE-containing interferon-stimulated genes (ISGs) commonly up-regulated by STAT2/IRF9 and ISGF3. The STAT2/IRF9-directed expression profile of these IFN-stimulated genes (ISGs) was prolonged as compared with the early and transient response mediated by ISGF3. In addition, we identified a group of 'STAT2/IRF9-specific' ISGs, whose response to IFNα was ISGF3-independent. Finally, STAT2/IRF9 was able to trigger an antiviral response upon encephalomyocarditis virus (EMCV) and vesicular stomatitis Indiana virus (VSV). Our results further prove that IFNα-activated STAT2/IRF9 induces a prolonged ISGF3-like transcriptome and generates an antiviral response in the absence of STAT1. Moreover, the existence of 'STAT2/IRF9-specific' target genes predicts a novel role of STAT2 in IFNα signalling.

Expression of pre-selected TMEMs with predicted ER localization as potential classifiers of ccRCC tumors.

BACKGROUND: VHL inactivation is the most established molecular characteristic of clear cell renal cell carcinoma (ccRCC), with only a few additional genes implicated in development of this kidney tumor. In recently published ccRCC gene expression meta-analysis study we identified a number of deregulated genes with limited information available concerning their biological role, represented by gene transcripts belonging to transmembrane proteins family (TMEMs). TMEMs are predicted to be components of cellular membranes, such as mitochondrial membranes, ER, lysosomes and Golgi apparatus. Interestingly, the function of majority of TMEMs remains unclear. Here, we analyzed expression of ten TMEM genes in the context of ccRCC progression and development, and characterized these proteins bioinformatically. METHODS: The expression of ten TMEMs (RTP3, SLC35G2, TMEM30B, TMEM45A, TMEM45B, TMEM61, TMEM72, TMEM116, TMEM207 and TMEM213) was measured by qPCR. T-test, Pearson correlation, univariate and multivariate logistic and Cox regression were used in statistical analysis. The topology of studied proteins was predicted with Metaserver, together with PSORTII, Pfam and Localizome tools. RESULTS: We observed significant deregulation of expression of 10 analyzed TMEMs in ccRCC tumors. Cluster analysis of expression data suggested the down-regulation of all tested TMEMs to be a descriptor of the most advanced tumors. Logistic and Cox regression potentially linked TMEM expression to clinical parameters such as: metastasis, Fuhrman grade and overall survival. Topology predictions classified majority of analyzed TMEMs as type 3 and type 1 transmembrane proteins, with predicted localization mainly in ER. CONCLUSIONS: The massive down-regulation of expression of TMEM family members suggests their importance in the pathogenesis of ccRCC and the bioinformatic analysis of TMEM topology implies a significant involvement of ER proteins in ccRCC pathology.

FUT11 as a potential biomarker of clear cell renal cell carcinoma progression based on meta-analysis of gene expression data.

In this paper, we provide a comprehensive summary of available clear cell renal cell carcinoma (ccRCC) microarray data in the form of meta-analysis of genes differentially regulated in tumors as compared to healthy tissue, using effect size to measure the strength of a relationship between the disease and gene expression. We identified 725 differentially regulated genes, with a number of interesting targets, such as TMEM213, SMIM5, or ATPases: ATP6V0A4 and ATP6V1G3, of which limited or no information is available in terms of their function in ccRCC pathology. Downregulated genes tended to represent pathways related to tissue remodeling, blood clotting, vasodilation, and energy metabolism, while upregulated genes were classified into pathways generally deregulated in cancers: immune system response, inflammatory response, angiogenesis, and apoptosis. One hundred fifteen deregulated genes were included in network analysis, with EGLN3, AP-2, NR3C1, HIF1A, and EPAS1 (gene encoding HIF2-α) as points of functional convergence, but, interestingly, 610 genes failed to join previously identified molecular networks. Furthermore, we validated the expression of 14 top deregulated genes in independent sample set of 32 ccRCC tumors by qPCR and tested if it could serve as a marker of disease progression. We found a correlation of high fucosyltransferase 11 (FUT11) expression with non-symptomatic course of the disease, which suggests that FUT11's expression might be potentially used as a biomarker of disease progression.

Data mining of atherosclerotic plaque transcriptomes predicts STAT1-dependent inflammatory signal integration in vascular disease.

Atherosclerotic plaque development involves multiple extra- and intra-cellular signals engaging cells from the immune system and from the vasculature. Pro-inflammatory pathways activated by interferon gamma (IFNγ) and toll-like receptor 4 (TLR4) ligands are profoundly involved in plaque formation and have been shown to involve cross-talk in all atheroma-interacting cell types leading to increased activation of signal transducer and activator of transcription-1 (STAT1) and elevated expression of pro-inflammatory mediators. Here we demonstrate that in Gene Expression Omnibus repository (GEO) deposited microarray datasets, obtained from human coronary and carotid atherosclerotic plaques, a significant increase in expression of pro-inflammatory and immunomodulatory genes can be detected. Moreover, increased expression of multiple chemokines, adhesion molecules and matrix-remodeling molecules was commonly detected in both plaque types and correlated with the presence of putative STAT1 binding sites in their promoters, suggesting strong involvement of STAT1 in plaque development. We also provide evidence to suggest that STAT1-nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) or STAT1-interferon-regulated factor (IRF) regulatory modules are over-represented in the promoters of these inflammatory genes, which points to a possible contribution of IFNγ and TLR4 cross-talk in the process of atherogenesis. Finally, a subset of these genes encodes for secreted proteins that could serve as a basis of a non-invasive diagnostic assay. The results of our in silico analysis in vitro provide potential evidence that STAT1-dependent IFNγ-TLR4 cross-talk plays a crucial role in coronary and carotid artery plaque development and identifies a STAT1-dependent gene signature that could represent a novel diagnostic tool to monitor and diagnose plaque progression in human atherosclerosis.

Erratum: Structural, topological, and functional characterization of transmembrane proteins TMEM213, 207, 116, 72 and 30B provides a potential link to ccRCC etiology.

[This corrects the article on p. 1863 in vol. 13, PMID: 37293153.].

Structural, topological, and functional characterization of transmembrane proteins TMEM213, 207, 116, 72 and 30B provides a potential link to ccRCC etiology.

Due to their involvement in the development of various cancers Transmembrane Proteins (TMEMs) are the focus of many recent studies. Previously we reported TMEM de-regulation in clear cell Renal Cell Carcinoma (ccRCC) with TMEM213, 207, 116, 72 and 30B being among the most downregulated on mRNA level. TMEM down-regulation was also more pronounced in advanced ccRCC tumors and was potentially linked to clinical parameters such as: metastasis (TMEM72 and 116), Fuhrman grade (TMEM30B) and overall survival (TMEM30B). To further investigate these findings, first, we set off to prove experimentally that selected TMEMs are indeed membrane-bound as predicted in silico, we verified the presence of signaling peptides on their N-termini, orientation of TMEMs within the membrane and validated their predicted cellular localization. To investigate the potential role of selected TMEMs in cellular processes overexpression studies in HEK293 and HK-2 cell lines were carried out. Additionally, we tested TMEM isoform expression in ccRCC tumors, identified mutations in TMEM genes and examined chromosomal aberrations in their loci. We confirmed the membrane-bound status of all selected TMEMs, assigned TMEM213, and 207 to early endosomes, TMEM72 to early endosomes and plasma membrane, TMEM116 and 30B to the endoplasmic reticulum. The N-terminus of TMEM213 was found to be exposed to the cytoplasm, the C-terminus of TMEM207, 116 and 72 were directed toward the cytoplasm, and both termini of TMEM30B faced the cytoplasm. Interestingly, TMEM mutations and chromosomal aberrations were infrequent in ccRCC tumors, yet we identified potentially damaging mutations in TMEM213 and TMEM30B and found deletions in the TMEM30B locus in nearly 30% of the tumors. Overexpression studies suggested selected TMEMs may take part in carcinogenesis processes such as cell adhesion, regulation of epithelial cell proliferation, and regulation of adaptive immune response, which could indicate a link to the development and progression of ccRCC.

SINBAD, structural, experimental and clinical characterization of STAT inhibitors and their potential applications.

The abnormal activation of signal transducer and activator of transcription (STAT) protein family is recognized as cause or driving force behind multiple diseases progression. Therefore, searching for potential treatment strategy is pursued by multiple scientific groups. We consider that providing comprehensive, integrated and unified dataset for STAT inhibitory compounds may serve as important tool for other researchers. We developed SINBAD (STAT INhbitor Biology And Drug-ability) in response to our experience with inhibitory compound research, knowing that gathering detailed information is crucial for effective experiment design and also for finding potential solutions in case of obtaining inconclusive results. SINBAD is a curated database of STAT inhibitors which have been published and described in scientific articles providing prove of their inhibitory properties. It is a tool allowing easy analysis of experimental conditions and provides detailed information about known STAT inhibitory compounds.

Dysregulated Interferon Response and Immune Hyperactivation in Severe COVID-19: Targeting STATs as a Novel Therapeutic Strategy.

A disease outbreak in December 2019, caused by a novel coronavirus SARS-CoV-2, was named COVID-19. SARS-CoV-2 infects cells from the upper and lower respiratory tract system and is transmitted by inhalation or contact with infected droplets. Common clinical symptoms include fatigue, fever, and cough, but also shortness of breath and lung abnormalities. Still, some 5% of SARS-CoV-2 infections progress to severe pneumonia and acute respiratory distress syndrome (ARDS), with pulmonary edema, acute kidney injury, and/or multiple organ failure as important consequences, which can lead to death. The innate immune system recognizes viral RNAs and triggers the expression of interferons (IFN). IFNs activate anti-viral effectors and components of the adaptive immune system by activating members of the STAT and IRF families that induce the expression of IFN-stimulated genes (ISG)s. Among other coronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV) and SARS-CoV, common strategies have been identified to antagonize IFN signaling. This typically coincides with hyperactive inflammatory host responses known as the "cytokine storm" that mediate severe lung damage. Likewise, SARS-CoV-2 infection combines a dysregulated IFN response with excessive production of inflammatory cytokines in the lungs. This excessive inflammatory response in the lungs is associated with the local recruitment of immune cells that create a pathogenic inflammatory loop. Together, it causes severe lung pathology, including ARDS, as well as damage to other vulnerable organs, like the heart, spleen, lymph nodes, and kidney, as well as the brain. This can rapidly progress to multiple organ exhaustion and correlates with a poor prognosis in COVID-19 patients. In this review, we focus on the crucial role of different types of IFN that underlies the progression of SARS-CoV-2 infection and leads to immune cell hyper-activation in the lungs, exuberant systemic inflammation, and multiple organ damage. Consequently, to protect from systemic inflammation, it will be critical to interfere with signaling cascades activated by IFNs and other inflammatory cytokines. Targeting members of the STAT family could therefore be proposed as a novel therapeutic strategy in patients with severe COVID-19.

STAT1-mediated signal integration between IFNγ and LPS leads to increased EC and SMC activation and monocyte adhesion.

Inflammation plays an important role in host defenses against infectious agents and injury, but it also contributes to the pathophysiology of atherosclerosis. Signal transducer and activated transcription 1 (STAT1) has been identified as a point of convergence for the cross talk between the pro-inflammatory cytokine interferon γ (IFNγ) and the Toll-like receptor-4 (TLR4) ligand LPS in immune cells. However, there is no information available on the role of STAT1 in TLR4-mediated progression of atherosclerosis and on potential synergism between lipopolysaccharides (LPS) and IFNγ signaling in cells from the vasculature. Cultured human microvascular endothelial cells (HMECs) exposed to LPS activated STAT1 in a delayed manner that was inhibited by cycloheximide treatment. Pretreatment of HMECs as well as primary vascular smooth muscle cells (VSMCs) with IFNγ followed by LPS resulted in a significant increase in STAT1 phosphorylation compared with both factors alone. Increased STAT1 protein levels, strictly mediated by IFNγ, correlated with the augmented STAT1 phosphorylation that was absent in TLR4(-/-) cells. As assessed by PCR, Western analysis, and ELISA, this coincided with increased expression of the chemokine interferon gamma-induced protein 10 kDa (IP-10) and the adhesion molecule ICAM-1 in a TLR4-dependent manner.The STAT1-inhibitor fludarabine markedly reduced these effects as well as IFNγ and LPS-dependent adhesion of U937 cells to endothelial cells, emphasizing the potential importance of STAT1 in the integration of both signals. With the established roles of IFNγ and TLRs in atherosclerotic pathology, the STAT1-dependent signal integration between IFNγ and TLR in ECs and VSMCs in response to exogenous and endogenous atherogenic ligands could result in amplification of pro-inflammatory responses in the damaged vessel and be a novel mechanism involved in the initiation and progression of atherosclerosis.

Identification of RCC Subtype-Specific microRNAs-Meta-Analysis of High-Throughput RCC Tumor microRNA Expression Data.

Renal cell carcinoma (RCC) is one of the most common cancers worldwide with a nearly non-symptomatic course until the advanced stages of the disease. RCC can be distinguished into three subtypes: papillary (pRCC), chromophobe (chRCC) and clear cell renal cell carcinoma (ccRCC) representing up to 75% of all RCC cases. Detection and RCC monitoring tools are limited to standard imaging techniques, in combination with non-RCC specific morphological and biochemical read-outs. RCC subtype identification relays mainly on results of pathological examination of tumor slides. Molecular, clinically applicable and ideally non-invasive tools aiding RCC management are still non-existent, although molecular characterization of RCC is relatively advanced. Hence, many research efforts concentrate on the identification of molecular markers that will assist with RCC sub-classification and monitoring. Due to stability and tissue-specificity miRNAs are promising candidates for such biomarkers. Here, we performed a meta-analysis study, utilized seven NGS and seven microarray RCC studies in order to identify subtype-specific expression of miRNAs. We concentrated on potentially oncocytoma-specific miRNAs (miRNA-424-5p, miRNA-146b-5p, miRNA-183-5p, miRNA-218-5p), pRCC-specific (miRNA-127-3p, miRNA-139-5p) and ccRCC-specific miRNAs (miRNA-200c-3p, miRNA-362-5p, miRNA-363-3p and miRNA-204-5p, 21-5p, miRNA-224-5p, miRNA-155-5p, miRNA-210-3p) and validated their expression in an independent sample set. Additionally, we found ccRCC-specific miRNAs to be differentially expressed in ccRCC tumor according to Fuhrman grades and identified alterations in their isoform composition in tumor tissue. Our results revealed that changes in the expression of selected miRNA might be potentially utilized as a tool aiding ccRCC subclass discrimination and we propose a miRNA panel aiding RCC subtype distinction.