Knockdown of vps54 aggravates tamoxifen-induced cytotoxicity in fission yeast

Tamoxifen (TAM) is an anticancer drug used to treat estrogen receptor (ER)‒positive breast cancer. However, its ER-independent cytotoxic and antifungal activities have prompted debates on its mechanism of action. To achieve a better understanding of the ER-independent antifungal action mechanisms of TAM, we systematically identified TAM-sensitive genes through microarray screening of the heterozygous gene deletion library in fission yeast (Schizosaccharomyces pombe). Secondary confirmation was followed by a spotting assay, finally yielding 13 TAM-sensitive genes under the drug-induced haploinsufficient condition. For these 13 TAM-sensitive genes, we conducted a comparative analysis of their Gene Ontology (GO) ‘biological process’ terms identified from other genome-wide screenings of the budding yeast deletion library and the MCF7breast cancer cell line. Several TAM-sensitive genes overlapped between the yeast strains and MCF7 in GO terms including ‘cell cycle’ (cdc2, rik1, pas1, and leo1), ‘signaling’ (sck2, oga1, and cki3), and ‘vesicle-mediated transport’ (SPCC126.08c, vps54, sec72, and tvp15), suggesting their roles in the ER-independent cytotoxic effects of TAM. We recently reported that the cki3 gene with the ‘signaling’ GO term was related to the ER-independent antifungal action mechanisms of TAM in yeast. In this study, we report that haploinsufficiency of the essential vps54 gene, which encodes the GARP complex subunit, significantly aggravated TAM sensitivity and led to an enlarged vesicle structure in comparison with the SP286 control strain. These results strongly suggest that the vesicle-mediated transport process might be another action mechanism of the ER-independent antifungal or cytotoxic effects of TAM.

Proteome Microarray Screening Identifies Human Polyphosphate-Binding Proteins in the Phosphatidylinositol Signaling Pathway

Polyphosphates are linear chains of orthophosphate residues that are present in all living cells. Polyphosphates are released from platelet d-granules and are also produced in bacteria. Polyphosphates are procoagulant in mammalian species and in bacteria are required for energy and phosphate storage, stress resistance, chelation of metal ions and escaping host immunity. Despite these pleiotropic effects, sparse information is available on molecular binding partners of polyphosphates. Here, we used a slide-based human proteome microarray screen for the search of polyphosphate-binding proteins. This approach suggested several novel proteins with relation to the phosphatidylinositol signaling pathway. The highest signals were obtained for Disabled-1 (DAB1) and phosphatidylinositol-5-phosphate 4-kinase 2B (PIP4K2B). Isothermal titration calorimetry was used for confirmation of DAB1 interactions with long-chain polyphosphates. These results offer new rationale to further investigate the interference of polyphosphates with intracellular signaling pathways.

A high-throughput pipeline for design and selection of peptides targeting the SARS-Cov-2 Spike protein

Rapid design, screening, and characterization of biorecognition elements (BREs) is essential for the development of diagnostic tests and antiviral therapeutics needed to combat the spread of viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To address this need, we developed a high-throughput pipeline combining in silico design of a peptide library specific for SARS-CoV-2 spike (S) protein and microarray screening to identify binding sequences. Our optimized microarray platform allowed the simultaneous screening of ~ 2.5 k peptides and rapid identification of binding sequences resulting in selection of four peptides with nanomolar affinity to the SARS-CoV-2 S protein. Finally, we demonstrated the successful integration of one of the top peptides into an electrochemical sensor with a clinically relevant limit of detection for S protein in spiked saliva. Our results demonstrate the utility of this novel pipeline for the selection of peptide BREs in response to the SARS-CoV-2 pandemic, and the broader application of such a platform in response to future viral threats.

Differential and Common Signatures of miRNA Expression and Methylation in Childhood Central Nervous System Malignancies: An Experimental and Computational Approach

Epigenetic modifications are considered of utmost significance for tumor ontogenesis and progression. Especially, it has been found that miRNA expression, as well as DNA methylation plays a significant role in central nervous system tumors during childhood. A total of 49 resected brain tumors from children were used for further analysis. DNA methylation was identified with methylation-specific MLPA and, in particular, for the tumor suppressor genes CASP8, RASSF1, MGMT, MSH6, GATA5, ATM1, TP53, and CADM1. miRNAs were identified with microarray screening, as well as selected samples, were tested for their mRNA expression levels. CASP8, RASSF1 were the most frequently methylated genes in all tumor samples. Simultaneous methylation of genes manifested significant results with respect to tumor staging, tumor type, and the differentiation of tumor and control samples. There was no significant dependence observed with the methylation of one gene promoter, rather with the simultaneous presence of all detected methylated genes’ promoters. miRNA expression was found to be correlated to gene methylation. Epigenetic regulation appears to be of major importance in tumor progression and pathophysiology, making it an imperative field of study.

CircRNA_0079586 and circRNA_RanGAP1 are involved in the pathogenesis of intracranial aneurysms rupture by regulating the expression of MPO

Several circRNAs have been reported to be dysregulated in human endothelial cells through sponging miRNAs. Previous reports demonstrated that MPO not only contributed to the formation and rupture of cerebral aneurysm but was also correlated with the degenerative remodeling predisposition to saccular intracranial aneurysm wall rupture, although its underlying mechanisms remain to be explored. Microarray screening was performed to compare the differential expression of circRNAs in the endothelial cells collected from UIAs and RIAs patients. Luciferase assays were used to explore the regulatory relationship between circRNAs and miRNAs, and between miRNAs and their target genes. Microarray screening analysis found a batch of up-regulated circRNAs in the endothelial cells harvested from RIAs patients, including circRNA-0079586 and circRNA-RanGAP1. Luciferase assays revealed the suppressive role of miR-183-5p/miR-877-3p in the expression of circRNA-0079586/circRNA-RanGAP1/MPO. And the expression of circRNA-0079586 and circRNA-RanGAP1 was respectively suppressed by the overexpression of miR-183-5p and miR-877-3p. And both the transfection of miR-183-5p and miR-877-3p mimics suppressed the relative expression level of MPO mRNA. The expression of circRNA-0079586, circRNA-RanGAP1 and MPO was significantly activated in the endothelial cells collected from RIAs patients when compared with UIAs patients, whereas the expression of miR-183-5p and miR-877-3p was remarkably suppressed in the endothelial cells collected from RIAs patients when compared with UIAs patients. We further altered the expression of circRNA-0079586 and circRNA-RanGAP1 using siRNA and overexpression in HUVECS, and the expression of circRNA-0079586 and circRNA-RanGAP1 was significantly and negatively correlated with the expression of miR-183-5p and miR-877-3p, but positively correlated with the expression of MPO under different conditions. In this study, we established two MPO-modulating signaling pathways of circRNA_0079586/miR-183-5p/MPO and circRNA_RanGAP1/miR-877-3p/MPO. These two signaling pathways are involved in the pathogenesis of intracranial aneurysms rupture.

Long Non-Coding RNA STARD13-AS Suppresses Glioma Cell Biological Activities

IntroductionIt has been unclear that STARD13-AS had effects in glioma. The aim of our research was to investigate the effects of STARD13-AS in glioma development and the mechanisms underlying these effects by vitro study.Material and methodsAdjacent normal and tumor tissues were collected for long non-coding RNA (lncRNA) microarray screening. STARD13-AS expression was measured by in situ hybridization (ISH) and reverse-transcription quantitative PCR (RT-qPCR) assays, and correlations between STARD13-AS and clinicopathological parameters and prognosis were analyzed. STARD13-AS transfection of glioma cell lines (U251 and U87) was used to evaluate biological activities of cells. Western blotting (WB) and RT-qPCR assays were used to investigate the underlying mechanisms.ResultsAccording to lncRNA microarray screening, ISH, and RT-qPCR, lncRNA STARD13-AS was significantly downregulated in tumor tissues. Low STARD13-AS expression was strongly correlated with poor prognosis and malignant clinicopathology. After STARD13-AS transfection, biological activities of glioma cells were significantly decreased (P＜0.001 for both cell types). WB and RT-qPCR assays showed that protein and mRNA expression levels of cyclin D, cyclin E, N-cadherin, E-cadherin, and vimentin were significantly related to STARD13-AS overexpression (P＜0.001 in all cases).ConclusionsSTARD13-AS overexpression suppresses the biological activities of glioma cells, indicating that STARD13-AS is a potential target for glioma treatment by vitro study.

Diagnostic and Prognostic Value of Long Noncoding RNAs as Potential Novel Biomarkers in Intrahepatic Cholestasis of Pregnancy

Long noncoding RNAs (lncRNAs) are a class of important regulators participating in various pathological processes. Until now, the role of lncRNAs in the occurrence and development of intrahepatic cholestasis of pregnancy (ICP) has rarely been investigated. The data from microarray screening revealed 58 upregulated and 85 downregulated lncRNAs and 47 upregulated and 71 downregulated mRNAs in ICP patients compared to healthy controls. Bioinformatics analysis revealed biological processes focused on lipid metabolism, apoptosis, cell cycle, cell differentiation, and oxidative stress. Furthermore, the expressions of three lncRNAs (ENST00000505175.1, ASO3480, and ENST00000449605.1) chosen for verification were significantly decreased and showed the diagnostic and prognostic value for ICP based on ROC analysis. This is the first study to report the specific role of lncRNAs in ICP, which may be helpful for the diagnosis and prognosis of ICP clinically.

Altered MicroRNA Expression in Intracranial Aneurysmal Tissues: Possible Role in TGF-β Signaling Pathway

The molecular mechanisms behind the rupture of intracranial aneurysms remain obscure. MiRNAs are key regulators of a wide array of biological processes altering protein synthesis by binding to target mRNAs. However, variations in miRNA levels in ruptured aneurysmal wall have not been completely examined. We hypothesized that altered miRNA signature in aneurysmal tissues could potentially provide insight into aneurysm pathophysiology. Using a high-throughput miRNA microarray screening approach, we compared the miRNA expression pattern in aneurysm tissues obtained during surgery from patients with aneurysmal subarachnoid hemorrhage (aSAH) with control tissues (GEO accession number GSE161870). We found that the expression of 70 miRNAs was altered. Expressions of the top 10 miRNA were validated, by qRT-PCR and results were correlated with clinical characteristics of aSAH patients. The level of 10 miRNAs (miR-24-3p, miR-26b-5p, miR-27b-3p, miR-125b-5p, miR-143-3p, miR-145-5p, miR-193a-3p, miR-199a-5p, miR-365a-3p/365b-3p, and miR-497-5p) was significantly decreased in patients compared to controls. Expression of miR-125b-5p, miR-143-3p and miR-199a-5p was significantly decreased in patients with poor prognosis and vasospasm. The target genes of few miRNAs were enriched in Transforming growth factor-beta (TGF-β) and Mitogen-activated protein kinases (MAPK) pathways. We found significant negative correlation between the miRNA and mRNA expression (TGF-β1, TGF-β2, SMAD family member 2 (SMAD2), SMAD family member 4 (SMAD4), MAPK1 and MAPK3) in aneurysm tissues. We suggest that miR-26b, miR-199a, miR-497and miR-365, could target multiple genes in TGF-β and MAPK signaling cascades to influence inflammatory processes, extracellular matrix and vascular smooth muscle cell degradation and apoptosis, and ultimately cause vessel wall degradation and rupture.

Microarray screening reveals a non-conventional SUMO-binding mode linked to DNA repair by non-homologous end-joining

SUMOylation is critical for a plethora of cellular signalling pathways including the repair of DNA double-strand breaks (DSBs). If misrepaired, DSBs can lead to cancer, neurodegeneration, immunodeficiency and premature ageing. Based on systematic proteome microarray screening combined with widely applicable carbene footprinting and high-resolution structural profiling, we define two non-conventional SUMO2-binding modules on XRCC4, a DNA repair protein important for DSB repair by non-homologous end-joining (NHEJ). Mechanistically, interaction of SUMO2 with XRCC4 is incompatible with XRCC4 binding to at least two other NHEJ proteins – XLF and DNA ligase 4 (LIG4). These findings are consistent with SUMO2 interactions of XRCC4 acting as backup pathways at different stages of NHEJ, in the absence of these factors or their dysfunctioning. Such scenarios are not only relevant for carcinogenesis, but also for the design of precision anti-cancer medicines and the optimisation of CRISPR/Cas9-based gene editing. This work reveals insights into topology-specific SUMO recognition and its potential for modulating DSB repair by NHEJ. Moreover, it provides a rich resource on binary SUMO receptors that can be exploited for uncovering regulatory layers in a wide array of cellular processes.

Modulation of Early Host Innate Immune Response by an Avipox Vaccine Virus’ Lateral Body Protein

The avian pathogen fowlpox virus (FWPV) has been successfully used as a vaccine vector in poultry and humans, but relatively little is known about its ability to modulate host antiviral immune responses in these hosts, which are replication-permissive and nonpermissive, respectively. FWPV is highly resistant to avian type I interferon (IFN) and able to completely block the host IFN-response. Microarray screening of host IFN-regulated gene expression in cells infected with 59 different, nonessential FWPV gene knockout mutants revealed that FPV184 confers immunomodulatory capacity. We report that the FPV184-knockout virus (FWPVΔ184) induces the cellular IFN response as early as 2 h postinfection. The wild-type, uninduced phenotype can be rescued by transient expression of FPV184 in FWPVΔ184-infected cells. Ectopic expression of FPV184 inhibited polyI:C activation of the chicken IFN-β promoter and IFN-α activation of the chicken Mx1 promoter. Confocal and correlative super-resolution light and electron microscopy demonstrated that FPV184 has a functional nuclear localisation signal domain and is packaged in the lateral bodies of the virions. Taken together, these results provide a paradigm for a late poxvirus structural protein packaged in the lateral bodies, capable of suppressing IFN induction early during the next round of infection.