Long non-coding RNA C5orf64 is a potential indicator for tumor microenvironment and mutation pattern remodeling in lung adenocarcinoma.

Understanding the synergistic and antagonistic effects of tumor microenvironment (TME) and tumor mutation pattern on lung adenocarcinoma (LUAD) is urgently needed. Herein, we applied ESTIMATE and CIBERSORT methods to calculate the ratio of immune and stromal components and TIICs proportion of LUAD samples from TCGA database. Immune-related genes were analyzed by Lasso regression analysis and used for ceRNA network construction. A 14-lncRNA immune-related signature was developed, among which C5orf64 was found to be positively correlated with abundances of M2 macrophages, monocytes, eosinophils and neutrophils, but negatively correlated with Tregs and plasma cells. PD-1, PD-L1 and CTLA-4 were demonstrated to be high expressed in high-level C5orf64 groups. However, C5orf64 had a negative correlation with TP53 mutation frequency. A novel model was built based on age, tumor stage and immune-related lncRNA signature. To conclude, lncRNA C5orf64 had potential to be an indicator for TME modulation and tumor mutation pattern remodeling in LUAD.

Expression profile analysis of a new testis-specifically expressed gene C17ORF64 and its association with cell apoptosis in MCF-7 cells.

With the increasing incidence of male infertility, identification and investigation the functions of new genes related to spermatogenesis are effective avenues to elucidate the decline of testicular function. In this study, a new gene, C17ORF64 (chromosome 17 open reading frame 64), was identified from mouse testes and its potential function was studied.RT-PCR and qRT-PCR assay showed that C17ORF64 mRNA was expressed exclusively in mouse testes and up-regulated from the 3-week old to 6-month old testes during postpartum development, which is consistent with C17ORF64 protein expression profile by western blotting analysis. Immunohistochemical analysis revealed that C17ORF64 protein was mainly localized in the cytoplasm of spermatogonia and spermatocytes, which is verified by GFP- labeled C17ORF64 gene expressed in GC-1 cells. C17ORF64 overexpression not only promoted cell apoptosis in MCF-7 cells, but also significantly decreased cell viability via MTT assay. Flow cytometric assay showed that C17ORF64 overexpression could inhibit cell cycle progression by arresting G1/S transition. Western blot and qRT-PCR analysis revealed that C17ORF64 overexpression inhibited the expression of anti-apoptotic protein bcl-2 and increased the expressions of pro-apoptotic protein caspase-3, caspase-8, caspase-9, Bax, P21 and P53. Taken together, our results confirmed C17ORF64 testis-specific expression pattern and, for the first time, demonstrated that C17ORF64 could inhibit cell viability and accelerate apoptosis in MCF-7 cells through caspase-3 regulatory pathways.

Exploring the molecular mechanisms of asthma across multiple datasets.

BACKGROUND: Asthma, a prevalent chronic respiratory disorder, remains enigmatic, notwithstanding considerable advancements in our comprehension. Continuous efforts are crucial for discovering novel molecular targets and gaining a comprehensive understanding of its pathogenesis. MATERIALS AND METHODS: In this study, we analyzed gene expression data from 212 individuals, including asthma patients and healthy controls, to identify 267 differentially expressed genes, among which C1orf64 and C7orf26 emerged as potential key genes in asthma pathogenesis. Various bioinformatics tools, including differential gene expression analysis, pathway enrichment, drug target prediction, and single-cell analysis, were employed to explore the potential roles of the genes. RESULTS: Quantitative PCR demonstrated differential expression of C1orf64 and C7orf26 in the asthmatic airway epithelial tissue, implying their potential involvement in asthma pathogenesis. GSEA enrichment analysis revealed significant enrichment of these genes in signaling pathways associated with asthma progression, such as ABC transporters, cell cycle, CAMs, DNA replication, and the Notch signaling pathway. Drug target prediction, based on upregulated and downregulated differential expression, highlighted potential asthma treatments, including Tyrphostin-AG-126, Cephalin, Verrucarin-a, and Emetine. The selection of these drugs was based on their significance in the analysis and their established anti-inflammatory and antiviral invasion properties. Utilizing Seurat and Celldex packages for single-cell sequencing analysis unveiled disease-specific gene expression patterns and cell types. Expression of C1orf64 and C7orf26 in T cells, NK cells, and B cells, instrumental in promoting hallmark features of asthma, was observed, suggesting their potential influence on asthma development and progression. CONCLUSION: This study uncovers novel genetic aspects of asthma, highlighting potential therapeutic pathways. It exemplifies the power of integrative bioinformatics in decoding complex disease patterns. However, these findings require further validation, and the precise roles of C1orf64 and C7orf26 in asthma warrant additional investigation to validate their therapeutic potential.

Small molecule screening identifies inhibitors of the Epstein-Barr virus deubiquitinating enzyme, BPLF1.

Herpesviral deubiquitinating enzymes (DUBs) were discovered in 2005, are highly conserved across the family, and are proving to be increasingly important players in herpesviral infection. EBV's DUB, BPLF1, is known to regulate both cellular and viral target activities, yet remains largely unstudied. Our work has implicated BPLF1 in a wide range of processes including infectivity, viral DNA replication, and DNA repair. Additionally, knockout of BPLF1 delays and reduces human B-cell immortalization and lymphoma formation in humanized mice. These findings underscore the importance of BPLF1 in viral infectivity and pathogenesis and suggest that inhibition of EBV's DUB activity may offer a new approach to specific therapy for EBV infections. We set out to discover and characterize small molecule inhibitors of BPLF1 deubiquitinating activity through high-throughput screening. An initial small pilot screen resulted in discovery of 10 compounds yielding >80% decrease in BPLF1 DUB activity at a 10 µM concentration. Follow-up dose response curves of top hits identified several compounds with an IC50 in the low micromolar range. Four of these hits were tested for their ability to cleave ubiquitin chains as well as their effects on viral infectivity and cell viability. Further characterization of the top hit, commonly known as suramin was found to not be selective yet decreased viral infectivity by approximately 90% with no apparent effects on cell viability. Due to the conserved nature of Herpesviral deubiquitinating enzymes, identification of an inhibitor of BPLF1 may prove to be an effective and promising new avenue of therapy for EBV and other herpesviral family members.

Expression Profile and Potential Functions of Circulating Long Noncoding RNAs in Acute Ischemic Stroke in the Southern Chinese Han Population.

Background: Long noncoding RNAs (lncRNAs) have been confirmed to be associated with ischemic stroke (IS); however, their involvement still needs to be extensively explored. Therefore, we aimed to study the expression profile of lncRNAs and the potential roles and mechanisms of lncRNAs in the pathogenesis of acute ischemic stroke (AIS) in the Southern Chinese Han population. Methods: In this study, lncRNA and mRNA expression profiles in AIS were analyzed using high-throughput RNA sequencing (RNA-Seq) and validated using quantitative real-time polymerase chain reaction (qRT-PCR). Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and network analyses were performed to predict the functions and interactions of the aberrantly expressed genes. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the diagnostic value of lncRNAs in AIS. Results: RNA-Seq analysis showed that 428 lncRNAs and 957 mRNAs were significantly upregulated, while 791 lncRNAs and 4,263 mRNAs were downregulated in patients with AIS when compared with healthy controls. GO enrichment and KEGG pathway analyses of differentially expressed genes showed that the apoptosis, inflammatory, oxidative and calcium signaling pathways were potentially implicated in AIS pathology. The PCR results showed that the selected lncRNA-C14orf64 and lncRNA-AC136007.2 were significantly downregulated in AIS. ROC curve analysis showed that the area under the ROC curve (AUC) values of lncRNA-C14orf64 and lncRNA-AC136007.2 between AIS and healthy controls were 0.74 and 0.94, respectively. Conclusion: This study provides evidence of altered expression of lncRNAs and their potential functions in AIS. Our findings may facilitate pathological mechanistic studies of lncRNAs in AIS and provide potential diagnostic biomarkers and therapeutic targets for AIS.

SRARP and HSPB7 are epigenetically regulated gene pairs that function as tumor suppressors and predict clinical outcome in malignancies.

Deletions of chromosome 1p36 are common in cancers; however, despite extensive studies, there has been limited success for discovering candidate tumor suppressors in this region. SRARP has recently been identified as a novel corepressor of the androgen receptor (AR) and is located on chromosome 1p36. Here, bioinformatics analysis of large tumor datasets was performed to study SRARP and its gene pair, HSPB7. In addition, using cancer cell lines, mechanisms of SRARP and HSPB7 regulation and their molecular functions were investigated. This study demonstrated that SRARP and HSPB7 are a gene pair located 5.2 kb apart on 1p36.13 and are inactivated by deletions and epigenetic silencing in malignancies. Importantly, SRARP and HSPB7 have tumor suppressor functions in clonogenicity and cell viability associated with the downregulation of Akt and ERK. SRARP expression is inversely correlated with genes that promote cell proliferation and signal transduction, which supports its functions as a tumor suppressor. In addition, AR exerts dual regulatory effects on SRARP, and although an increased AR activity suppresses SRARP transcription, a minimum level of AR activity is required to maintain baseline SRARP expression in AR+ cancer cells. Furthermore, as observed with SRARP, HSPB7 interacts with the 14-3-3 protein, presenting a shared molecular feature between SRARP and HSPB7. Of note, genome- and epigenome-wide associations of SRARP and HSPB7 with survival strongly support their tumor suppressor functions. In particular, DNA hypermethylation, lower expression, somatic mutations, and lower copy numbers of SRARP are associated with worse cancer outcome. Moreover, DNA hypermethylation and lower expression of SRARP in normal adjacent tissues predict poor survival, suggesting that SRARP inactivation is an early event in carcinogenesis. In summary, SRARP and HSPB7 are tumor suppressors that are commonly inactivated in malignancies. SRARP inactivation is an early event in carcinogenesis that is strongly associated with worse survival, presenting potential translational applications.

C1orf64 is a novel androgen receptor target gene and coregulator that interacts with 14-3-3 protein in breast cancer.

This study investigated the network of genes that are co-expressed with androgen receptor (AR) to discover novel AR targets in breast cancer. Bioinformatics analysis of two datasets from breast cancer cell lines resulted in the identification of an AR-gene signature constituted of 98 genes that highly correlated with AR expression. Notably, C1orf64 showed the highest positive correlation with AR across the datasets with a correlation coefficient (CC) of 0.737. In addition, C1orf64 closely correlated with AR expression in primary and metastatic breast tumors and C1orf64 expression was relatively higher in breast tumors with a lower grade and lobular histology. Furthermore, there is a functional interplay between AR and C1orf64 in breast cancer. In this process, AR activation directly represses C1orf64 transcription and C1orf64, in turn, interacts with AR as a corepressor and negatively regulates the AR-mediated induction of prolactin-induced protein (PIP) and AR reporter activity. Moreover, the corepressor effect of C1orf64 results in a reduction of AR binding to PIP promoter. The other aspect of this interplay involves a cross-talk between AR and estrogen receptor (ER) signaling in which C1orf64 silencing intensifies the AR-mediated down-regulation of ER target gene, progesterone receptor. Therefore, the repression of C1orf64 by AR provides an underlying mechanism for the AR inhibitory effects on ER signaling. To elucidate the biochemical mechanisms of C1orf64 function, this study demonstrates that C1orf64 is a phosphothreonine protein that interacts with the chaperone protein 14-3-3. In summary, C1orf64 is a novel AR coregulator and a 14-3-3 binding partner in breast cancer.

The Chromatin Remodelling Protein CHD1 Contains a Previously Unrecognised C-Terminal Helical Domain.

The packaging of eukaryotic DNA into nucleosomes, and the organisation of these nucleosomes into chromatin, plays a critical role in regulating all DNA-associated processes. Chromodomain helicase DNA-binding protein 1 (CHD1) is an ATP-dependent chromatin remodelling protein that is conserved throughout eukaryotes and has an ability to assemble and organise nucleosomes both in vitro and in vivo. This activity is involved in the regulation of transcription and is implicated in mammalian development and stem cell biology. CHD1 is classically depicted as possessing a pair of tandem chromodomains that directly precede a core catalytic helicase-like domain that is then followed by a SANT-SLIDE DNA-binding domain. Here, we have identified an additional conserved domain C-terminal to the SANT-SLIDE domain and determined its structure by multidimensional heteronuclear NMR spectroscopy. We have termed this domain the CHD1 helical C-terminal (CHCT) domain as it is comprised of five α-helices arranged in a variant helical bundle topology. CHCT has a conserved, positively charged surface and is able to bind DNA and nucleosomes. In addition, we have identified another group of proteins, the as yet uncharacterised C17orf64 proteins, as also containing a conserved CHCT domain. Our data provide new structural insights into the CHD1 enzyme family.

Molecular characterization of Brazilian equid herpesvirus type 1 strains based on neuropathogenicity markers.

Partial nucleotide sequences of ORF72 (glycoprotein D, gD), ORF64 (infected cell protein 4, ICP4) and ORF30 (DNA polymerase) genes were compared with corresponding sequences of EHV-1 reference strains to characterize the molecular variability of Brazilian strains. Virus isolation assays were applied to 74 samples including visceral tissue, total blood, cerebrospinal fluid (CSF) and nasal swabs of specimens from a total of 64 animals. Only one CSF sample (Iso07/05 strain) was positive by virus isolation in cell culture. EHV-1 Iso07/05 neurologic strain and two abortion visceral tissues samples (Iso11/06 and Iso33/06) were PCR-positive for ORF33 (glycoprotein B, gB) gene of EHV-1. A sequence analysis of the ORF72, ORF64 and ORF30 genes from three EHV-1 archival strains (A3/97, A4/72, A9/92) and three clinical samples (Iso07/05, Iso11/06 and Iso33/06) suggested that among Brazilian EHV-1 strains, the amplified region of the gD gene sequence is highly conserved. Additionally, the analysis of ICP4 gene showed high nucleotide and amino acid identities when compared with genotype P strains, suggesting that the EHV-1 Brazilian strains belonged to the same group. All the EHV-1 Brazilian strains were classified as non-neuropathogenic variants (N752) based on the ORF30 analysis. These findings indicate a high conservation of the gD-, ICP4- and ORF30-encoding sequences. Different pathotypes of the EHV-1 strain might share identical genes with no specific markers, and tissue tropism is not completely dependent on the gD envelope, immediate-early ICP4 and DNA polymerase proteins.

Molecular characterization of Brazilian equid herpesvirus type 1 strains based on neuropathogenicity markers

Partial nucleotide sequences of ORF72 (glycoprotein D, gD), ORF64 (infected cell protein 4, ICP4) and ORF30 (DNA polymerase) genes were compared with corresponding sequences of EHV-1 reference strains to characterize the molecular variability of Brazilian strains. Virus isolation assays were applied to 74 samples including visceral tissue, total blood, cerebrospinal fluid (CSF) and nasal swabs of specimens from a total of 64 animals. Only one CSF sample (Iso07/05 strain) was positive by virus isolation in cell culture. EHV-1 Iso07/05 neurologic strain and two abortion visceral tissues samples (Iso11/06 and Iso33/06) were PCR-positive for ORF33 (glycoprotein B, gB) gene of EHV-1. A sequence analysis of the ORF72, ORF64 and ORF30 genes from three EHV-1 archival strains (A3/97, A4/72, A9/92) and three clinical samples (Iso07/05, Iso11/06 and Iso33/06) suggested that among Brazilian EHV-1 strains, the amplified region of the gD gene sequence is highly conserved. Additionally, the analysis of ICP4 gene showed high nucleotide and amino acid identities when compared with genotype P strains, suggesting that the EHV-1 Brazilian strains belonged to the same group. All the EHV-1 Brazilian strains were classified as non-neuropathogenic variants (N752) based on the ORF30 analysis. These findings indicate a high conservation of the gD-, ICP4- and ORF30-encoding sequences. Different pathotypes of the EHV-1 strain might share identical genes with no specific markers, and tissue tropism is not completely dependent on the gD envelope, immediate-early ICP4 and DNA polymerase proteins.