Cuproptosis-related gene DLAT is a biomarker of the prognosis and immune microenvironment of gastric cancer and affects the invasion and migration of cells.

OBJECTIVE: Cuproptosis is a novel cell death dependent on mitochondrial respiration and regulated by copper. This study aimed to investigate the cuproptosis-related gene DLAT potential value in gastric cancer (GC). METHODS: Bioinformatics was used to analyze DLAT expression. DLAT expression in GC cell lines was detected using qRT-PCR. Cell proliferation ability was assessed using CCK8 and cell cycle assay. Cell migration and invasion were assessed using wound healing and transwell assay. A prognostic assessment was performed through survival and Cox regression analysis. DLAT protein expression was analyzed through HPA immunohistochemistry. Biological functions and processes were analyzed through GO and KEGG enrichment analysis and PPI. Correlation with immune cell infiltration and immune checkpoint genes was analyzed for DLAT. RESULTS: DLAT expression was upregulated in GC tissues and cells and correlated with shorter survival for patients. Age, gender, histological typing, lymph node metastasis, and distant metastasis were identified as independent prognostic factors affecting OS in GC. DLAT protein was upregulated in GC. The biological functions and pathways enriched in DLAT were mainly linked to mitochondrial respiration and the TCA cycle. The expression of DLAT was found to be positively correlated with the infiltration of Th and Th2 immune cells and only positively correlated with the expression of the BTN2A1 immune checkpoint gene. CONCLUSION: DLAT has the potential to serve as a prognostic assessment factor in GC. The expression of DLAT was correlated with immune infiltration and tumor immune escape, providing a new target for immunotherapy of GC.

The Up-Regulated Expression of Mitochondrial Membrane Molecule RHOT1 in Gastric Cancer Predicts the Prognosis of Patients and Promotes the Malignant Biological Behavior of Cells.

Gastric cancer (GC) remains a major disease of high morbidity and mortality worldwide despite advances in diagnosis and treatment. Ras homolog family member T1 (RHOT1) plays an important role in several cancers. Our study aimed to analyze RHOT1 expression, to assess the relationship between its expression and the prognosis of patients, and know the impact of RHOT1 on GC cells. The Cancer Genome Atlas (TCGA) RNA-seq data was used for gene expression analysis, survival and prognostic analysis. Nomograms were created to analyze the pathological factors of GC patients. RHOT1 expression was up-regulated by analyzed TCGA-Stomach adenocarcinoma (STAD) data and verified by Polymerase Chain Reaction (PCR) assay in GC tissues and cell lines. Furthermore, RHOT1 up-regulation was significantly associated with shorter survival of GC patients. At last, after silencing the expression of RHOT1 in AGS cell lines, we found that the proliferative ability of the cells was significantly reduced, the cell invasion ability was significantly inhibited, the cell migration ability was also significantly weakened, the cell cycle was arrested in the G0/G1 phase, and apoptosis was significantly increased. So RHOT1 could impact the apoptosis, proliferation, invasion, and migration behavior of GC cells. We trust RHOT1 has the potential to become a new oncogene biomarker for diagnosis and prognosis as well as a new therapeutic target in GC.

A controllable SERS biosensor for ultrasensitive detection of miRNAs based on porous MOFs and subject-object recognition ability.

In this work, a simple and sensitive SERS-based miRNA biosensor was constructed based on porous MOFs nanoparticles and efficient subject-object recognition ability. MOFs as a container can package lots of signal probe neutral red (NR) for the advantages of three dimensional structure and porosity. The partially complementary duplex DNA can as a "lock" to lock up the hole for obtaining a weak Raman signal. In the present of miRNA, miRNA just like a "key" to open the duplex structure with the results of releasing NR. At this time, the released NR can be captured by SERS substrate AuNS@CB[7] for the subject-object recognition ability to produce a strong Raman signal which was positive correlation to target miRNA. By this way, the proposed SERS biosensor can achieve sensitively and selectively detect miRNA with a detection limit of 0.562 fM. This MOF-based SERS biosensor also be hopeful application for clinical diagnostics.

Hsa_circ_0005230 is up-regulated and promotes gastric cancer cell invasion and migration via regulating the miR-1299/RHOT1 axis.

Gastric cancer (GC) is one of the most common cancers in the world. Circular RNAs (circRNAs) are a class of non-coding RNAs that are widely expressed in eukaryotic cells. However, their role has been poorly understood in GC. This report aimed to explore the biological functions of hsa_circ_0005230 and its action mechanism in GC. This study validated that hsa_circ_0005230 was significantly up-regulated in 130 cases of GC tissues using qRT-PCR, and clinicopathological feature analysis revealed that its high expression was positively associated with histological grade, lymph node metastasis, TNM stages, and poor prognosis. In vitro, functional experiments showed that silencing hsa_circ_0005230 significantly decreased GC cell proliferation, invasion and migration capabilities. In addition, the major proteins of EMT (epithelial-mesenchymal transition) relevance have changed. In mechanism studies, bioinformatics analyses were used to predict the hsa_circ_0005230/miR-1299/RHOT1 axis and hsa_circ_0005230 may serve as a sponge for miR-1299 and indirectly regulate the expression of RHOT1. The regulated relationships between the molecules on the axis were verified using qRT-PCR and correlation analysis. Dual-luciferase reporter gene assay has been used to verify the binding site between miR-1299 and RHOT1. WB (Western blotting) and IHC (Immunohistochemical) were used to verify that RHOT1 may play the role of oncoprotein and affect the biological behavior of GC. Overall, hsa_circ_0005230 could enhance the EMT phenotype by promoting RHOT1 expression through sponging miR-1299, thus affecting the biological behavior of GC. Hsa_circ_0005230 can be easily identified as a potential diagnostic biomarker and assessment prognosis target for GC.

OSlgg: An Online Prognostic Biomarker Analysis Tool for Low-Grade Glioma.

Glioma is the most frequent primary brain tumor that causes high mortality and morbidity with poor prognosis. There are four grades of gliomas, I to IV, among which grade II and III are low-grade glioma (LGG). Although less aggressive, LGG almost universally progresses to high-grade glioma and eventual causes death if lacking of intervention. Current LGG treatment mainly depends on surgical resection followed by radiotherapy and chemotherapy, but the survival rates of LGG patients are low. Therefore, it is necessary to use prognostic biomarkers to classify patients into subgroups with different risks and guide clinical managements. Using gene expression profiling and long-term follow-up data, we established an Online consensus Survival analysis tool for LGG named OSlgg. OSlgg is comprised of 720 LGG cases from two independent cohorts. To evaluate the prognostic potency of genes, OSlgg employs the Kaplan-Meier plot with hazard ratio and p value to assess the prognostic significance of genes of interest. The reliability of OSlgg was verified by analyzing 86 previously published prognostic biomarkers of LGG. Using OSlgg, we discovered two novel potential prognostic biomarkers (CD302 and FABP5) of LGG, and patients with the elevated expression of either CD302 or FABP5 present the unfavorable survival outcome. These two genes may be novel risk predictors for LGG patients after further validation. OSlgg is public and free to the users at http://bioinfo.henu.edu.cn/LGG/LGGList.jsp.

OSlihc: An Online Prognostic Biomarker Analysis Tool for Hepatocellular Carcinoma.

Liver hepatocellular carcinoma (LIHC) is one of the most common malignant tumors in the world with an increasing number of fatalities. Identification of novel prognosis biomarker for LIHC may improve treatment and therefore patient outcomes. The availability of public gene expression profiling data offers the opportunity to discover prognosis biomarkers for LIHC. We developed an online consensus survival analysis tool named OSlihc using gene expression profiling and long-term follow-up data to identify new prognosis biomarkers. OSlihc consists of 637 cases from four independent cohorts. As a risk assessment tool, OSlihc generates the Kaplan-Meier survival plot with hazard ratio (HR) and p value to evaluate the prognostic value of a gene of interest. To test the reliability of OSlihc, we analyzed 65 previous reported prognostic biomarkers in OSlihc and showed that all of which have significant prognostic values. Furthermore, we identified four novel potential prognostic biomarkers (ATG9A, WIPI1, CXCL1, and CSNK2A2) for LIHC, the elevated expression of which predict the unfavorable survival outcomes. These genes (ATG9A, WIPI1, CXCL1, and CSNK2A2) may be potentially new biomarkers to identify at-risk LIHC patients when further validated. By OSlihc, users can evaluate the prognostic abilities of genes of their interest, which provides a platform for researchers to identify prognostic biomarkers to further develop targeted therapy strategies for LIHC patients. OSlihc is public and free to the users at http://bioinfo.henu.edu.cn/LIHC/LIHCList.jsp.

Acute effects of endothelin receptor antagonists on hepatic hemodynamics of cirrhotic and noncirrhotic rats.

Hepatic and circulating endothelin-1 (ET-1) are increased in patients with cirrhosis and in cirrhotic animals. However, the distinct roles of ET receptor subtypes ETA and ETB in cirrhosis and portal hypertension (PHT) have not been clearly elucidated. Thus, we studied the effects of selective ET-1 antagonists (ETA-ant or ETB-ant) and nonselective ET-1 antagonist (ETA/B-ant) on hepatic hemodynamics in cirrhotic rats. Liver fibrosis and PHT were induced by complete bile duct ligation (BDL) in rats. Two weeks after BDL or sham surgery, hemodynamic responses were measured during intraportal infusion of incremental doses of the following ET-ants: (i) BQ-123, (ii) BQ-788, and (iii) bosentan. After equilibration with vehicle, doses of ET-ants were infused for 30 min periods, and steady-state systemic and hepatic hemodynamics, portal venous pressure (PVP), and hepatic blood flow (HBF) were measured. BDL induced significant PHT and elevated concentrations of plasma ET-1 compared with sham. ETA-ant decreased PVP of cirrhotic rats but had no effect on sham, whereas ETB-ant increased PVP in sham but had no effect in BDL. Nonselective ETA/B-ant decreased PVP of BDL similarly to ETA-ant. Both ETA-ant and ETB-ant decreased local HBF, whereas a nonselective antagonist did not change HBF in sham; however no significant changes were observed in HBF of BDL rats with any of the antagonists. These findings suggest ETA activation contributes to PHT in cirrhotic rats, whereas ETB-mediated portal depressor effects are attenuated in cirrhotic rats compared with noncirrhotic rats.

Agonist-induced nuclear export of GFP-HDAC5 in isolated adult rat ventricular myocytes.

INTRODUCTION: Histone acetylation/deacetylation represents a central mechanism for the control of gene expression. Histone deacetylases (HDACs) deacetylate histones and transcription factors, causing transcriptional repression critical to the maintenance of the normal adult cardiac myocyte phenotype. Export of HDAC5 from the nucleus is associated with derepression of the fetal gene program and induction of hypertrophy in the cardiac myocyte. Prior studies of HDAC5 localization in adult cardiomyocytes have employed rabbit cells. METHODS: Because many laboratories are restricted from working with USDA-regulated species such as rabbits, we sought to develop a quantitative assay to monitor signal-dependent nuclear export of HDAC5 in adult rat ventricular myocytes (ARVMs). RESULTS: We demonstrate that GFP-tagged HDAC5 expressed from an adenoviral vector appropriately localizes in nuclei of cultured ARVMs within 24 h post infection. Nuclear localized GFP-HDAC5 undergoes quantitative nuclear export (approximately 20-30% within 2 h) in response to hypertrophic agonists such as endothelin-1 (ET-1) and prostaglandin-F2alpha (PGF2alpha), as well as the direct protein kinase C (PKC) activator and phorbol myristate acetate (PMA). Nuclear export of HDAC5 in ARVMs is blocked by Gö6976, a small molecule inhibitor of PKC and protein kinase D (PKD). DISCUSSION: These data suggest that GFP-HDAC5 is appropriately functional in ARVMs 24 h post infection, and that translocation events can be quantitated for the study of hypertrophy or the identification of antihypertrophic agents in adult cardiac myocytes.

A phosphorimager-based filter binding thyroid hormone receptor competition assay for chemical screening.

INTRODUCTION: A phosphorimager-based filter binding thyroid hormone receptor (THR) competition assay has been developed for use in verifying hits from compound library screens. METHODS: This method employs in vitro translated ligand binding domains (LBDs) of THRalpha and THRbeta, separation through nitrocellulose via a 96-well vacuum manifold, and analysis of receptor-bound radioactivity by phosphorimaging. RESULTS: A standard curve of [I(125)]T3 showed a linear response over the dynamic range of a competition assay, and a comparison of Sephadex G-25 column separation and gamma counting with en masse filtration and phosphorimaging revealed similar IC(50) and K(i) values when using unlabeled T3 as competitor. In addition, this method produced IC(50) and K(i) values for the known T3 competitors [3,5-Dimethyl-4-(4'-hydoxy-3'-isopropylbenzyl) phenoxy] acetic acid (GC-1) and 3,5-diiodothyropropionic acid (DITPA) similar to those reported elsewhere. DISCUSSION: These data suggest that filtration and phosphorimaging adequately and properly reproduces binding values associated with THR competition. Further, this method gave a 3-fold reduction in time and a 40-fold reduction in radioactive waste over the column-based method. These reductions allow for a substantial increase in assay throughput. Taken together, these data suggest that en masse filtration and phosphorimaging is an efficient and tractable method for verifying large groups of putative T3 competitors in vitro.

Small deletion variants of the replication protein, pi, and their potential for over-replication-based antimicrobial activity.

The emergence of multiply antibiotic-resistant microorganisms in the environment has become a serious public health threat. To address this, our lab has devised a methodology in which antimicrobial agents are transferred into unwanted cells using the process of bacterial conjugation. In the work described here, we pursued proteins that cause plasmid over-replication as potential antimicrobial agents. Our focus was on the pir-encoded pi protein of plasmid R6K that possesses both positive and negative functions in controlling gamma origin-based replication. We observed that three of four pir mutations examined, including two in-frame deletions, severely impaired negative plasmid-replication control. The resulting over-replication phenotype was particularly strong when a pir mutant was placed in cis to gamma origin. In conjugative mating experiments with several representatives of the family Enterobacteriaceae, the plasmids expressed postconjugational antimicrobial activity. The potential utility of a conjugation-based antimicrobial approach is discussed. Additionally, we describe the replication inhibitory function of a novel and useful Rep protein variant, pi*M36A;M38A, which binds iteron DNA exclusively as dimers.