Higher mRNA Expression Levels Research Articles

Effects of dietary n-3 LC-PUFA levels on the growth, immunity, and lipid metabolism of freshwater carnivorous teleost largemouth bass (Micropterus salmoides) juveniles

The n-3 long chain polyunsaturated fatty acids (n-3 LC-PUFA) play important roles in the normal growth and metabolism of fish . In order to explore the appropriate requiremnts of n-3 LC-PUFA in juvenile largemouth bass Micropterus salmoides, a freshwater carnivorous teleost, four isoproteic (50 %) and isolipidic (11 %) diets were formulated. The control diet used fish oil (FO) as the sole lipid source, and the other three diets used mixed vegetable oil as the main lipid source and supplemented with 0.50 %, 0.80 % and 1.10 % n-3 LC-PUFA, respectively. After the juvenile largemouth bass (initial weight 12.11 ± 0.01 g) fed with the four diets for 75 days, the growth, serum and heptic biochemical indices, as well as hepatic lipid metabolism were investigated. The results showed that the growth performance of 0.50 %-1.10 % groups were high that of control group; comparing to the 1.10 % group, high weight gain, specific growth rate and daily gain, as well as muscle hardness, chewiness and gumminess were detected in the 0.80 % group (P < 0.05). With the increase of dietary n-3 LC-PUFA levels, the serum biochemical indices (total cholesterol, triglyceride, and aspartate aminotransferase activity) and hepatic triglyceride were significantly decreased, while the serum acid phosphatase and alkaline phosphatase, as well as hepatic and muscle n-3 LC-PUFA contents were significantly increased (P < 0.05). Compared with the 0.50 % group, high hepatic mRNA expression levels of anti-inflammatory genes (il-10, tgf-β) were observed in the 0.80 % group (P < 0.05); and the relatively high transcript levels of genes related to lipid transport and catabolism (hsl, pparα, fatp and mtp) and low transcript levels of genes related to lipid anabolism (accα and pparγ) were detected in the 0.80 % and 1.10 % groups, as well as high mRNA levels of accα and pparγ were measured in the 0.80 % group (P < 0.05). The results indicated that 0.80 % dietary n-3 LC-PUFA level is suitable for largemouth bass concerning the growth performance, muscle quality and hepatic lipid metabolism. When subjected the weight gain to the second-order polynomial regression analysis, the maximum value was achieved at 0.76 % n-3 LC-PUFA level, which provide a basis for the development of formula feed for largemouth bass.

ACE2 ve TMPRSS2 Genlerinin Farklı Hücre Hatlarındaki İfade Düzeyleri

Objective: ACE2 and TMPRSS2 proteins have received increased attention gained emphasis together with the pandemic COVID-19. These proteins have roles in respiratory and hypertension disorders as well as cardiovascular and renal diseases. The objective of this work was to examine the mRNA and protein levels of ACE2 and TMPRSS2 in cell lines derived from various tissue origins.&#x0D; Methods: After the growth of 14 different cell lines, protein and mRNA were isolated from the cell pellets. The amounts of mRNAs and proteins were then determined and quantified using RT-PCR and ELISA.&#x0D; Results: Findings showed that VERO, HEK293T, and VERO E6 cell lines significantly differed from others in the mRNA levels of both the ACE2 and TMPRSS2 genes. In protein levels obtained using ELISA, PNT1A cell line had the highest level of ACE2 protein expression, while for TMPRSS2, A549 had the highest level of protein expression. &#x0D; Conclusion: It was showed in this study how the expressions of ACE2 and TMPRSS2 depend on the cell type. This may be an explanation for why virulence entrance differs in different types of tissues. It is thought that HEK293T cells with high levels of both genes may be a suitable option for studies at the RNA level by using these two genes. MCF7 may be a good option for studies at the protein level. Given the high levels of mRNA expression of both genes, it may be inferred that cells derived from the kidney were among those that were most susceptible to virus entry.

INVESTIGATING NEW THERAPEUTIC OPPORTUNITIES FOR HYPERTENSION THROUGH TRANSCRIPTOMIC AND PROTEOMIC ANALYSIS OF THE KIDNEY RENIN-ANGIOTENSIN SYSTEM

Objective: We systematically characterised the expression of all renin-angiotensin system (RAS) genes in the human kidney and searched in silico for new pharmacological therapeutic opportunities to modify RAS. Design and method: We compiled data from open access resources to characterise all components of the RAS pathway. We used transcriptomic profiles of 73 human kidney tissues from the Genotype-Tissue Expression Project, integrated genome-wide DNA genotypes and RNA-sequencing-derived transcriptome profiles of 478 human kidneys from Human Kidney Tissue Resource and, kidney tissue proteomics from 72 samples recruited by Clinical Proteomic Tumour Analysis Consortium. Additionally, we used an independent single-cell RNA sequencing data and gene expression datasets curated from the Kidney Precision Medicine Project (KPMP) and Enrichr (web-based enrichment analysis tool). Lastly, we integrated the Connectivity Map and Drug Gene Interaction Database to identify drug repurposing opportunities for treatment of hypertension targeting the RAS genes. Results: We identified 37 genes encoding components of the RAS pathway with measurable expression in human kidney tissue. 15% of these gene-pairs demonstrated a positive (r&gt;0.5) correlation in kidney expression most consistent with a functionally integrated network of interacting molecules. Angiotensinogen (AGT), the key component of the pathway - showed the highest level of mRNA expression in the liver, and we demonstrated that its hepatic mRNA expression correlates most strongly with its mRNA expression in the kidney (r = 0.71, P = 2.60e-06). Expression levels of mRNA for 92% of RAS genes correlated positively with their protein abundance in the kidney, where renin had the strongest positive correlation (r = 0.64, P&lt;0.01). Enrichr and KPMP revealed that RAS gene expression was enriched in mesangial and epithelial cell types. Existing genetic evidence for correlation between glutamyl aminopeptidase (ENPEP) and systolic blood pressure might relate to its capacity to generate angiotensin III acting on dilator angiotensin II type 2 receptors. This raises potential repurposing opportunities for ENPEP inhibitors like firibastat and tosedostat for the treatment of hypertension. Conclusions: Collectively, this study confirmed synchronized expression of RAS genes in the kidney at both mRNA and protein levels and identified new therapeutic opportunities for treatment of hypertension, through drug repurposing.

Epigenetic regulation of SST2 expression in small intestinal neuroendocrine tumors.

Somatostatin receptor type 2 (SST2) expression is critical for the diagnosis and treatment of neuroendocrine tumors and is associated with improved patient survival. Recent data suggest that epigenetic changes such as DNA methylation and histone modifications play an important role in regulating SST2 expression and tumorigenesis of NETs. However, there are limited data on the association between epigenetic marks and SST2 expression in small intestinal neuroendocrine tumors (SI-NETs). Tissue samples from 16 patients diagnosed with SI-NETs and undergoing surgical resection of the primary tumor at Erasmus MC Rotterdam were analysed for SST2 expression levels and epigenetic marks surrounding the SST2 promoter region, i.e. DNA methylation and histone modifications H3K27me3 and H3K9ac. As a control, 13 normal SI-tissue samples were included. The SI-NET samples had high SST2 protein and mRNA expression levels; a median (IQR) of 80% (70-95) SST2-positive cells and 8.2 times elevated SST2 mRNA expression level compared to normal SI-tissue (p=0.0042). In comparison to normal SI-tissue, DNA methylation levels and H3K27me3 levels were significantly lower at five out of the eight targeted CpG positions and at two out of the three examined locations within the SST2 gene promoter region of the SI-NET samples, respectively. No differences in the level of activating histone mark H3K9ac were observed between matched samples. While no correlation was found between histone modification marks and SST2 expression, SST2 mRNA expression levels correlated negatively with DNA methylation within the SST2 promoter region in both normal SI-tissue and SI-NETs (p=0.006 and p=0.04, respectively). SI-NETs have lower SST2 promoter methylation levels and lower H3K27me3 methylation levels compared to normal SI-tissue. Moreover, in contrast to the absence of a correlation with SST2 protein expression levels, significant negative correlations were found between SST2 mRNA expression level and the mean level of DNA methylation within the SST2 promoter region in both normal SI-tissue and SI-NET tissue. These results indicate that DNA methylation might be involved in regulating SST2 expression. However, the role of histone modifications in SI-NETs remains elusive.

Molecular Characterization of Nine TRAF Genes in Yellow Catfish (Pelteobagrus fulvidraco) and Their Expression Profiling in Response to Edwardsiella ictaluri Infection.

The yellow catfish (Pelteobagrus fulvidraco) is an economic fish with a large breeding scale, and diseases have led to huge economic losses. Tumor necrosis factor receptor-associated factors (TRAFs) are a class of intracellular signal transduction proteins that play an important role in innate and adaptive immune responses by mediating NF-κB, JNK and MAPK signaling pathways. However, there are few studies on the TRAF gene family in yellow catfish. In this study, the open reading frame (ORF) sequences of TRAF1, TRAF2a, TRAF2b, TRAF3, TRAF4a, TRAF4b, TRAF5, TRAF6 and TRAF7 genes were cloned and identified in yellow catfish. The ORF sequences of the nine TRAF genes of yellow catfish (Pf_TRAF1-7) were 1413-2025 bp in length and encoded 470-674 amino acids. The predicted protein structures of Pf_TRAFs have typically conserved domains compared to mammals. The phylogenetic relationships showed that TRAF genes are conserved during evolution. Gene structure, motifs and syntenic analyses of TRAF genes showed that the exon-intron structure and conserved motifs of TRAF genes are diverse among seven vertebrate species, and the TRAF gene family is relatively conserved evolutionarily. Among them, TRAF1 is more closely related to TRAF2a and TRAF2b, and they may have evolved from a common ancestor. TRAF7 is quite different and distantly related to other TRAFs. Real-time quantitative PCR (qRT-PCR) results showed that all nine Pf_TRAF genes were constitutively expressed in 12 tissues of healthy yellow catfish, with higher mRNA expression levels in the gonad, spleen, brain and gill. After infection with Edwardsiella ictaluri, the expression levels of nine Pf_TRAF mRNAs were significantly changed in the head kidney, spleen, gill and brain tissues of yellow catfish, of which four genes were down-regulated and one gene was up-regulated in the head kidney; four genes were up-regulated and four genes were down-regulated in the spleen; two genes were down-regulated, one gene was up-regulated, and one gene was up-regulated and then down-regulated in the gill; one gene was up-regulated, one gene was down-regulated, and four genes were down-regulated and then up-regulated in the brain. These results indicate that Pf_TRAF genes might be involved in the immune response against bacterial infection. Subcellular localization results showed that all nine Pf_TRAFs were found localized in the cytoplasm, and Pf_TRAF2a, Pf_TRAF3 and Pf_TRAF4a could also be localized in the nucleus, uncovering that the subcellular localization of TRAF protein may be closely related to its structure and function in cellular mechanism. The results of this study suggest that the Pf_TRAF gene family plays important roles in the immune response against pathogen invasion and will provide basic information to further understand the roles of TRAF gene against bacterial infection in yellow catfish.

INHA acts as a novel and potential biomarker in lung adenocarcinoma and shapes the immune-suppressive tumor microenvironment

BackgroundINHA expression has been correlated with the development, growth, and progression of multiple cancer types. However, the biological role of INHA has not been investigated in patients with lung adenocarcinoma (LUAD). Here, we performed a comprehensive bioinformatics analysis of the LUAD dataset to determine the mechanisms underlying the regulation of tumorigenesis by INHA. Materials and methodsINHA expression and clinical information of patients with LUAD were obtained from The Cancer Genome Atlas (TCGA) database. Protein levels in LUAD cell lines and human lung epithelial cells were examined by western blotting. Next, the prognostic value of INHA in LUAD was assessed using Cox regression analysis, while the potential biological functions and the impact on the immune microenvironment of INHA were investigated using gene set enrichment analysis (GSEA) and single sample GSEA (ssGSEA). Finally, the effect of INHA on LUAD cell proliferation and invasion was determined in vitro and in vivo. ResultsWe found significantly high mRNA and protein expression levels of INHA in LUAD tissues and cell lines. Additionally, a higher expression of INHA was linked to a shorter overall survival (OS) and a worse pathological stage, while INHA expression was associated with immune cell infiltration and immune-related markers in the LUAD tumor microenvironment. LUAD with high INHA expression tends to be a cold tumor. Furthermore, GO and KEGG enrichment analysis indicated that INHA-related genes were enriched in the cell adhesion and immune signaling pathways of LUAD. INHA promoted LUAD cell proliferation and invasion, in vitro and in vivo, by inducing the EGFR pathway. ConclusionOur findings revealed that INHA is overexpressed in LUAD and is linked to a poor prognosis. Our study demonstrates the potential of INHA as an immunotherapeutic and predictive biomarker in LUAD.

Aberrant methylation of dipeptidyl peptidase‑like 6 as a potential prognostic biomarker for lung adenocarcinoma.

We previously performed the genome-wide screening of aberrantly methylated CpG islands (CGIs) using the paired tumorous and non-tumorous tissues of 12 lung adenocarcinomas (LADC). In comparisons with paired normal lung tissues, dipeptidyl peptidase-like 6 (DPP6) has been identified as the most significantly hypermethylated CGI in LADC. DPP6 is a protein that modulates A-type potassium channels in the somatodendritic compartments of neurons, which play a role in synaptic plasticity. Previous studies have showed that DPP6 is downregulated in cancers, such as acute myeloid leukemia and melanoma, but upregulated in colon cancer, which is attributed to hyper- and hypomethylation, respectively. The present study investigated the methylation and expression levels of DPP6 and its prognostic value in patients with LADC. The DNA methylation and mRNA expression levels of DPP6 in surgically resected LADC tissues were examined by bisulfite pyrosequencing and reverse transcription-quantitative PCR, respectively. The DNA methylation and mRNA expression levels of DPP6 were both significantly higher in LADC tissues compared with in normal lung tissues (n=25; P<0.0001). Overall and disease-free survival rates were significantly higher in LADC with high mRNA expression levels compared with those with low levels. In conclusion, epigenetic alterations in DPP6 were significantly higher in LADC tissues compared with in normal lung tissues, which may contribute to the malignant features and worse prognosis of these patients.

GABA shunt pathway is stimulated in response to early defoliation-induced carbohydrate limitation in Mandarin fruits

The regulation of sugar and organic acid metabolism during fruit development has a major effect on high-quality fruit production. The reduction of leaf area is a common feature in plant growth, induced by abiotic and biotic stresses and disturbing source/sink ratio, thus impacting fruit quality. Here, we induced carbohydrate limitation by partial leaf defoliation at the beginning of the second stage of mandarin development (before the citrate peak). Resulting changes were monitored in the short-term (48 h and 1 week) and long-term (7 weeks) after the defoliation. Short-term response to early defoliation implied metabolic settings to re-feed TCA for sustaining respiration rate. These features involved (i) vacuolar sucrose degradation (high acid invertase activity and mRNA expression level) and enhanced glycolytic flux (high ATP-phosphofructokinase activity), (ii) malic and citric acid utilization (increased phosphoenolpyruvate kinase and NADP-Isocitrate dehydrogenase) associated with vacuolar citric acid release (high mRNA expression of the transporter CsCit1) and (iii) stimulation of GABA shunt pathway (low GABA content and increased mRNA expression of succinate semialdehyde dehydrogenase). A steady-state proline level was found in ED fruits although an increase in P5CS mRNA expression level. These results contribute to a better knowledge of the molecular basis of the relationship between defoliation and sugar and organic acid metabolism in mandarin fruit.

CCNE1 is a predictive and immunotherapeutic indicator in various cancers including UCEC: a pan-cancer analysis

BackgroundCCNE1 plays an important oncogenic role in several tumors, especially high-stage serous ovarian cancer and endometrial cancer. Nevertheless, the fundamental function of CCNE1 has not been explored in multiple cancers. Therefore, bioinformatics analyses of pan-cancer datasets were carried out to explore how CCNE1 regulates tumorigenesis.MethodsA variety of online tools and cancer databases, including GEPIA2, SangerBox, LinkedOmics and cBioPortal, were applied to investigate the expression of CCNE1 across cancers. The pan-cancer datasets were used to search for links between CCNE1 expression and prognosis, DNA methylation, m6A level, genetic alterations, CCNE1-related genes, and tumor immunity. We verified that CCNE1 has biological functions in UCEC cell lines using CCK-8, EdU, and Transwell assays.ResultsIn patients with different tumor types, a high mRNA expression level of CCNE1 was related to a poor prognosis. Genes related to CCNE1 were connected to the cell cycle, metabolism, and DNA damage repair, according to GO and KEGG enrichment analyses. Genetic alterations of CCNE1, including duplications and deep mutations, have been observed in various cancers. Immune analysis revealed that CCNE1 had a strong correlation with TMB, MSI, neoantigen, and ICP in a variety of tumor types, and this correlation may have an impact on the sensitivity of various cancers to immunotherapy. CCK-8, EdU and Transwell assays suggested that CCNE1 knockdown can suppress UCEC cell proliferation, migration and invasion.ConclusionOur study demonstrated that CCNE1 is upregulated in multiple cancers in the TCGA database and may be a promising predictive biomarker for the immunotherapy response in some types of cancers. Moreover, CCNE1 knockdown can suppress the proliferation, migration and invasion of UCEC cells.

The specific phagocytosis regulators could predict recurrence and therapeutic effect in thyroid cancer: A study based on bioinformatics analysis.

Thyroid cancer (TC) is one of the growing cancers and is prone to recurrence. Meanwhile, in immunotherapy, antibody-dependent cellular phagocytosis (ADCP) phagocytosis related regulators (PRs) play an important role. This study aims to investigate the prognostic value of specific PRs in TC. The purpose of this study was to identify specific PRs in TC patients by retrieving RNA-seq and Clustered Regularly Interspaced Short Palindromic Repeats-cas9 data and an algorithm based on LASSO was used to construct the PRs-signature. Subsequently, prognosis value of PRs-signature for recurrence-free survival (RFS) was explored through various statistical analysis, including Cox regression analysis, Kaplan-Meier analysis, and receiver operating characteristic curve. Additionally, an analysis of immune cell content by risk group was conducted using CIBERSORT, single sample gene set enrichment analysis and MCP-counter algorithms, with a particular focus on the correlation between macrophages and specific PRs. We identified 36 specific PRs, and a PRs-signature was constructed using 5-prognostic PRs (CAPN6, MUC21, PRDM1, SEL1L3, and CPQ). Receiver operating characteristic analysis showed that predictive power of PRs-signature was decent, and the PRs risk score as an independent prognostic factor was found to be correlated with RFS showed by multivariate cox regression analysis. Meanwhile, a lower RFS was observed in the high-risk group than in the low-risk group. The results of the 3 algorithms suggested that our PRs-signature may have certain significance for macrophage content and ADCP. Interestingly, the low-risk group had higher levels of mRNA expression than the high-risk group at PDCD1, CTLA4, and pro-inflammatory factors from macrophage. For the purpose of prognostic management, this study developed a prediction model. And the cross-talk between certain PRs and TC patients was revealed in this study. Besides, the PRs-signature can predict the immunotherapy response, macrophage content, and ADCP status. TC patients will benefit from these developments by gaining insight into novel therapeutic strategies.

Knockdown Experiment Reveals an Essential GTPase CgtA's Involvement in Growth, Viability, Motility, Morphology, and Persister Phenotypes in Vibrio cholerae.

CgtA is an essential bacterial GTPase consisting of a highly conserved N-terminal Spo0B-associated GTP-binding protein (Obg) domain, a central GTPase domain, and a variable C-terminal domain (CTD). This study reports global changes in the proteome and transcriptome of wild-type (Wt) versus full-length CgtA-depleted Vibrio cholerae in minimal media. Comparative transcriptome sequencing (RNA-Seq), followed by comparative proteomic analyses, revealed that the knockdown of cgtA significantly altered expressions of 311 proteins involved in diverse cellular activities, many of which are associated with the survival of V. cholerae. Various intracellular functional roles of CgtA in growth, viability, motility, morphology, and persister phenotypes in V. cholerae are revealed based on subsequent confirmatory experiments. Furthermore, a more sustained mRNA expression pattern of cgtA in a minimal medium than in a rich medium was also observed for Wt V. cholerae, where the highest level of mRNA expression of cgtA was observed during the logarithmic growth phase. Thereby, we propose that minimal medium-associated reduced growth rate coupled with cgtA depletion aggravates the intracellular stress in V. cholerae, interrupting vital cellular processes. The functional role of the CTD in V. cholerae is not fully understood. Hence, to specifically investigate the intracellular role of the 57-amino-acid-long CTD of CgtAVC, the CTD-only portion of CgtA was deleted. Subsequent proteomics studies revealed an altered expression of 240 proteins in the CgtA(ΔCTD) mutant, having major overlap with the full-length cgtA-deleted condition. Overall, our study reveals an alternative facet of the survival mechanism of V. cholerae during nutritional downshift as per the concomitant consequences of cgtA depletion. IMPORTANCE It is very important that we must find new drug target proteins from multidrug-resistant human-pathogenic organisms like V. cholerae. CgtA is among such potential candidates, and here, we are reporting about some newly identified cellular roles of it that are important for the survival of V. cholerae. Briefly, we knocked down the full-length cgtA gene, as well as did a partial deletion of this gene from the V. cholerae genome followed by RNA-Seq and proteomics studies. Results from our study revealed up- and downregulation of several known and unknown genes and proteins as the effect of the cgtA knockdown experiment. Also, we have presented some interesting observations that are linked with cgtA for growth, viability, motility, morphology, and persister phenotypes in V. cholerae. Our study enhances the importance of CgtA and paves the way for further exploration based on our provided data.

Effect of Atorvastatin on Angiogenesis-Related Genes VEGF-A, HGF and IGF-1 and the Modulation of PI3K/AKT/mTOR Transcripts in Bone-Marrow-Derived Mesenchymal Stem Cells.

Stem cell transplantation represents a unique therapeutic tool in tissue engineering and regenerative medicine. However, it was shown that the post-injection survival of stem cells is poor, warranting a more comprehensive understanding of activated regenerative pathways. Numerous studies indicate that statins improve the therapeutic efficacy of stem cells in regenerative medicine. In the present study, we investigated the effect of the most widely prescribed statin, atorvastatin, on the characteristics and properties of bone-marrow-derived mesenchymal stem cells (BM-MSCs) cultured in vitro. We found that atorvastatin did not decrease the viability of BM-MSCs, nor did it change the expression of MSC cell surface markers. Atorvastatin upregulated the mRNA expression levels of VEGF-A and HGF, whereas the mRNA expression level of IGF-1 was decreased. In addition, the PI3K/AKT signaling pathway was modulated by atorvastatin as indicated by the high mRNA expression levels of PI3K and AKT. Moreover, our data revealed the upregulation of mTOR mRNA levels; however, no change was observed in the BAX and BCL-2 transcripts. We propose that atorvastatin benefits BM-MSC treatment due to its ability to upregulate angiogenesis-related genes expression and transcripts of the PI3K/AKT/mTOR pathway.

High Intra-Tumor Transforming Growth Factor Beta 2 Level as a Predictor of Poor Treatment Outcomes in Pediatric Diffuse Intrinsic Pontine Glioma

Here, we report that tumor samples from newly diagnosed pediatric diffuse intrinsic pontine glioma (DIPG) patients express significantly higher levels of transforming growth factor beta 2 (TGFB2) messenger ribonucleic acid (mRNA) than control pons samples, which correlated with augmented expression of transcription factors that upregulate TGFB2 gene expression. Our study also demonstrated that RNA sequencing (RNAseq)-based high TGFB2 mRNA level is an indicator of poor prognosis for DIPG patients, but not for pediatric glioblastoma (GBM) patients or pediatric diffuse midline glioma (DMG) patients with tumor locations outside of the pons/brainstem. Notably, DIPG patients with high levels of TGFB2 mRNA expression in their tumor samples had significantly worse overall survival (OS) and progression-free survival (PFS). By comparison, high levels of transforming growth factor beta 3 (TGFB3) mRNA expression in tumor samples was associated with significantly better survival outcomes of DIPG patients, whereas high levels of transforming growth factor beta 1 (TGFB1) expression was not prognostic. Our study fills a significant gap in our understanding of the clinical significance of high TGFB2 expression in pediatric high-grade gliomas.

ARL9 is upregulated and serves as a biomarker for a poor prognosis in colon adenocarcinoma

BackgroundARL9 is a newly identified member of the ARF family, and the clinical significance of ARL9 in colon adenocarcinoma is unknown. In this study, we aimed to explore the expression of ARL9 mRNA in colon adenocarcinoma, and its effect on the prognosis of patients with colon adenocarcinoma.MethodsWe investigated the differential expression of ARL9 between colon adenocarcinoma tissue and adjacent tissues through a bioinformatics analysis using The Cancer Genome Atlas (TCGA) database. The correlation between clinical characteristics and the mRNA expression level of ARL9 were analyzed. A survival analysis and a Cox regression analysis were used to determine the prognostic significance of ARL9. Finally, we conducted a gene set enrichment analysis (GSEA) to explore the ARL9 signaling pathways involved in the development of colon adenocarcinoma. The effect of the expression of ARL9 on the proliferation and migration of colon adenocarcinoma was analyzed by the CCK8 method and a cell scratch test, respectively.ResultsThe mRNA expression of ARL9 in colon adenocarcinoma tissues was higher in comparison to the level in normal adjacent tissues (P < 0.05). The mRNA expression of ARL9 was not related to sex, tumor stage, T stage, N stage, M stage, but to age. The 5-year survival rate of colon adenocarcinoma patients with high ARL9 mRNA expression levels was significantly lower than that of patients with low ARL9 mRNA expression levels (P < 0.05). Age and the high mRNA expression of ARL9 were independent risk factors for a poor prognosis in patients with colon adenocarcinoma. The GSEA suggested that ARL9 may be able to upregulate cell adhesion, extracellular matrix receptor interactions, tumor-associated pathways, and downregulate the citrate cycle and tricarboxylic acid cycle pathway, which are involved in the development of colon adenocarcinoma. After knocking down ARL9, the proliferation and migration abilities of colon adenocarcinoma cells were decreased (P < 0.01).ConclusionThe mRNA expression of ARL9 is upregulated in colon adenocarcinoma, and higher mRNA expression levels are associated with a poor prognosis. Knocking down ARL9 can reduce the proliferation and migration of colon adenocarcinoma cells. ARL9 mRNA can be used as a prognostic biomarker in patients with colon adenocarcinoma.

Host defence peptide LEAP2 contributes to antimicrobial activity in a mustache toad (Leptobrachium liui)

BackgroundThe liver-expressed antimicrobial peptide 2 (LEAP2) is essential in host immunity against harmful pathogens and is only known to act as an extracellular modulator to regulate embryonic development in amphibians. However, there is a dearth of information on the antimicrobial function of amphibian LEAP2. Hence, a LEAP2 homologue from Leptobrachium liui was identified, characterized, and chemically synthesized, and its antibacterial activities and mechanisms were determined.ResultsIn this study, LEAP2 gene (Ll-LEAP2) cDNA was cloned and sequenced from the Chong’an Moustache Toad (Leptobrachium liui). The predicted amino acid sequence of Ll-LEAP2 comprises a signal peptide, a mature peptide, and a prodomain. From sequence analysis, it was revealed that Ll-LEAP2 belongs to the cluster of amphibian LEAP2 and displays high similarity to the Tropical Clawed Frog (Xenopus tropicalis)‘s LEAP2. Our study revealed that LEAP2 protein was found in different tissues, with the highest concentration in the kidney and liver of L. liui; and Ll-LEAP2 mRNA transcripts were expressed in various tissues with the kidney having the highest mRNA expression level. As a result of Aeromonas hydrophila infection, Ll-LEAP2 underwent a noticeable up-regulation in the skin while it was down-regulated in the intestines. The chemically synthesized Ll-LEAP2 mature peptide was selective in its antimicrobial activity against several in vitro bacteria including both gram-positive and negative bacteria. Additionally, Ll-LEAP2 can kill specific bacteria by disrupting bacterial membrane and hydrolyzing bacterial gDNA.ConclusionsThis study is the first report on the antibacterial activity and mechanism of amphibian LEAP2. With more to uncover, the immunomodulatory functions and wound-healing activities of Ll-LEAP2 holds great potential for future research.

Characterization of a novel corticosterone response gene in Xenopus tropicalis tadpole tails.

Corticosteroids are critical for development and for mediating stress responses across diverse vertebrate taxa. Study of frog metamorphosis has made significant breakthroughs in our understanding of corticosteroid signaling during development in non-mammalian vertebrate species. However, lack of adequate corticosterone (CORT) response genes in tadpoles make identification and quantification of CORT responses challenging. Here, we characterized a CORT-response gene frzb (frizzled related protein) previously identified in Xenopus tropicalis tadpole tail skin by an RNA-seq study. We validated the RNA-seq results that CORT and not thyroid hormone induces frzb in the tails using quantitative PCR. Further, maximum frzb expression was achieved by 100-250 nM CORT within 12-24 hours. frzb is not significantly induced in the liver and brain in response to 100 nM CORT. We also found no change in frzb expression across natural metamorphosis when endogenous CORT levels peak. Surprisingly, frzb is only induced by CORT in X. tropicalis tails and not in Xenopus laevis tails. The exact downstream function of increased frzb expression in tails in response to CORT is not known, but the specificity of hormone response and its high mRNA expression levels in the tail render frzb a useful marker of exogenous CORT-response independent of thyroid hormone for exogenous hormone treatments and in-vivo endocrine disruption studies.

Characterization of a glutamate-cysteine ligase in Bombyx mori.

Glutamate-cysteine ligase (GCL) is a crucial enzyme involved in the synthesis of glutathione (GSH). Despite various studies on glutathione transferase, and its essential role in detoxification and resistance to oxidative stress, GSH synthesis has not been described in Bombyx mori (silkworms) to date. Silkworms form part of the lepidopterans that are considered as a model of agricultural pests. This study aimed to understand the GSH synthesis by GCL in silkworms, which may help in developing insecticides to tackle agricultural pests. Based on the amino acid sequence and phylogenetic tree, the B. mori GCL belongs to group 2, and is designated bmGCL. Recombinant bmGCL was overexpressed and purified to ensure homogeneity. Biochemical studies revealed that bmGCL uses ATP and Mg2+ to ligate glutamate and cysteine. High expression levels of bmgcl mRNA and GSH were observed in the silkworm fat body after exposure to insecticides and UV-B irradiation. Moreover, we found an increase in bmgcl mRNA and GSH content during pupation in the silkworm fat body. In this study, we characterized the B. mori GCL and analyzed its biochemical properties. These observations indicate that bmGCL might play an important role in the resistance to oxidative stress in the silkworms.

The Interplay between RNA Editing Regulator ADAR1 and Immune Environment in Colorectal Cancer.

An abnormality in the regulation of adenosine deaminase acting on RNA (ADAR) enzymes, which catalyzed adenosine-to-inosine (A-to-I) RNA editing, was closely associated with the highly aggressive biologic behavior and poor prognosis in many malignancies. In the present study, we aimed to investigate the relationship among transcript factors-microRNAs regulatory network, immune environment, and ADAR gene in colorectal carcinoma (CRC). The association among the expression levels of ADAR mRNA and copy number variation, methylation, and mutation status were comprehensively analyzed using cBioPortal, Wanderer, and UALCAN databases in CRC datasets. ADAR-transcript factors (TFs) and ADAR-miRNA regulation networks were constructed by Cistrome Cancer and miRWalk2.0, respectively. The full network and subnetworks for ADAR coexpression genes were constructed using the STRING database and visualized by the MCODE module of the Cytoscape app. The relationship between ADAR mRNA expression and the abundance of infiltrating immune cells in CRC patients was explored by the Tumor Immune Estimation Resource, CIBERSORT, and single-gene gene set enrichment analysis (GSEA). ADAR mRNA was elevated and was a cancer essential gene in CRC. ADAR mRNA and transcripts P110 were significantly elevated in CRC compared to normal controls. Low-level methylation in the promoter region and high copy number amplification of ADAR were responsible for high levels of ADAR mRNA expression. ADAR coexpression genes were mainly involved in immunoregulation, especially T-lymphocyte activation. Hub genes, including CD2, CD274, and FASLG, were also significantly upregulated in the ADAR-high group compared to the control group. Besides, M1 macrophages were enriched in the ADAR-high group compared to the control group. This study demonstrated that ADAR, a new essential gene, was involved in the immune regulator and was a novel immune treatment target in CRC.

Comparative analysis of two cathepsin L genes in Asiatic hard clam (Meretrix meretrix): Similar in sequence features, different in expression profiles

Cathepsin L is widely found in eukaryotes and prokaryotes, and it plays important roles in innate immunity. In the present study, we cloned two cathepsin L genes (designated as MmCTSL1 and MmCTSL2, respectively) from Asiatic hard clam (Meretrix meretrix). The complete sequence of MmCTSL1 cDNA contained a 5′ untranslated region (UTR) of 31 bp, a 3′ UTR of 228 bp with a poly (A) tail, and an open reading frame (ORF) of 1005 bp encoding 334 amino acids with predicted molecular weight of 37.5 kDa and theoretical isoelectric point of 5.27, and contained a signal peptide (from M1 to A16), a protease inhibitor I29 family domain (from W27 to F87), and a papain family cysteine protease domain (from L118 to T333). The complete sequence of MmCTSL2 cDNA contained a 5′ UTR of 50 bp, a 3’ UTR of 162 bp with a poly (A) tail, and an ORF of 996 bp encoding a polypeptide of 331 amino acids with predicted molecular weight of 36.8 kDa and theoretical isoelectric point of 7.07. It contained a signal peptide (from M1 to A16), a protease inhibitor I29 family domain (from W30 to F89), and a papain family cysteine protease domain (from L115 to T330). Real-time quantitative PCR analysis demonstrated that MmCTSL1 and MmCTSL2 were widely expressed in all the tested tissues, including adductor muscle, foot, gill, hemocytes, hepatopancreas and mantle, with the highest mRNA expression level in hepatopancreas and hemocytes, respectively. After Vibrio splendidus challenge, the mRNA expression levels of MmCTSL1 and MmCTSL2 in hemocytes and hepatopancreas were both significantly up-regulated with different expression profiles. In hemocytes, the expression levels of MmCTSL1 and MmCTSL2 reached their respective peaks (3.4-fold and 13.0-fold compared with the control, respectively) at 12 h after bacterial challenge, and MmCTSL2 responds earlier than MmCTSL1. In hepatopancreas, the expression levels of MmCTSL1 and MmCTSL2 reached their respective peaks at 6 h (9.0-fold compared with the control) and 24 h (2.8-fold compared with the control) after bacterial challenge, meaning that MmCTSL1 responds earlier than MmCTSL2. At the same time, whether in hepatopancreas or hemocytes, MmCTSL1 persist for a while after the bacterial challenge peak, while MmCTSL2 would quickly return to the initial level after the bacterial challenge peak. These results indicate that cathepsin L may be involved in the immune process of hard clam against V. splendidus with different potential roles.

Collagen type VII α1 chain: A promising prognostic and immune infiltration biomarker of pancreatic cancer.

Pancreatic cancer (PC) is a stubborn malignancy with high lethality and a low 5-year overall survival (OS) rate. Collagen type VII α1 chain (COL7A1), a major component of the extracellular matrix, serves important roles in numerous physiological processes and various illnesses. COL7A1 protein acts as an anchoring fibril between the external epithelial cells and the underlying stroma, and mutation of COL7A1 could cause recessive dystrophic epidermolysis bullosa. Raw data for PC were acquired from The Cancer Genome Atlas and the Gene Expression Omnibus database, and raw data for the normal pancreas were obtained from the Genotype-Tissue Expression database. COL7A1 mRNA expression in PC tissues was compared with that in either paired (GSE15471 dataset) or unpaired (all other data) normal pancreas tissues. The association between COL7A1 mRNA expression and clinicopathological factors was assessed using logistic regression analysis. Cox analysis and Kaplan-Meier analysis were used to evaluate the role of COL7A1 mRNA expression in prognosis and nomograms were constructed. Gene Ontology analysis, Kyoto Encyclopedia of Genes and Genomes analysis, Gene Set Enrichment Analysis (GSEA) and single-sample GSEA (ssGSEA) were performed to evaluate the relevant functions of COL7A1 and correlation with immune cell infiltration. Furthermore, reverse transcription-quantitative PCR was used to assess the mRNA expression levels of COL7A1 in PC. The present study demonstrated that COL7A1 mRNA expression was higher in PC tissues compared with in normal pancreas tissues. The Kaplan-Meier survival analysis indicated that patients with PC with high COL7A1 mRNA expression had shorter overall survival (OS), disease-specific survival (DSS) and progression-free interval (PFI) times compared with patients with PC with low COL7A1 mRNA expression. Multivariate analysis demonstrated that COL7A1 mRNA expression was an independent risk factor for OS, DSS and PFI. Nomogram and calibration plots were constructed to predict the prognosis of patients with PC. GSEA demonstrated that high mRNA expression levels of COL7A1 were associated with multiple cancer-related pathways. ssGSEA analysis indicated that COL7A1 expression was positively associated with natural killer CD56bright cells and T helper (Th)2 cells, and negatively associated with Th17 cells and eosinophils. The results of the present study suggested that COL7A1 could be an independent biomarker and an influential moderator of immune infiltration in PC.