Real-time Polymerase Chain Reaction Experiments Research Articles

Lentinan Regulates Glioma Cell Proliferation and Apoptosis by Activating p53 and Caspases Pathways

Background Gliomas are highly lethal malignancies that develop in the central nervous system. The primary treatment for gliomas involves surgical resection followed by chemoradiotherapy. However, due to the infiltrative growth nature of gliomas, surgical resection is often incomplete. Moreover, the efficacy of chemotherapeutic drugs is constrained by their ability to cross the blood–brain barrier, and the currently utilized agents can lose effectiveness, particularly with prolonged administration. Lentinan, an active compound in Lentinula edodes, exhibits various pharmacological activities. Purpose This study aims to investigate the anti-tumor effects of lentinan on glioma U251 cells. Methods Cell proliferation assays, cell fluorescence staining, scratch healing experiments, and transwell chamber experiments were conducted to assess the anti-tumor activity of lentinan on U251 cells. Additionally, quantitative real-time polymerase chain reaction (qPCR) and Western blot experiments were performed to validate the anti-tumor mechanism of lentinan. Results The findings revealed that lentinan significantly suppressed the proliferation of U251 cells, induced robust apoptosis, and decreased the cells’ migration and invasion capabilities. Furthermore, lentinan notably influenced the gene and protein expression of P53, Bcl-2, Cyto-c, Bax, Caspases, and MMP-9 in U251 cells. Conclusion These findings suggest that lentinan may inhibit glioma cells by activating P53 and caspase-related apoptosis pathways.

Crocin ameliorates neuroinflammation and cognitive impairment in mice with Alzheimer's disease by activating PI3K/AKT pathway.

Crocin has a good prospect in the treatment of Alzheimer's disease (AD), but the mechanisms underlying its neuroprotective effects remain elusive. This study aimed to investigate the neuroprotective effects of Crocin and its underlying mechanisms in AD. AD mice were set up by injecting Aβ25-35 solution into the hippocampus. Then, the AD mice were injected intraperitoneally with 40mg/kg/day of Crocin for 14 days. Following the completion of Crocin treatment, an open-field test, Y-maze test and Morris water maze test were conducted to evaluate the impact of Crocin on spatial learning and memory deficiency in mice. The effects of Crocin on hippocampal neuron injury, proinflammatory cytokine expressions (IL-1β, IL-6, and TNF-α), and PI3K/AKT signaling-related protein expressions were measured using hematoxylin and eosin staining, Western blot, and quantitative real-time polymerase chain reaction (qRT-PCR) experiments, respectively. Crocin attenuated Aβ25-35-induced spatial learning and memory deficiency and hippocampal neuron injury. Furthermore, the Western blot and qRT-PCR results showed that Crocin effectively suppressed inflammation and activated the PI3K/AKT pathway in Aβ25-35-induced mice. Crocin restrained neuroinflammation via the activation of the PI3K/AKT pathway, thereby ameliorating the cognitive dysfunction of AD mice.

Sijunzi decoction enhances sensitivity of colon cancer cells to NK cell destruction by modulating P53 expression

Ethnopharmacological relevanceSijunzi Decoction (SJZD), a traditional Chinese herbal remedy, is frequently employed in the treatment of various cancers, including colon cancer. Previous research suggests that SJZD plays a pivotal role in modulating the immune system and enhancing immunity against tumors. However, the precise role of SJZD in combating colon cancer and its potential molecular functions in regulating natural killer cells remain elusive. Aims of the studyTo elucidate the potential mechanism underlying the anticolon cancer effects of SJZD in synergy with natural killer (NK) cells through both in vivo and in vitro experiments. Materials and methodsIn vivo experiments: A subcutaneous tumor mouse model of colon cancer and in vivo NK cell depletion experiments were conducted to observe the anticolon cancer effects of SJZD. Flow cytometry assessed immune cell depletion in mouse spleens, while immunohistochemical (IHC) staining detected the expression of apoptotic genes in tumor tissues. In vitro experiments: The mechanism by which SJZD regulates the sensitization of colon cancer cells to NK cells was investigated using real-time polymerase chain reaction (RT-PCR), western blotting (WB), and co-culture experiments with NK cells. ResultsSijunzi Decoction (SJZD) significantly impeded tumor growth in mice; however, NK cell depletion markedly attenuated the tumor-suppressive effect of SJZD. Immunohistochemical (IHC) results indicated that SJZD increased the expression of P53, death receptor 4 (DR4), and death receptor 5 (DR5) in tumor tissues. In vitro experiments, 24 h SJZD-pretreated colon cancer cells showed a substantial elevation in P53, DR4, and DR5 levels, and the activity of colon cancer cells significantly diminished after co-culture with NK cells. These effects of SJZD were reversed with the addition of the P53 inhibitor pifithrin-α (PFT-α), resulting in reduced inhibition of colon cancer cells by NK cells. ConclusionSJZD enhances the levels of DR4 and DR5 through the modulation of P53 expression, consequently increasing the sensitivity of colon cancer cells to NK cell-mediated killing. These findings provide a theoretical foundation for the clinical application of SJZD in patients with colon cancer.In this study, we first investigated the effect of SJZD on subcutaneous tumor growth in mice with colon cancer using in vivo assays and assessed the impact of NK cells on the anticolon cancer effect of SJZD in vivo through NK cell depletion. In vitro experiments were conducted to explore the potential mechanism of action of SJZD in NK cell-mediated anticolon cancer effects.

A novel risk signature based on liquid-liquid phase separation-related genes reveals prognostic and tumour microenvironmental features in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma(ccRCC) is one of the most common malignancies. However, there are still many barriers to its underlying causes, early diagnostic techniques and therapeutic approaches. The Cancer Genome Atlas (TCGA)- Kidney renal clear cell (KIRC) cohort differentially analysed liquid-liquid phase separation (LLPS)-related genes from the DrLLPS website. Univariate and multivariate Cox regression analyses and LASSO regression analyses were used to construct prognostic models. The E-MTAB-1980 cohort was used for external validation. Then, potential functions, immune infiltration analysis, and mutational landscapes were analysed for the high-risk and low-risk groups. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) experiments as well as single-cell analyses validated the genes key to the model. We screened 174 LLPS-related genes in ccRCC and constructed a risk signature consisting of five genes (CLIC5, MXD3, NUF2, PABPC1L, PLK1). The high-risk group was found to be associated with worse prognosis in different subgroups. A nomogram constructed by combining age and tumour stage had a strong predictive power for the prognosis of ccRCC patients. In addition, there were differences in pathway enrichment, immune cell infiltration, and mutational landscapes between the two groups. The results of qRT-PCR in renal cancer cell lines and renal cancer tissues were consistent with the biosignature prediction. Three single-cell data of GSE159115, GSE139555, and GSE121636 were analysed for differences in the presence of these five genes in different cells. We developed a risk signature constructed based on the five LLPS-related genes and can have a high ability to predict the prognosis of ccRCC patients, further providing a strong support for clinical decision-making.

Cuproptosis-related molecular classification and gene signature of hepatocellular carcinoma and experimental verification.

Hepatocellular carcinoma (HCC) is a highly heterogeneous malignancy with poor overall prognosis. Cuproptosis, a recently proposed mode of copper-dependent cell death, plays a critical role in the malignant progression of various tumors; however, the expression and prognostic value of cuproptosis-related regulatory genes in HCC remain unclear. Genomic, genetic, and expression profiles of ten key cuproptosis-related regulatory genes were analyzed using The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) dataset and protein expression data from the Human Protein Atlas (HPA) database. Unsupervised clustering of HCC patients based on these ten key cuproptosis-related regulatory genes was used to identify different HCC subtypes and analyze the differences in clinical and immune characteristics among subtypes. Subsequently, univariate Cox and least absolute shrinkage and selection operator (LASSO) Cox analyses were used to establish a cuproptosis-related prognostic signature, and the accuracy of prognostic signature prediction was internally validated by Kaplan-Meier survival analysis and time-dependent receiver operating characteristic curve in TCGA training and testing cohorts. The prognostic signature was externally validated using TCGA-LIHC entire cohort and International Cancer Genome Consortium Liver Cancer (ICGC-LIRI) cohorts. Finally, the expression landscape of cuproptosis-related regulatory genes in prognostic signature was explored by quantitative real-time polymerase chain reaction (qRT-PCR), western blotting and immunohistochemistry (IHC) experiments. Ten cuproptosis-related genes were differentially expressed in normal and HCC tissues. Unsupervised clustering identified two subtypes and HCC patients with these two subtypes had different clinical prognoses and immune characteristics, as well as different degrees of response to immunotherapy. Lipoyltransferase 1 (LIPT1), dihydrolipoamide s-acetyltransferase (DLAT), and cyclin dependent kinase inhibitor 2A (CDKN2A) were selected to construct a prognostic signature, which significantly distinguished HCC patients with different survival periods in the TCGA training and testing cohorts and was well validated in both the TCGA-LIHC entire cohort and ICGC-LIRI cohort. The risk score of the prognostic signature was confirmed to be an independent prognostic factor, and nomograms were generated to effectively predict the probability of HCC patient survival. The qRT-PCR, western blotting and IHC results also revealed a significant imbalance in the expression of these cuproptosis-related genes in HCC. The classification and prognostic signature based on cuproptosis-related regulatory genes helps to explain the heterogeneity of HCC, which may contribute to the individualized treatment of patients with the disease.

Metabolomics responses and tolerance of Pseudomonas aeruginosa under acoustic vibration stress.

Sound has been shown to impact microbial behaviors. However, our understanding of the chemical and molecular mechanisms underlying these microbial responses to acoustic vibration is limited. In this study, we used untargeted metabolomics analysis to investigate the effects of 100-Hz acoustic vibration on the intra- and extracellular hydrophobic metabolites of P. aeruginosa PAO1. Our findings revealed increased levels of fatty acids and their derivatives, quinolones, and N-acylethanolamines upon sound exposure, while rhamnolipids (RLs) showed decreased levels. Further quantitative real-time polymerase chain reaction experiments showed slight downregulation of the rhlA gene (1.3-fold) and upregulation of fabY (1.5-fold), fadE (1.7-fold), and pqsA (1.4-fold) genes, which are associated with RL, fatty acid, and quinolone biosynthesis. However, no alterations in the genes related to the rpoS regulators or quorum-sensing networks were observed. Supplementing sodium oleate to P. aeruginosa cultures to simulate the effects of sound resulted in increased tolerance of P. aeruginosa in the presence of sound at 48 h, suggesting a potential novel response-tolerance correlation. In contrast, adding RL, which went against the response direction, did not affect its growth. Overall, these findings provide potential implications for the control and manipulation of virulence and bacterial characteristics for medical and industrial applications.

LATS1 Promotes B-ALL Tumorigenesis by Regulating YAP1 Phosphorylation and Subcellular Localization.

YAP1 plays a dual role as an oncogene and tumor suppressor gene in several tumors; differentiating between these roles may depend on the YAP1 phosphorylation pattern. The specific function of YAP1 in B cell acute lymphoblastic leukemia (B-ALL), however, is currently unclear. Thus, in the present study, the role of YAP1 in B-ALL was investigated using relevant cell lines and patient datasets. The effects of shRNA-mediated knockdown on YAP1 and LATS1 levels in the NALM6 and MOLT-4 cell lines were examined using Western blotting, quantitative real-time polymerase chain reaction, flow cytometry, immunostaining, and nude mouse subcutaneous tumorigenesis experiments. Gene expression levels of Hippo pathway-related molecules before and after verteporfin (VP) treatment were compared using RNA-Seq to identify significant Hippo pathway-related genes in NALM6 cells. Patients with ALL showing high YAP1 expression and low YAP1-Ser127 phosphorylation levels had worse prognoses than those with low YAP1 protein expression and high YAP1-Ser127 phosphorylation levels. YAP1-Ser127 phosphorylation levels were lower in NALM6 cells than in MOLT-4 and control cells; YAP1 was distributed in the nuclei in NALM6 cells. Knockdown of YAP1 inhibited MOLT-4 and NALM6 cell proliferation and arrested the NALM6 cell cycle in the G0/G1 phase. Before and after VP treatment, the expression of the upstream gene LATS1 was upregulated; its overexpression promoted YAP1-Ser127 phosphorylation. Further, YAP1 was distributed in the plasma. LATS1 may downregulate YAP1-Ser127 phosphorylation and maintain B-ALL cell function; thus, VP, which targets this axis, may serve as a new therapeutic method for improving the outcomes for B-ALL patients.

A prognostic aging-related lncRNA risk model correlates with the immune microenvironment in HCC

<p style="margin-top: 12.0pt; text-align: justify; text-justify: inter-ideograph;"><strong><span lang="EN-US" style="font-family: Nunito; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: minor-bidi; color: #212529; background: white;">Background: </span></strong><span lang="EN-US" style="font-size: 10.5pt; font-family: Nunito; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: minor-bidi; color: #212529; background: white;">Hepatocellular carcinoma (HCC) stands out as one of the most lethal cancers globally, given its complexity, recurrence following surgical resection, metastatic potential, and inherent heterogeneity.</span><strong><span lang="EN-US" style="font-family: Nunito; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: minor-bidi; color: #212529; background: white;"> </span></strong><span lang="EN-US" style="font-family: Nunito; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: minor-bidi; color: #212529; background: white;">In<strong> </strong></span><span lang="EN-US" style="font-size: 10.5pt; font-family: Nunito; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: minor-bidi; color: #212529; background: white;">recent years, researchers have systematically elucidated the significance of long non-coding RNA (lncRNA) in the initiation and progression of HCC. The introduction of The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases has significantly enhanced the prognostic assessment of HCC. However, the association between HCC and cell senescence has been infrequently explored in the literature. </span><strong><span lang="EN-US" style="font-family: Nunito; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: minor-bidi; color: #212529; background: white;">Method:</span></strong><span lang="EN-US" style="font-size: 10.5pt; font-family: Nunito; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: minor-bidi; color: #212529; background: white;"> We downloaded liver hepatocellular carcinoma (LIHC)-related messenger RNA and lncRNA expression levels from TCGA. Correlation analysis, Cox regression, and least absolute shrinkage and selection operator (LASSO) regression analysis were employed to validate the lncRNA risk model associated with cellular aging. Comparing the infiltration of diverse immune cells enabled the identification of distinct differences in the immunological microenvironments of the two risk groups. Subsequently, we conducted a real-time polymerase chain reaction (qPCR) experiment to confirm the accuracy of the selected lncRNAs. </span><strong><span lang="EN-US" style="font-family: Nunito; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: minor-bidi; color: #212529; background: white;">Results:</span></strong><span lang="EN-US" style="font-size: 10.5pt; font-family: Nunito; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: minor-bidi; color: #212529; background: white;"> A predictive framework for HCC was constructed based on the expression levels of five lncRNAs. Multivariate and univariate Cox regression analyses revealed that lncRNA signatures associated with senescence were independently correlated with an increased risk of HCC. Additionally, the nomogram also provides a more refined and sensitive model. Further investigation into the variations in immune cells and functions between the high-risk and low-risk groups was conducted. Subsequently, a qPCR experiment results revealed underexpression of AC068756.1, AC090578.1, AC145343.1, and LINC0022 in Huh7 and LM3 cells. In contrast, AP003392.4 did not exhibit a significant difference between Huh7 and control cells. </span><strong><span lang="EN-US" style="font-family: Nunito; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: minor-bidi; color: #212529; background: white;">Conclusion:</span></strong><span lang="EN-US" style="font-size: 10.5pt; font-family: Nunito; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: minor-bidi; color: #212529; background: white;"> The prognostic features and nomogram, consisting of five aging-related lncRNAs (AC068756.1, AC090578.1, AC145343.1, AP003392.4, and LINC00221), may be useful in predicting the overall survival of HCC.</span></p>

Real-Time PCR-Based Screening for Homozygous SMN2 Deletion Using Residual Dried Blood Spots.

The survival motor neuron 2 (SMN2) gene is a recognized modifier gene of spinal muscular atrophy (SMA). However, our knowledge about the role of SMN2-other than its modification of SMA phenotypes-is very limited. Discussions regarding the relationship between homozygous SMN2 deletion and motor neuron diseases, including amyotrophic lateral sclerosis, have been mainly based on retrospective epidemiological studies of the diseases, and the precise relationship remains inconclusive. In the present study, we first estimated that the frequency of homozygous SMN2 deletion was ~1 in 20 in Japan. We then established a real-time polymerase chain reaction (PCR)-based screening method using residual dried blood spots to identify infants with homozygous SMN2 deletion. This method can be applied to a future prospective cohort study to clarify the relationship between homozygous SMN2 deletion and motor neuron diseases. In our real-time PCR experiment, both PCR (low annealing temperatures) and blood (high hematocrit values and low white blood cell counts) conditions were associated with incorrect results (i.e., false negatives and positives). Together, our findings not only help to elucidate the role of SMN2, but also aid in our understanding of the pitfalls of current SMA newborn screening programs for detecting homozygous SMN1 deletions.

Full-Length Transcriptome Sequencing Analysis and Characterization of WRKY Transcription Factors Responsive to Cadmium Stress in Arabis paniculata.

Arabis paniculata is a newly discovered hyperaccumulator known for its ability to accumulate multiple metals. WRKY proteins play a significant role in plant responses to various stresses, including cadmium (Cd) stress. However, there is limited research on the molecular biology of Arabis paniculata, especially regarding the WRKY family. In this study, we conducted third-generation sequencing for functional annotation and structural analysis of Arabis paniculata. We obtained 41,196 high-quality isoforms from the full-length transcriptome, with an average length of 1043 bp. A total of 26,670 genes were predicted against NR, Swissprot, KOG, and KEGG databases. Functional comparison using the KOG database revealed excellent annotation in 25 functional categories, with general function prediction (1822 items) being the most predominant. MISA analysis identified 12,593 SSR loci, with single nucleotide repeats being the largest category (44.83% of the total). Moreover, our predictions provide insights into 20,022 coding sequences (CDS), 811 transcription factors, and 17,963 LncRNAs. In total, 34 WRKY gene sequences were identified in Arabis paniculata. Bioinformatics analysis revealed diverse numbers of amino acids in these WRKYs (113 to 545 aa), and a conserved WRKYGQK sequence within the N-terminus of the WRKY protein. Furthermore, all WRKYs were found to be localized in the nucleus. Phylogenetic analysis classified the WRKY genes into three categories: I (14 members), II (17 members), and III (3 members). Category II was subsequently divided into four sub-categories: II-a (8 members), II-b (1 member), II-c (1 member), and II-d (7 members). Our quantitative real-time polymerase chain reaction (qRT-PCR) experiments revealed that ApWRKY23 and ApWRKY34 exhibited the highest expression levels at the 24-h time point, suggesting their potential role as the candidate genes for Cd stress response. These findings contribute to our understanding of the genomic information of Arabis paniculata and provide a basis for the analysis of its genetic diversity. Additionally, this study paves the way for a comprehensive exploration of the molecular mechanisms underlying the WRKY genes in Arabis paniculata under Cd stress conditions.

Active compounds of Caodoukou () inhibit the migration, invasion and metastasis of human pancreatic cancer cells by targeting phosphoinosmde-3-kinase/ protein kinase B/mammalian target of rapamycin pathway.

To detect the effects of active compounds of Caodoukou () (ACAK) on the proliferation, migration and invasion of pancreatic cancer, and explain the possible molecular mechanism of ACAK interacting with these processes. Cell counting kit-8 method, cell scratch repair experiment, Transwell migration and invasion experiment, immunohistochemistry, western blot assay and real-time polymerase chain reaction experiment were used to evaluate the effect of ACAK on the proliferation, migration and invasion of pancreatic cancer cells. The levels of active molecules involved in the phosphoinosmde-3-kinase (PI3K)/Akt/the mammalian target of rapamycin (mTOR) signal transduction were detected by Western blot assay. In addition, the function of ACAK was evaluated by xenotransplantation tumor model in nude mice. The inhibitory effect of ACAK on the proliferation of pancreatic cancer cells showed certain time-dose dependence. The results of scratch repair test, Transwell test, Western blotting and real time polymerase chain reaction assay showed that ACAK could inhibit the migration and invasion of pancreatic cancer cells . In addition, the regulatory effect of ACAK on epithelial-mesenchymal transition (EMT) is partly attributed to PI3K/Akt/mTOR signaling pathway. The experimental results showed that ACAK regulated the development of pancreatic cancer. ACAK can partly inhibit the activity of EMT and matrix metallopeptidases by down-regulating the downstream proteins of PI3K/Akt/mTOR signal pathway, thus inhibiting the ability of migration and invasion of pancreatic cancer.

Deciphering the immunological and prognostic features of bladder cancer through platinum-resistance-related genes analysis and identifying potential therapeutic target P4HB.

To identify the molecular subtypes and develop a scoring system for the tumor immune microenvironment (TIME) and prognostic features of bladder cancer (BLCA) based on the platinum-resistance-related (PRR) genes analysis while identifying P4HB as a potential therapeutic target. In this study, we analyzed gene expression data and clinical information of 594 BLCA samples. We used unsupervised clustering to identify molecular subtypes based on the expression levels of PRR genes. Functional and pathway enrichment analyses were performed to understand the biological activities of these subtypes. We also assessed the TIME and developed a prognostic signature and scoring system. Moreover, we analyzed the efficacy of immune checkpoint inhibitors. Then we conducted real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) experiments to detect the expression level of prolyl 4-hydroxylase subunit beta (P4HB) in BLCA cell lines. Transfection of small interference ribonucleic acid (siRNA) was performed in 5637 and EJ cells to knock down P4HB, and the impact of P4HB on cellular functions was evaluated through wound-healing and transwell assays. Finally, siRNA transfection of P4HB was performed in the cisplatin-resistant T24 cell to assess its impact on the sensitivity of BLCA to platinum-based chemotherapy drugs. In a cohort of 594 BLCA samples (TCGA-BLCA, n=406; GSE13507, n=188), 846 PRR-associated genes were identified by intersecting BLCA expression data from TCGA and GEO databases with the PRR genes from the HGSOC-Platinum database. Univariate Cox regression analysis revealed 264 PRR genes linked to BLCA prognosis. We identified three molecular subtypes (Cluster A-C) and the PRR scoring system based on PRR genes. Cluster C exhibited a better prognosis and lower immune cell infiltration compared to the other Clusters A and B. The high PRR score group was significantly associated with an immunosuppressive tumor microenvironment, poor clinical-pathological features, and a poor prognosis. Furthermore, the high PRR group showed higher expression of immune checkpoint molecules and a poorer response to immune checkpoint inhibitors than the low PRR group. The key PRR gene P4HB was highly expressed in BLCA cell lines, and cellular functional experiments in vitro indicate that P4HB may be an important factor influencing BLCA migration and invasion. Our study demonstrates that the PRR signatures are significantly associated with clinical-pathological features, the TIME, and prognostic features. The key PRR gene, P4HB, s a biomarker for the individualized treatment of BLCA patients.

Genetic and histological relationship between pheromone-secreting tissues of the musk gland and skin of juvenile Chinese forest musk deer (Moschus berezovskii Flerov, 1929).

BACKGROUND: The musk glands of adult male Chinese forest musk deer (Moschus berezovskii Flerov, 1929) (FMD), which are considered as special skin glands, secrete a mixture of sebum, lipids, and proteins into the musk pod. Together, these components form musk, which plays an important role in attracting females during the breeding season. However, the relationship between the musk glands and skin of Chinese FMD remains undiscovered. Here, the musk gland and skin of Chinese FMD were examined using histological analysis and RNA sequencing (RNA-seq), and the expression of key regulatory genes was evaluated to determine whether the musk gland is derived from the skin. METHODS: A comparative analysis of musk gland anatomy between juvenile and adult Chinese FMD was conducted. Then, based on the anatomical structure of the musk gland, skin tissues from the abdomen and back as well as musk gland tissues were obtained from three juvenile FMD. These tissues were used for RNA-seq, hematoxylin-eosin (HE) staining, immunohistochemistry (IHC), western blot (WB), and quantitative real-time polymerase chain reaction (qRT-PCR) experiments. RESULTS: Anatomical analysis showed that only adult male FMD had a complete glandular organ and musk pod, while juvenile FMD did not have any well-developed musk pods. Transcriptomic data revealed that 88.24% of genes were co-expressed in the skin and musk gland tissues. Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analysis found that the genes co-expressed in the abdomen skin, back skin, and musk gland were enriched in biological development, endocrine system, lipid metabolism, and other pathways. Gene Ontology (GO) enrichment analysis indicated that the genes expressed in these tissues were enriched in biological processes such as multicellular development and cell division. Moreover, the Metascape predictive analysis tool demonstrated that genes expressed in musk glands were skin tissue-specific. qRT-PCR and WB revealed that sex-determining region Y-box protein 9 (Sox9),Caveolin-1 (Cav-1), andandrogen receptor (AR) were expressed in all three tissues, although the expression levels differed among the tissues. According to the IHC results, Sox9 and AR were expressed in the nuclei of sebaceous gland, hair follicle, and musk gland cells, whereas Cav-1 was expressed in the cell membrane. CONCLUSIONS: The musk gland of Chinese FMD may be a derivative of skin tissue, and Sox9, Cav-1, and AR may play significant roles in musk gland development.

Development and validation a prognostic model based on natural killer T cells marker genes for predicting prognosis and characterizing immune status in glioblastoma through integrated analysis of single-cell and bulk RNA sequencing.

Glioblastoma (GBM) is an aggressive and unstoppable malignancy. Natural killer T (NKT) cells, characterized by specific markers, play pivotal roles in many tumor-associated pathophysiological processes. Therefore, investigating the functions and complex interactions of NKT cells is great interest for exploring GBM. We acquired a single-cell RNA-sequencing (scRNA-seq) dataset of GBM from Gene Expression Omnibus (GEO) database. The weighted correlation network analysis (WGCNA) was employed to further screen genes subpopulations. Subsequently, we integrated the GBM cohorts from The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) databases to describe different subtypes by consensus clustering and developed a prognostic model by least absolute selection and shrinkage operator (LASSO) and multivariate Cox regression analysis. We further investigated differences in survival rates and clinical characteristics among different risk groups. Furthermore, a nomogram was developed by combining riskscore with the clinical characteristics. We investigated the abundance of immune cells in the tumor microenvironment (TME) by CIBERSORT and single sample gene set enrichment analysis (ssGSEA) algorithms. Immunotherapy efficacy assessment was done with the assistance of Tumor Immune Dysfunction and Exclusion (TIDE) and The Cancer Immunome Atlas (TCIA) databases. Real-time quantitative polymerase chain reaction (RT-qPCR) experiments and immunohistochemical profiles of tissues were utilized to validate model genes. We identified 945 NKT cells marker genes from scRNA-seq data. Through further screening, 107 genes were accurately identified, of which 15 were significantly correlated with prognosis. We distinguished GBM samples into two distinct subtypes and successfully developed a robust prognostic prediction model. Survival analysis indicated that high expression of NKT cell marker genes was significantly associated with poor prognosis in GBM patients. Riskscore can be used as an independent prognostic factor. The nomogram was demonstrated remarkable utility in aiding clinical decision making. Tumor immune microenvironment analysis revealed significant differences of immune infiltration characteristics between different risk groups. In addition, the expression levels of immune checkpoint-associated genes were consistently elevated in the high-risk group, suggesting more prominent immune escape but also a stronger response to immune checkpoint inhibitors. By integrating scRNA-seq and bulk RNA-seq data analysis, we successfully developed a prognostic prediction model that incorporates two pivotal NKT cells marker genes, namely, CD44 and TNFSF14. This model has exhibited outstanding performance in assessing the prognosis of GBM patients. Furthermore, we conducted a preliminary investigation into the immune microenvironment across various risk groups that contributes to uncover promising immunotherapeutic targets specific to GBM.

A new marker constructed from immune-related lncRNA pairs can be used to predict clinical treatment effects and prognosis: in-depth exploration of underlying mechanisms in HNSCC

BackgroundLong non-coding RNA (lncRNA) plays a vital role in tumor proliferation, migration, and treatment. Since it is challenging to standardize the gene expression levels detected by different platforms, the signatures composed of many immune-related single lncRNAs are still inaccurate. Utilizing a gene pair formed of two immune-related lncRNAs and strategically assigning values can effectively meet the demand for a higher-accuracy dual biomarker combination.MethodsCo-expression and differential expression analyses were performed on immune genes and lncRNAs data from The Cancer Genome Atlas and the ImmPort database to obtain differentially expressed immune-related lncRNAs for pairwise pairing. The prognostic-related differentially expressed immune-related lncRNAs (PR-DE-irlncRNAs) pairs were then identified by univariate Cox regression and used for lasso regression to construct a prognostic model. Various methods were used to validate the predictive prognostic performance of the model. Additionally, we explored the potential guiding value of the model in immunotherapy and chemotherapy and constructed a nomogram suitable for efficient prognosis prediction. Mechanistic exploration of anti-tumor immunity and mutational perspectives are also included. We also analyzed the correlation between the model and immune checkpoint inhibitors (ICIs)-related, N6-methyadenosine (m6A)-related, and multidrug resistance genes.ResultsWe used a total of 20 pairs of PR-DE-irlncRNAs to create a prognosis model. Quantitative real-time polymerase chain reaction experiments further verified the abnormal expression of 11 lncRNAs in HNSCC cells. Various methods have confirmed the excellent performance of the model in predicting patient prognosis. We reasoned that lncRNAs/TP53 mutation might play a positive/negative anti-tumor role through the immune system by multi-perspective analyses. Finally, it was found that the prognostic model was closely related to immunotherapy and chemotherapy as well as the expression of ICIs/m6A/multidrug resistance-related genes.ConclusionThe prognostic model performs excellently in predicting the prognosis of patients and provides the potential value of practical guidance for treatment.

E3 ubiquitin ligase PbrATL18 is a positive factor in pear resistance to drought and Colletotrichum fructicola infection

The Arabidopsis Tóxicos en Levadura (ATL) protein is a subfamily of the E3 ubiquitin ligases, which exists widely in plants and is extensively involved in plant growth and development. Although the ATL family has been identified in other species, such as Arabidopsis, Oryza sativa, and grapevine, few reports on pear ATL gene families have been reported. In this study, 92 PbrATL genes were identified and analyzed from the Pyrus breschneideri genome. Motif analysis and phylogenetic tree generation divided them into nine subgroups, and chromosome localization analysis showed that the 92 PbrATL genes were distributed in 16 of 17 pear chromosomes. Transcriptome data and quantitative real-time polymerase chain reaction (qRT-PCR) experiments demonstrated that PbrATL18, PbrATL41, and PbrATL88 were involved in both pear drought resistance and Colletotrichum fructicola infection. In addition, Arabidopsis thaliana overexpressing PbrATL18 showed greater resistance to drought stress than the wild type (WT), and PbrATL18-silenced pear seedlings showed greater sensitivity to drought and C. fructicola infection than the controls. PbrATL18 regulated plant resistance by regulating chitinase (CHI), phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO), catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) activities. This study provided a reference for further exploring the functions of the PbrATL gene in drought resistance and C. fructicola infection.

Effect of NETs/COX-2 pathway on immune microenvironment and metastasis in gastric cancer.

Neutrophil extracellular traps (NETs) are crucial in the progression of several cancers. The formation of NETs is closely related to reactive oxygen species (ROS), and the granule proteins involved in nucleosome depolymerization under the action of ROS together with the loosened DNA compose the basic structure of NETs. This study aims to investigate the specific mechanisms of NETs promoting gastric cancer metastasis in order to perfect the existing immunotherapy strategies. In this study, the cells and tumor tissues of gastric cancer were detected by immunological experiments, real-time polymerase chain reaction and cytology experiments. Besides, bioinformatics analysis was used to analyze the correlation between cyclooxygenase-2 (COX-2) and the immune microenvironment of gastric cancer, as well as its effect on immunotherapy. Examination of clinical specimens showed that NETs were deposited in tumor tissues of patients with gastric cancer and their expression was significantly correlated with tumor staging. Bioinformatics analysis showed that COX-2 was involved in gastric cancer progression and was associated with immune cell infiltration as well as immunotherapy. In vitro experiments, we demonstrated that NETs could activate COX-2 through Toll-like receptor 2 (TLR2) and thus enhance the metastatic ability of gastric cancer cells. In addition, in a liver metastasis model of nude mice we also demonstrated the critical role of NETs and COX-2 in the distant metastasis of gastric cancer. NETs can promote gastric cancer metastasis by initiating COX-2 through TLR2, and COX-2 may become a target for gastric cancer immunotherapy.

Intestinal epithelial cell-specific deletion of cytokine-inducible SH2- containing protein alleviates experimental colitis in ageing mice.

The incidence of inflammatory bowel disease (IBD) in the elderly has increased in recent years. However, the mechanisms underlying the ageing-related IBD susceptibility remain elusive. Cytokine-inducible SH2-containing protein (CISH) involved in regulating metabolism, the expansion of intestinal tuft cells and type-2 innate lymphoid cells, and ageing-related airway inflammation. Here, we investigated the role of CISH in ageing-related colitis susceptibility. CISH and phosphorylated signal transducer and activator of transcription-3 (p-STAT3) levels were evaluated in the colons of ageing mice and older ulcerative colitis (UC) patients. Mice with intestinal epithelial cell-specific knockout of Cish (CishΔIEC) and Cish-floxed mice were administered dextran sodium sulfate (DSS) or trinitrobenzene sulfonic acid (TNBS) to induce colitis. Colonic tissues were analysed in quantitative real-time polymerase chain reaction, immunoblotting, immunohistochemical, and histological staining experiments. Differentially expressed genes from colonic epithelia were analysed by RNA-sequencing. Ageing increased the severity of DSS-induced colitis and the expression of colonic epithelial CISH in mice. CishΔIEC prevented against the DSS or TNBS-induced colitis in middle-aged mice but not, in the young mice. RNA-sequencing analysis revealed that CishΔIEC significantly suppressed DSS-induced oxidative stress and proinflammatory responses. During ageing CCD841 cell model, knockdown of CISH decreased ageing-induced oxidative stress and proinflammatory responses, whereas these effects were compromised by knocking down or inhibiting STAT3. The increase in CISH expression was higher in the colonic mucosa of older patients with UC than that in healthy controls. CISH might be a proinflammatory regulator in ageing; therefore, targeted therapy against CISH may provide a novel strategy for treating ageing-related IBD.

An integrated strategy to explore the wine-processed mechanism of Corni Fructus on chronic renal failure based on metabolomics, network analysis and bioinformatics approaches.

Corni Fructus is one of the most famous traditional Chinese medicines (TCMs) for the treatment of various chronic kidney diseases. Wine-processed Corni Fructus (WCF) is the main processed form of Crude Corni Fructus (CCF). In this study, potential mechanisms of action of CCF and WCF on chronic renal failure (CRF) model were developed to explore wine-processed mechanism of Corni Fructus. An integrated strategy combining metabolomics, network analysis and bioinformatics analysis has been established to investigate the therapeutic mechanisms of WCF and CCF in rats with CRF. The histopathological results showed that both WCF and CCF improved kidney injury and dysfunction of CRF rats, but WCF was more effective than CCF. Metabolic pathway analysis indicated that 24 metabolites and 5 major disturbed pathways associated with CCF, while WCF regulated 27 metabolites and 2 metabolic pathways. Bioinformatic analysis and network analysis revealed that 8 genes and 7 genes were regulated by CCF and WCF on CRF rats, respectively. The quantitative real-time polymerase chain reaction experiments verified the regulatory ability of CCF and WCF on the expression of 4 genes. An integrated strategy combined metabolomics, network analysis and bioinformatics was established to provide valuable holistic insight to explore the processing mechanism of TCMs.

Identification of an unrecognized circRNA associated with development of renal fibrosis.

Backgroud: Renal fibrosis is the common characteristic of chronic kidney disease. Circular RNA plays an essential role in the occurrence and development of Renal fibrosis, but its regulative mechanism remains elusive. Methods: The animal and cell model of Renal fibrosis was established, and RNA-sequencing and real-time polymerase chain reaction (qRT-PCR) experiments were implemented. Subsequently, experiments for detecting apoptosis and proliferation of cell, were carried out, and the isobaric tags for relative and absolute quantification proteomics analyses were performed accordingly. Results: It was found that a newly discovered Circular RNA (circRNA_0002158), is highly expressed in kidneys or cells with fibrosis, implying that this Circular RNA might be associated with the occurrence and development of Renal fibrosis. Subsequently, the overexpression and knockdown of circRNA_0002158 were conducted in the human kidney epithelial cell line (HK-2) cells, and the results indicated that the circRNA_0002158 could inhibit apoptosis, and promote proliferation of cells. The kidney injury-related factors, including Fibronectin and plasminogen activator inhibitor-1 (PAI-1), were decreased in HK-2 cells with overexpression of circRNA_0002158, while the results were reversed in cells with knockdown of circRNA_0002158. Finally, to explore the regulative mechanism of circRNA_0002158, the iTRAQ proteomics analyses were implemented for the cell samples with OE of circRNA_0002158 and its control, it showed that multiple genes and functional pathways were associated with the occurrence and development of Renal fibrosis. Conclusion: CircRNA_0002158 is associated with regulating Renal fibrosis, and may contribute to ameliorating the progression of Renal fibrosis in the future.