Bioinformatics Methods Research Articles

Pan-cancer Analysis Combined with Experiments Deciphers PHB Regulation for Breast Cancer Cell Survival and Predicts Biomarker Function.

Breast carcinoma has become the leading fatal disease among women. The location of prohibitin in the chromosome is close to the breast cancer susceptibility gene 1 (BRCA1). Accumulated research reported that prohibitin could interact with a variety of transcription factors and cell cycle-regulating proteins. This present study aims to comprehensively explore and reveal the biological functions of prohibitin on breast cancer via The Cancer Genome Atlas (TCGA) and validation experiment in vitro. Exploring the expression level of prohibitin across 27 tumors based on the TGGA database by bioinformatic methods and its relationship with tumor immune infiltration. Furthermore, we thus analyzed the biological roles of prohibitin on human breast cancer cell line MCF- 7 with pEGFP-prohibitin overexpression plasmid by western blotting and transwell-assay. Firstly, we found prohibitin is overexpressed in most tumors based on The Cancer Genome Atlas database, and the negative relationships between prohibitin and tumors infiltrating lymphocytes including B lymphocyte, CD4 T lymphocyte, CD8 T lymphocyte, Neutrophil, Macrophage and Dendritic, and its significant correlation with the prognosis of human cancer. In vitro, expression not only inhibited cell viability and invasive abilities but also increased the apoptosis percentage of cells with a decreased percentage of the S phase and an increased G2 phase. The reduction of Bcl-2 was observed when prohibitin was upregulated, although the expression of E2F-1 did not change. Although prohibitin is over-expressed in various cancer types, it functions as an important tumor suppressor that may suppress breast cancer cell proliferation and the invasive ability of MCF-7 by influencing its DNA synthesis and promoting cell apoptosis. All these may be likely associated with P53, erbB-2, and Bcl-2.

Molecular characterization of PANoptosis-related genes in chronic kidney disease.

Chronic kidney disease (CKD) is characterized by fibrosis and inflammation in renal tissues. Several types of cell death have been implicated in CKD onset and progression. Unlike traditional forms of cell death, PANoptosis is characterized by the crosstalk among programmed cell death pathways. However, the interaction between PANoptosis and CKD remains unclear. Here, we used bioinformatics methods to identify differentially expressed genes and differentially expressed PANoptosis-related genes (DE-PRGs) using data from the GSE37171 dataset. Following this, we further performed gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, and gene set enrichment analysis using the data. We adopted a combined approach to select hub genes, using the STRING database and CytoHubba plug-in, and we used the GSE66494 as a validation dataset. In addition, we constructed ceRNA, transcription factor (TF)-gene, and drug-gene networks using Cytoscape. Lastly, we conducted immunohistochemical analysis and western blotting to validate the hub genes. We identified 57 PANoptosis-associated genes as DE-PRGs. We screened nine hub genes from the 57 DE-PRGs. We identified two hub genes (FOS and PTGS2) using the GSE66494 database, Nephroseq, immunohistochemistry, and western blotting. A common miRNA (Hsa-miR-101-3p) and three TFs (CREB1, E2F1, and RELA) may play a crucial role in the onset and progression of PANoptosis-related CKD. In our analysis of the drug-gene network, we identified eight drugs targeting FOS and 52 drugs targeting PTGS2.

DLAT promotes triple-negative breast cancer progression via YAP1 activation

ABSTRACT Background Breast cancer (BC) is the most prevalent malignant tumor in women globally. Triple-negative breast cancer (TNBC) represents the most malignant and invasive subtype of BC. New therapeutic targets are urgently needed for TNBC owing to its receptor expression characteristics, which render it insensitive to traditional targeted and endocrine therapies for BC. The role and mechanisms of dihydrolipoamide S-acetyltransferase (DLAT) as a crucial molecule in glycometabolism and cuproptosis-related biological processes in tumors remain to be explored. Methods DLAT expression was investigated using bioinformatics methods and quantitative real-time polymerase chain reaction. Subsequently, the MTT assay, colony formation assay, and migration-invasion assay were performed to validate the effect of DLAT on TNBC cell viability, proliferation, and migration. Cytoplasmic-nuclear separation experiments, western blot analysis, and co-immunoprecipitation assays were performed to elucidate the underlying molecular mechanisms. Results This study revealed a robust correlation between elevated DLAT expression in BC and unfavorable prognosis in patients, with higher expression of DLAT compared to other subtypes in TNBC. Functional cytology experiments indicated that DLAT plays a tumor-promoting role in TNBC. Mechanistic studies showed that DLAT directly interacts with YAP1, leading to the dephosphorylation and activation of YAP1 and its increased nuclear translocation, thereby transcriptionally activating and regulating downstream oncogenes, promoting the malignant phenotype of TNBC. Rescue experiments indicated that DLAT promotes the malignant behavior of TNBC through a YAP1-dependent pathway. Conclusions Our research unveiled the significant involvement of DLAT in TNBC, along with the potential for modulating DLAT/YAP1 activity as a targeted treatment strategy for TNBC.

Identification of the HbZAR1 Gene and Its Potential Role as a Minor Gene in Response to Powdery Mildew and Anthracnose of Hevea brasiliensis

Powdery mildew and anthracnose are the main diseases of rubber trees. In recent years, there have been large outbreaks in the rubber-planting areas of Asia, seriously affecting the yield and quality of rubber latex. ZAR1 is a conserved and distinctive coiled-coil nucleotide-binding leucine-rich (CNL) repeat in the plant kingdom, playing a crucial role in disease-resistance processes. To elucidate the function of the HbZAR1 gene in rubber trees (Hevea brasiliensis), three candidate HbZAR1 genes were identified using bioinformatics methods and comprehensively analyzed. The results indicate that the HbZAR1 protein is conserved in different plant species. Examination of cis-regulatory element sequences of HbZAR1genes reveals that the HbZAR1 gene promoter exhibits a remarkable enrichment of stress, light, and hormone elements. An expression analysis shows that the expression levels of the three HbZAR1 genes are highest in the bark and lowest in latex. Three HbZAR1 genes can respond to both rubber tree Erysiphe quercicola and Colletotrichum siamense infection; especially, HbZAR1.1 and HbZAR1.2 show significant upregulation in expression levels during the early stages of infection. These findings suggest that the three HbZAR1 genes may be involved in rubber tree susceptibility to E. quercicola and C. siamense through different immune mechanisms. Subcellular localization results indicate that the HbZAR1 genes are expressed in the nucleus and plasma membrane. This study also shows that the three HbZAR1 genes and activated mutant HbZAR1.1D481V do not induce stable ROS production and cell death, suggesting possible gene degradation, functional redundancy, or acting as minor genes in disease resistance. This research provides valuable insights for further studying the function of HbZAR1 genes in rubber trees and the mechanisms of immune molecules.

Identification and validation of a prognostic model based on three TLS-Related genes in oral squamous cell carcinoma

BackgroundThe tertiary lymphoid structures (TLSs) have an immunomodulatory function and have a positive impact on the survival outcomes of patients with oral squamous cell carcinoma (OSCC). However, there is a lack of standard approaches for quantifying TLSs and prognostic models using TLS-related genes (TLSRGs). These limitations limit the widespread use of TLSs in clinical practice.MethodsA convolutional neural network was used to automatically detect and quantify TLSs in HE-stained whole slide images. By employing bioinformatics and diverse statistical methods, this research created a prognostic model using TCGA cohorts and explored the connection between this model and immune infiltration. The expression levels of three TLSRGs in clinical specimens were detected by immunohistochemistry. To facilitate the assessment of individual prognostic outcomes, we further constructed a nomogram based on the risk score and other clinical factors.ResultsTLSs were found to be an independent predictor of both overall survival (OS) and disease-free survival in OSCC patients. A larger proportion of the TLS area represented a better prognosis. After analysis, we identified 69 differentially expressed TLSRGs and selected three pivotal TLSRGs to construct the risk score model. This model emerged as a standalone predictor for OS and exhibited close associations with CD4 + T cells, CD8 + T cells, and macrophages. Immunohistochemistry revealed high expression levels of CCR7 and CXCR5 in TLS + OSCC samples, while CD86 was highly expressed in TLS- OSCC samples. The nomogram demonstrates excellent predictive ability for overall survival in OSCC patients.ConclusionsThis is the first prognostic nomogram based on TLSRGs, that can effectively predict survival outcomes and contribute to individual treatment strategies for OSCC patients.

Association of Spexin Hormone Levels with Metabolic Disturbance in Women with Polycystic Ovarian Syndrome

Background: Polycystic ovarian syndrome is affecting around 5–15% of females. Spexin hormone, identified as neuropeptide Q, has been newly recognized by bioinformatics methods. Objective: To evaluate the relationship concerning levels of spexin hormone with metabolic disruption in women with polycystic ovarian syndrome. Patients and Methods: A case-control study carried out in the department of Obstetrics and Gynecology in Al-Imamain Al-Khademain medical city/Baghdad, Iraq, from Jan 1, 2022, to the end of Dec 31, 2022. The study sample comprises, 192 participants aged 18-45 years were joined and allocated into a case group (96 women with PCOS) and a control group (96 women without PCOS). Results: Mean value of spexin hormone was (2.7±0.3 ng/mL) in the PCOS group, while it was (3.5±0.7 ng/mL) in the control group; fasting blood sugar shows significant association with a negative, weak correlation with Spexin in patients (P-value= 0.005), Insulin shows significant association with inverse correlation with Spexin in patients (P-value= 0.04), Homeostasis model valuation of insulin resistance (HOMA-IR) shows significant association with inverse correlation with Spexin in patients (P-value= 0.003). Spexin had a significant inverse correlation with LH, SHBG, testosterone, FAI, and Dehydroepiandrosterone. Conclusion: Serum level of spexin hormone was meaningfully decreased in patients with PCOS than that in healthy women.

Gene polymorphisms of miR-323b and miR-1343 that regulate kininogen L are associated with schizophrenia susceptibility: A preliminary population‑based study.

miR-SNP is a type of functional single nucleotide polymorphism (SNP) that affects the regulatory functions of miRNA genes, miRNA binding sites, or components of miRNA biogenesis. This study aimed to explore the relationship between miRNA gene polymorphisms that regulate the kininogen L protein and schizophrenia (SCZ). Bioinformatics methods predicted miRNA gene polymorphism sites regulating the kininogen L protein. The polymorphisms of rs56103835, rs6513496, rs651349, and rs2986407 were detected using imLDR multiple SNP typing technologies in 513 SCZ patients and 509 controls. The association of miR-SNP variations with SCZ susceptibility and symptoms was evaluated using SNPstat to determine the optimal inheritance model. Generalized multifactor dimensionality reduction (GMDR) analysis and logistic regression were used to calculate miR-SNP interactions. The association between hsa-miR-323b-rs56103835 and SCZ was statistically significant under the dominant model. The result of gene-gene interaction showed that the three-factor model (rs56103835 / rs2986407 / rs2155248) was the best, but it could not be considered significantly related to SCZ. Additionally, SCZ patients with the CC or CT genotype on rs2986407 were more likely to experience auditory hallucinations than those with the TT genotype. Our data revealed that the mutation of hsa-miR-323b-rs56103835 from C to T was associated with susceptibility to SCZ. The mutation of hsa-miR-1343-rs2986407 from T to C increases the risk of auditory hallucinations in SCZ patients.

Overexpression of miR-451a Aggravates Renal Ischemia–Reperfusion Injury by Targeting KLF1-ACSL4 to Promote Ferroptosis

Ischemia–reperfusion injury (IRI) is a predominant factor leading to delayed graft function (DGF) following kidney transplantation. MicroRNAs (miRNAs) play a pivotal role in the pathogenesis of renal IRI, with ferroptosis being a critical driving force throughout the process. In this study, we utilized bioinformatics methods to construct a network diagram of differentially expressed miRNAs, transcription factors (TFs), and ferroptosis-related genes. An I/R-induced renal injury model in mice and an in vitro H/R-induced HK-2 cell injury model were established. Quantitative real-time PCR (qRT-PCR) and Western blot analysis were used to measure the mRNA and miRNA levels in cells and tissues. The MDA concentration, iron levels, and GSH concentration were measured to evaluate the ferroptosis levels. CCK-8 assays were performed to assess cell viability. Luciferase reporter assays were conducted to validate the downstream targets of miRNA, and chromatin immunoprecipitation assays were performed to verify the interaction between TFs and mRNAs. Both the in vivo and in vitro results demonstrate that miR-451a was significantly enriched in the IRI renal tissues and cells, exacerbating ferroptosis. MiR-451a was found to reduce the expression of Kruppel-like factor 1 (KLF1) by directly binding to the 3′UTR of KLF1 mRNA. Additionally, KLF1 was identified as a negative transcription factor for acyl-CoA synthetase long-chain family member 4 (ACSL4). We demonstrated that IRI induced the upregulation of miR-451a, which reduced KLF1 expression, thereby promoting ferroptosis by upregulating ACSL4 expression, ultimately aggravating IRI-induced renal damage.

Bioinformatics design of peptide binding to the human cardiac troponin I (cTnI) in biosensor development for myocardial infarction diagnosis.

Cardiovascular disease has reached a mortality rate of 470,000 patients each year. Myocardial infarction accounts for 49.2% of these deaths, and the cTnI protein is a crucial target in diagnosing myocardial infarction. A peptide-based bioreceptor design using a computational approach is a good candidate to be developed for a rapid, effective, and selective detection method for cTnI although it is still lacking in study. Hence, to address the scientific gap, we develop a new candidate peptide for the cTnI biosensor by bioinformatics method and present new computational approaches. The sequential point mutations were made to the selected peptide to increase its stability and affinity for cTnI. Next, molecular docking was performed to select the mutated peptide, and one of the best results was subjected to the molecular dynamics simulation. Finally, the results showed that the best peptide showed the lowest affinity and good stability among other mutated peptide designs for interacting with the cTnI protein. In addition, the peptide has been tested to have a higher specificity towards cTnI than its major isomer, sTnI, through molecular docking and molecular dynamics simulation. Therefore, the peptide is considered a good potential bioreceptor for diagnosing myocardial infarction diseases.

Genome-Wide Identification and Expression Analysis of the Melon Aldehyde Dehydrogenase (ALDH) Gene Family in Response to Abiotic and Biotic Stresses

Through the integration of genomic information, transcriptome sequencing data, and bioinformatics methods, we conducted a comprehensive identification of the ALDH gene family in melon. We explored the impact of this gene family on melon growth, development, and their expression patterns in various tissues and under different stress conditions. Our study discovered a total of 17 ALDH genes spread across chromosomes 1, 2, 3, 4, 5, 7, 8, 11, and 12 in the melon genome. Through a phylogenetic analysis, these genes were classified into 10 distinct subfamilies. Notably, genes within the same subfamily exhibited consistent gene structures and conserved motifs. Our study discovered a pair of fragmental duplications within the melon ALDH gene. Furthermore, there was a noticeable collinearity relationship between the melon’s ALDH gene and that of Arabidopsis (12 times), and rice (3 times). Transcriptome data reanalysis revealed that some ALDH genes consistently expressed highly across all tissues and developmental stages, while others were tissue- or stage-specific. We analyzed the ALDH gene’s expression patterns under six stress types, namely salt, cold, waterlogged, powdery mildew, Fusarium wilt, and gummy stem blight. The results showed differential expression of CmALDH2C4 and CmALDH11A3 under all stress conditions, signifying their crucial roles in melon growth and stress response. RT-qPCR (quantitative reverse transcription PCR) analysis further corroborated these findings. This study paves the way for future genetic improvements in melon molecular breeding.

Genomic Characterization and Probiotic Properties of Lactiplantibacillus pentosus Isolated from Fermented Rice.

The aim of the study was the preliminary genetic and phenotypic characterization of a potential probiotic strain of Lactiplantibacillus pentosus (strain krglsrbmofpi2) obtained from traditionally fermented rice. Genome sequencing revealed that the strain has a 3.7-Mb genome with a GC content of 46 and a total of 3192 protein-coding sequences. Using bioinformatic methods, we have successfully identified phage genes, plasmids, pathogenicity, antibiotic resistance and a variety of bacteriocins. Through comprehensive biochemical and biophysical analyses, we have gained valuable insights into its auto-aggregation, co-aggregation, antibiotic resistance, hydrophobicity, antioxidant activity and tolerance to simulated gastrointestinal conditions. The safety evaluation of the isolated L. pentosus was performed on the basis of its haemolytic activity. Our studies have shown that this strain has a strong antagonistic activity against the priority pathogens identified by the World Health Organization such as Vibrio cholerae, Clostridium perfringens, Salmonella enterica subsp. enterica ser. Typhi, Escherichia coli, Listeria monocytogenes and Staphylococcus aureus. It is essential to fully understand the genetic and functional properties of the L. pentosus strain before considering its use as a useful probiotic in the food industry.

Exploring the Blood Biomarkers and Potential Therapeutic Agents for Human Acute Mountain Sickness Based on Transcriptomic Analysis, Inflammatory Infiltrates and Molecular Docking.

A high-altitude, low-pressure hypoxic environment has severe effects on the health and work efficiency of its residents, and inadequate preventive measures and adaptive training may lead to the occurrence of AMS. Acute exposure to hypoxia conditions can have a less-favorable physiological effect on the human immune system. However, the regulation of the immune system in high-altitude environments is extremely complex and remains elusive. This study integrated system bioinformatics methods to screen for changes in immune cell subtypes and their associated targets. It also sought potential therapeutically effective natural compound candidates. The present study observed that monocytes, M1 macrophages and NK cells play a crucial role in the inflammatory response in AMS. IL15RA, CD5, TNFSF13B, IL21R, JAK2 and CXCR3 were identified as hub genes, and JAK2 was positively correlated with monocytes; TNFSF13B was positively correlated with NK cells. The natural compound monomers of jasminoidin and isoliquiritigenin exhibited good binding affinity with JAK2, while dicumarol and artemotil exhibited good binding affinity with TNFSF13B, and all are expected to become a potential therapeutic agents.

From Diabetes to Dementia: Identifying Key Genes in the Progression of Cognitive Impairment.

To provide a basis for further research on the molecular mechanisms underlying type 2 diabetes-associated mild cognitive impairment (DCI) using two bioinformatics methods to screen key genes involved in the progression of mild cognitive impairment (MCI) and type 2 diabetes. RNA sequencing data of MCI and normal cognition groups, as well as expression profile and sample information data of clinical characteristic data of GSE63060, which contains 160 MCI samples and 104 normal samples, were downloaded from the GEO database. Hub genes were identified using weighted gene co-expression network analysis (WGCNA). Protein-protein interaction (PPI) analysis, combined with least absolute shrinkage and selection operator (LASSO) and receiver operating characteristic (ROC) curve analyses, was used to verify the genes. Moreover, RNA sequencing and clinical characteristic data for GSE166502 of 13 type 2 diabetes samples and 13 normal controls were downloaded from the GEO database, and the correlation between the screened genes and type 2 diabetes was verified by difference and ROC curve analyses. In addition, we collected clinical biopsies to validate the results. Based on WGCNA, 10 modules were integrated, and six were correlated with MCI. Six hub genes associated with MCI (TOMM7, SNRPG, COX7C, UQCRQ, RPL31, and RPS24) were identified using the LASSO algorithm. The ROC curve was screened by integrating the GEO database, and revealed COX7C, SNRPG, TOMM7, and RPS24 as key genes in the progression of type 2 diabetes. COX7C, SNRPG, TOMM7, and RPS24 are involved in MCI and type 2 diabetes progression. Therefore, the molecular mechanisms of these four genes in the development of type 2 diabetes-associated MCI should be studied.

Mask wearing impacts skin barrier function and microbiome profile in sensitive skin

Mask-wearing behavior, common in the post-COVID-19 era, raises concerns for sensitive skin. This split-face study investigated mask-related changes in skin barrier function and microbiome composition among 30 female volunteers with sensitive skin and assessed the mitigating effects of a moisturizer containing biological lipids and probiotics. Skin physiological indicators (transepidermal water loss, erythema index, stratum corneum hydration, pH, temperature) of masked and unmasked areas were collected at baseline, after three hours of mask-wearing, post-tape stripping, and after 24 h, respectively. Microbiome samples collected from the masked areas before and after wearing a medical mask were analyzed with bioinformatics methods. Mask-wearing significantly weakened barrier function in both masked and adjacent unmasked areas, while reducing bacterial diversity. It was also associated with an increase in Cutibacterium (P = 0.110) and decreases in Streptococcus (P = 0.032) and Prevotella (P = 0.026) abundance. Moisturizer application prior to mask-wearing significantly reduced transepidermal water loss and erythema (both P < 0.001) and further improved erythema after 24 h (P = 0.048). These findings demonstrate that mask-wearing can disrupt the skin barrier and microbiome in individuals with sensitive skin and applying a moisturizer beforehand can mitigate mask-related discomforts by aiding barrier repair and reducing sensitivity.

Understanding the relationship between inflammation, apoptosis, and diabetes osteoporosis: A bioinformatics approach and experimental verification.

Diabetes osteoporosis (DOP) is a chronic metabolic bone disease. This study aimed to identify potential biomarkers of DOP and explore their underlying mechanisms through bioinformatics methods and experimental verification. Bioinformatics methods were used to identify differentially expressed genes (DEGs) for DOP based on GEO data and the GeneCards database. GO and KEGG enrichment analyses were used to search the key pathways. The STRING website was used to construct a protein-protein interaction (PPI) network and identify key genes. Then, 50 mg/mL glucose was used to interveneosteoblasts (OBs).CCK-8 and Alizarin Red staining were used to investigate the proliferation and differentiation changes in OBs. Flowcytometry was used to investigate apoptosis. The membrane protein chip, WB, and RT-PCR were used to verify the expression of key targets or pathways about DOP. Forty-two common genes were screened between DOP-related targets and DEGs. GO and KEGG enrichment analysis showed that DOP was mainly associated with cytokine-cytokine receptor interactions, and apoptosis. PPI network analysis showed that TNF, IL1A, IL6, IL1B, IL2RA, Fas ligand (FASLG), and Fas cell surface death receptor (FAS) were key up-regulated genes in the occurrence of DOP. The experiment results show that 50 mg/mL glucose significantly inhibited OBs proliferation but presented an increase in apoptosis. Membrane protein chip, WB, and RT-PCR-verified a significantly active in the expression of TNF/FASLG/FAS pathway. High glucose activated the TNF-α/FAS/FASLG pathway and induced the inflammatory microenvironment and apoptosis, then impaired osteogenic differentiation of OBs. These may be an important mechanism for the occurrence and development of DOP.

Innovation in cancer pharmacotherapy through integrative consideration of germline and tumor genomes.

Precision cancer medicine is widely established, and numerous molecularly targeted drugs for various tumor entities are approved or in development. Personalized pharmacotherapy in oncology has so far been based primarily on tumor characteristics, e.g., somatic mutations. However, the response to drug treatment also depends on pharmacological processes summarized under the term ADME (absorption, distribution, metabolism, and excretion). Variations in ADME genes have been the subject of intensive research for more than five decades, considering individual patients' genetic makeup, referred to as pharmacogenomics (PGx). The combined impact of a patient's tumor and germline genome is only partially understood and often not adequately considered in cancer therapy. This may be attributed, in part, to the lack of methods for combined analysis of both data layers. Optimized personalized cancer therapies should, therefore, aim to integrate molecular information about the tumor and the germline, taking into account existing PGx guidelines for drug therapy. Moreover, such strategies should provide the opportunity to consider genetic variants of previously unknown functional significance. Bioinformatic analysis methods and corresponding algorithms for data interpretation need to be developed to consider PGx data in interdisciplinary molecular tumor boards, where cancer patients are discussed to provide evidence-based recommendations for clinical management based on individual tumor profiles. Significance Statement The era of personalized oncology has seen the emergence of drugs tailored to genetic variants associated with cancer biology. However, full potential of targeted therapy remains untapped due to the predominant focus on acquired tumor-specific alterations. Optimized cancer care must integrate tumor and patient genomes, guided by pharmacogenomic principles. An essential prerequisite for realizing truly personalized drug treatment of cancer patients is the development of bioinformatic tools for comprehensive analysis of all data layers generated in modern precision oncology programs.

Identification of necroptosis-related gene expression and the immune response in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a common reproductive and endocrine disorder; however, the understanding of the pathogenesis of PCOS remains unclear. Necroptosis is a newly discovered mechanism of cell death, and it is closely related to reproductive endocrine-related diseases. This study aimed to investigate the hub necroptosis-related genes in PCOS patients and its correlation with immune cell infiltration by bioinformatics methods. The gene expression chip result matrix and the annotation matrix files of the GSE34526, GSE8157, and GSE5090 datasets were downloaded from the GEO database. We analyzed the expression and correlation of the necroptosis-related genes in all samples, constructed a diagnostic model based on all necroptosis-related genes and genes with significant differences, performed unsupervised clustering of samples and gene enrichment analysis, and evaluated the correlations between the hub gene and immune cell infiltration levels by the R packages GSVA and CIBERSORT. Finally, PPI networks were constructed using the Cytoscape software GeneMANIA plug-in, and the miRNA, transcription factors, RBP, and drugs were predicted. Necroptosis-related genes have important relationships in the development of PCOS and are potentially associated with immune infiltration in PCOS patients.

In silico and in vitro evaluation of a PE38 and Nb-based recombinant immunotoxin targeting the GRP78 receptor in cancer cells.

Cancer is a global health problem despite the most developed therapeutic modalities. The delivery of specific therapeutic agents to a target increases the effectiveness of cancer treatment by reducing side effects and post-treatment issues. Our aim in this study was to design a recombinant protein consisting of nanobody molecules and exotoxin that targets the surface GRP78 receptor on tumor cells. Bioinformatics methods make drug design and recombinant protein evaluation much easier before the laboratory steps. Two constructs were designed from a single-variable domain on heavy chain nanobody domains and PE toxin domains II, Ib, and III. The physicochemical properties, secondary structure, and solubility of the chimeric protein were analyzed using different software. Prostate cancer DU-145 and breast cancer MDA-MB-468 cell lines were used as GRP78-positive and negative controls, respectively. Accordingly, the cytotoxicity, binding affinity, cell internalization, and apoptosis were evaluated using MTT, enzyme-linked immunosorbent assay, and western blot. The results showed that in the DU-145 cell line, the cytotoxicity of two recombinant immunotoxins is dose and time-dependent. In MDA-MB-468 and HEK-293 cells, such an event does not occur. It is possible that two constructs designed for immunotoxins can attach to GRP78-positive cancer cells and then eradicate cancer cells by internalization and apoptosis. As our in vitro results were in line with in silico data confirming the Bioinformatics predictions, it can be concluded that the designed recombinant immunotoxins may exhibit therapeutic potential against GRP78-positive tumor cells.

Genome-Wide Identification and Expression Analysis of SNAP Gene Family in Wheat

Background/Objectives: The SNAP gene family is a class of proteins containing a SNAP domain, which plays a crucial role in the growth and development of plants. Methods: Bioinformatics methods were used to systematically analyze the gene structure, phylogenetic evolution, chromosomal distribution, physicochemical properties, conserved motifs, and cis-acting elements of the TaSNAP family members. Results: The TaSNAP family comprises members that encode proteins ranging between 120 and 276 amino acids, with isoelectric points spanning from 4.87 to 7.92. Phylogenetic analysis elucidated the categorization of the eight TaSNAP into three distinct subfamilies, wherein members of the same subfamily display marked similarities in their gene structures. Chromosomal mapping revealed the distribution of TaSNAP family members across chromosomes 2A, 2B, 2D, 7A, 7B, and 7D. Utilizing the Plant CARE tool, we identified ten elements linked to plant hormones and four associated with stress responses. Expression analysis via qRT-PCR was performed to assess the levels of the eight TaSNAP genes in various tissues and under diverse abiotic stress conditions. The results indicated heightened expression of most genes in roots compared to spikes. Notably, under ABA stress, the majority of genes exhibited upregulation, whereas certain genes were downregulated under PEG stress, implying a substantial role for SNAP protein in wheat growth and development. Conclusions: This study conducted a comprehensive bioinformatics analysis of each member of the wheat SNAP family, laying a crucial foundation for future functional investigations.

KLF14 directly downregulates the expression of GPX4 to exert antitumor effects by promoting ferroptosis in cervical cancer

BackgroundCervical cancer is the fourth leading cause of cancer-related death among women worldwide, and effective therapeutic strategies for its treatment are limited. Recent studies have indicated that ferroptosis, a form of regulated cell death, is a promising therapeutic strategy. KLF14 has been shown to regulate both cell proliferation and apoptosis in cervical cancer. However, its role in modulating lipid peroxidation and ferroptosis remains largely unexplored and enigmatic.MethodsSiHa and HeLa cells were transduced with lentiviral vectors to overexpress KLF14. Protein levels were analyzed via western blotting and immunohistochemistry (IHC). LDH assays, calcein-AM/propidium iodide (PI) staining, and generation of cell growth curves using a real-time cell analysis (RTCA) system were used to detect cell damage and proliferation. Cellular ROS, lipid ROS, transmission electron microscopy (TEM), and Fe2+ assays and a xenograft mouse model were used to measure the level of ferroptosis. Proteomics combined with bioinformatics methods was used to screen target genes regulated by KLF14, and CUT&Tag and dual-luciferase assays confirmed the repression of GPX4 by KLF14 via direct binding to its promoter.ResultsKLF14 is abnormally expressed in various tumors and downregulated in cervical cancer. Overexpression of KLF14 induced ferroptosis and inhibited cell proliferation in vitro as well as xenograft tumorigenicity in vivo. Mechanistic studies revealed that KLF14 binds to the promoter of GPX4, suppressing its transcriptional activity and thereby decreasing its expression, which contributes to the induction of ferroptosis. Truncation and point mutation analyses of the GPX4 promoter revealed multiple binding sites for KLF14 within the − 1000 bp to + 35 bp region, which are responsible for its inhibitory effect on GPX4 transcription. Additionally, deletion of the zinc finger motif in KLF14 abolished its inhibitory effect on GPX4 promoter activity and cell proliferation.ConclusionOur data revealed a previously unidentified function of KLF14 in promoting ferroptosis, which results in the suppression of cell proliferation. Mechanistically, we revealed a novel regulatory mechanism by which KLF14 targets GPX4. These findings suggest a novel strategy to induce ferroptosis through the targeting of KLF14 in human cervical cancer cells.