Regulatory Signaling Pathways Research Articles

Role of HOXA10 in pathologies of the endometrium.

HOXA10 belongs to the homeobox gene family and is essential for uterine biogenesis, endometrial receptivity, embryo implantation, and stromal cell decidualization. Available evidence suggests that the expression of HOXA10 is dysregulated in different endometrial disorders like endometrial hyperplasia, endometrial cancer, adenomyosis, endometriosis, recurrent implantation failure, and unexplained infertility. The downregulation of HOXA10 occurs by genetic changes in the HOXA10 gene, methylation of the HOXA10 locus, or selected miRNAs. Endocrine disruptors and organic pollutants also cause the reduced expression of HOXA10 in these conditions. In vivo experiments in mouse models and in vitro studies in human cell lines demonstrate that downregulation of HOXA10 leads to endometrial epithelial cell proliferation, failure of stromal cell decidualization, altered expression of genes involved in cell cycle regulation, immunomodulation, and various signaling pathways. These disruptions are speculated to cause infertility associated with the disorders of the endometrium.

Comprehensive circular RNA profiling in various sheep tissues

Despite the scientific relevance of circular RNAs (circRNAs), the study of these RNAs in non-model organisms, especially in sheep, is still in its infancy. On the other hand, while some studies have focused on sheep circRNA identification in a limited number of tissues, there is a lack of comprehensive analysis that profile circRNA expression patterns across the tissues not yet investigated. In this study, 61 public RNA sequencing datasets from 12 different tissues were uniformly analyzed to identify circRNAs, profile their expression and investigate their various characteristics. We reported for the first time a circRNA expression landscape with functional annotation in sheep tissues not yet investigated including hippocampus, BonMarrowMacrophage, left-ventricle, thymus, ileum, reticulum and 23-day-embryo. A stringent computational pipeline was employed and 8919 exon-derived circRNAs with high confidence were identified, including 88 novel circRNAs. Tissue-specificity analysis revealed that 3059 circRNAs were tissue-specific, which were also more specific to the tissues than linear RNAs. The highest number of tissue-specific circRNAs was found in kidney, hippocampus and thymus, respectively. Co-expression analysis revealed that expression of circRNAs may not be affected by their host genes. While most of the host genes produced more than one isoform, only one isoform had dominant expression across the tissues. The host genes of the tissue-specific circRNAs were significantly enriched in biological/pathways terms linked to the important functions of their corresponding tissues, suggesting potential roles of circRNAs in modulating physiological activity of those tissues. Interestingly, functional terms related to the regulation and various signaling pathways were significantly enriched in all tissues, suggesting some common regulatory mechanisms of circRNAs to modulate the physiological functions of tissues. Finding of the present study provide a valuable resource for depicting the complexity of circRNAs expression across tissues of sheep, which can be useful for the field of sheep genomic and veterinary research.

Bromodomain and extraterminal domain (BET) promote autophagy in buffalo sertoli cells

Sertoli cells (SCs) play a pivotal role in spermatogenesis, with autophagy modulation being an evolutionarily conserved mechanism for maintaining cellular homeostasis and protecting spermatogenic cells against apoptosis. The bromodomain and extraterminal domain (BET) family are transcriptional regulators of autophagy. This study investigated the relationship between BET inhibition and autophagy in buffalo SCs. Our findings reveal that BET inhibition suppresses cell proliferation and alters the biological characteristics of SCs. RNA-seq analysis demonstrated significant downregulation of autophagy-related genes upon BET inhibition. Moreover, our bioinformatics analysis suggested the involvement of the PI3K-AKT signaling pathway in autophagy regulation within buffalo SCs. Immunofluorescence and Transmission electron microscopy observations indicated that BET inhibition results in autophagosome accumulation and impedes autophagosome-lysosome degradation, thereby compromising autophagy activity and flux. In summary, this study sheds light on the indispensable role of BET proteins in autophagy and paves the way for further investigations into the mechanisms governing BET protein-mediated autophagy regulation and its implications for male reproduction.

Targeting the VEGFR2 signaling pathway for angiogenesis and fibrosis regulation in neovascular age-related macular degeneration

Neovascular age-related macular degeneration (nAMD) is characterized by abnormal blood vessel growth from the choroid, leading to complications and eventual blindness. Despite anti-VEGF therapy, subretinal fibrosis remains a major concern, as VEGF/VEGF receptor-2 (VEGFR2) signaling can contribute to both angiogenesis and fibrosis. For the identification of the aqueous humor proteome, we performed liquid chromatography with tandem mass spectrometry analysis. To investigate the potential therapeutic effects of targeting the VEGF signaling pathway using apatinib, a highly selective VEGFR2 tyrosine kinase inhibitor, this study employed in vitro (THP-1 conditioned media-treated ARPE-19 cells) and in vivo (laser-induced choroidal neovascularization mouse) models of nAMD. This study revealed elevated VEGFR2 protein levels in the aqueous humor of nAMD patients, suggesting a potential target to mitigate neovascularization and fibrosis in nAMD. Apatinib effectively reduced VEGFA and αSMA levels in both in vitro and in vivo models. Moreover, apatinib showed improvement in laser-induced subretinal hyper-reflective lesions. The action mechanism was linked to the inhibition of VEGFR2 activation, leading to the suppression of both angiogenesis and fibrosis through the downregulation of STAT3 phosphorylation. Therefore, the VEGFR2 signaling pathway appears to play a central role in the development of nAMD by regulating both angiogenesis and fibrosis.

Chondrocyte autophagy mechanism and therapeutic prospects in osteoarthritis.

Osteoarthritis (OA) is the most common type of arthritis characterized by progressive cartilage degradation, with its pathogenesis closely related to chondrocyte autophagy. Chondrocytes are the only cells in articular cartilage, and the function of chondrocytes plays a vital role in maintaining articular cartilage homeostasis. Autophagy, an intracellular degradation system that regulates energy metabolism in cells, plays an incredibly important role in OA. During the early stages of OA, autophagy is enhanced in chondrocytes, acting as an adaptive mechanism to protect them from various environmental changes. However, with the progress of OA, chondrocyte autophagy gradually decreases, leading to the accumulation of damaged organelles and macromolecules within the cell, prompting chondrocyte apoptosis. Numerous studies have shown that cartilage degradation is influenced by the senescence and apoptosis of chondrocytes, which are associated with reduced autophagy. The relationship between autophagy, senescence, and apoptosis is complex. While autophagy is generally believed to inhibit cellular senescence and apoptosis to promote cell survival, recent studies have shown that some proteins are degraded by selective autophagy, leading to the secretion of the senescence-associated secretory phenotype (SASP) or increased SA-β-Gal activity in senescent cells within the damaged region of human OA cartilage. Autophagy activation may lead to different outcomes depending on the timing, duration, or type of its activation. Thus, our study explored the complex relationship between chondrocyte autophagy and OA, as well as the related regulatory molecules and signaling pathways, providing new insights for the future development of safe and effective drugs targeting chondrocyte autophagy to improve OA.

Role of SIK1 in tumors: Emerging players and therapeutic potentials (Review).

Salt‑induced kinase 1 (SIK1) is a serine/threonine protein kinase that is a member of the AMP‑activated protein kinase family. SIK is catalytically activated through its phosphorylation by the upstream kinase LKB1. SIK1 has been reported to be associated with numerous types of cancer. The present review summarizes the structure, regulatory factors and inhibitors of SIK1, and also describes how SIK1 is a signal regulatory factor that fulfills connecting roles in various signal regulatory pathways. Furthermore, the anti‑inflammatory effects of SIK1 during the early stage of tumor occurrence and its different regulatory effects following tumor occurrence, are summarized, and through collating the tumor signal regulatory mechanisms in which SIK1 participates, it has been demonstrated that SIK1 acts as a necessary node in cancer signal transduction. In conclusion, SIK1 is discussed independent of the SIKs family, its research results and recent progress in oncology are summarized in detail with a focus on SIK1, and its potential as a therapeutic target is highlighted, underscoring the need for SIK1‑targeted regulatory strategies in future cancer therapy.

Screening and identification of microRNAs mediating cartilage endplate degeneration in human intervertebral disks.

This study aimed to discover micro-ribonucleic acids (microRNAs) involved in the degeneration of cartilage endplates through next-generation sequencing and lay the foundation for further research. The cartilage endplate was obtained from patients who underwent interbody fusion surgery at the Department of Spine Surgery, Shanghai East Hospital Affiliated to Tongji University, from 1 January 2020 to 1 January 2023. Total RNA was extracted from the cartilage endplate tissue. Discover differential genes through NGS. To annotate gene functions, all target genes were aligned against the Kyoto Encyclopedia of Genes (KEGG) and Gene Ontology (GO) databases. The GO enrichment and KEGG enrichment analyses of target genes were performed using phyper, a function of R. The p-value was corrected using the Bonferroni method, and a corrected p-value of ≤0.05 was taken as the threshold. GO terms or KEGG terms fulfilling this condition were defined as significantly enriched terms. The screened miRNAs and their target protein were verified in vitro using quantitative polymerase chain reaction (qPCR) and Western blotting (WB). RNA was extracted from normal and degenerated cartilage endplate tissues for NGS. Eight downregulated differentially expressed genes (DEGs) and 22 upregulated DEGs were found. The KEGG pathway analysis of these target genes revealed that differential microRNAs and target genes were enriched in different signaling pathways, and the regulated signaling pathways were mainly mitochondrial autophagy and autophagy. The qPCR results demonstrated a significant upregulation of miR-25-3p and miR-345-5p in degenerative cartilage endplate tissues (p ≤ 0.001). Western blot analysis revealed that BRD4 exhibited a marked increase in protein expression levels in degenerative cartilage endplate tissues (p ≤ 0.0001), while BECN1 showed a significant decrease in protein expression levels within these samples (p ≤ 0.0001). We found that DEG hsa-miR-25-3p and hsa-miR-345-5p can be used as diagnostic and therapeutic targets for IDD. The significant target proteins of miR-25-3p and miR-345-5p were BRD4 and BECN1, respectively.

The soluble factor milieu in idiopathic pulmonary fibrosis dysregulates epithelial differentiation.

In idiopathic pulmonary fibrosis (IPF), epithelial abnormalities are present including bronchiolization and alveolar cell dysfunction. We hypothesized that the IPF microenvironment disrupts normal epithelial growth and differentiation. We mimicked the soluble factors within an IPF microenvironment using an IPF cocktail (IPFc), composed of nine factors which are increased in IPF lungs (CCL2, IL-1β, IL-4, IL-8, IL-13, IL-33, TGF-β, TNFα, and TSLP). Using IPFc, we asked whether the soluble factor milieu in IPF affects epithelial growth and differentiation and how IPFc compares to TGF-β alone. Epithelial growth and differentiation were studied using mouse lung organoids (primary Epcam+ epithelial cells co-cultured with CCL206 fibroblasts). Organoids exposed to IPFc and TGF-β were re-sorted into epithelial and fibroblast fractions and subjected to RNA sequencing. IPFc did not affect the number of organoids formed. However, pro-surfactant protein C expression was decreased. On these parameters, TGF-β alone had similar effects. However, RNA sequencing of re-sorted organoids revealed that IPFc and TGF-β had distinct effects on both epithelial cells and fibroblasts. IPFc upregulated goblet cell markers, whereas these were inhibited by TGF-β. Although both IPFc and TGF-β increased extracellular matrix gene expression, only TGF-β increased myofibroblast markers. VEGF-C and Wnt signaling were among the most differentially regulated signaling pathways by IPFc versus TGF-β. Interestingly, Wnt pathway activation rescued Sftpc downregulation induced by IPFc. In conclusion, IPFc alters epithelial differentiation in a way that is distinct from TGF-β. Alterations in Wnt signaling contribute to these effects. IPFc may be a more comprehensive representation of the soluble factor microenvironment in IPF.

Transfer RNAs and transfer RNA-derived small RNAs in cerebrovascular diseases

This article explores the important functions of transfer RNA and - transfer RNA derived small RNAs (tsRNAs) in cellular processes and disease pathogenesis, with a particular emphasis on their involvement in cerebrovascular disorders. It discusses the biogenesis and structure of tsRNAs, including types such as tRNA halves and tRNA-derived fragments, and their functional significance in gene regulation, stress response, and cell signaling pathways. The importance of tsRNAs in neurodegenerative diseases, cancer, and cardiovascular diseases has already been highlighted, while their role in cerebrovascular diseases is in early phase of exploration. This paper presents the latest advancements in the field of tsRNAs in cerebrovascular conditions, such as ischemic stroke, intracerebral hemorrhage, and moyamoya disease. Furthermore, revealing the aptitude of tsRNAs as biomarkers for the prediction of cerebrovascular diseases and as targets for therapeutic intervention. It provides insights into the role of tsRNAs in these conditions and proposes directions for future research.

MicroRNAs and human viral diseases: A focus on the role of microRNA-29

The viral replication can impress through cellular miRNAs. Indeed, either the antiviral responses or the viral infection changes through cellular miRNAs resulting in affecting many regulatory signaling pathways. One of the microRNA families that is effective in human cancers, diseases, and viral infections is the miR-29 family. Members of miR-29 family are effective in different viral infections as their roles have appeared in regulation of immunity pathways either in innate immunity including interferon and inflammatory pathways or in adaptive immunity including activation of T-cells and antibodies production. Although miR-29a affects viral replication by suppressing antiviral responses, it can inhibit the expression of viral mRNAs via binding to their 3′UTR. In the present work, we discuss the evidence related to miR-29a and viral infection through host immunity regulation. We also review roles of other miR-29 family members by focusing on their role as biomarkers for diagnosing and targets for viral diseases management.

Versatile whey acidic protein four-disulfide core domain proteins: biology and role in diseases.

The Whey acidic protein four-disulfide core (WFDC) protein family consists of proteins with one or more WFDC domains which are ubiquitously expressed throughout the body of human and perform a wide range of functions, including antiprotease, antibacterial, and immunomodulatory functions. Aberrant expression of WFDC proteins is associated with human diseases. However, review on the WFDC protein family is limited and insufficient. Furthermore, a systematic summary of the underlying mechanisms of WFDC protein activity is lacking. In this review, we give a summary of the structural basis and molecular function of these proteins and review the immune regulatory mechanisms and signaling pathways of WFDC proteins in the development of certain diseases. Furthermore, we discuss the diagnostic and prognostic potential of multiple WFDC proteins in the aforementioned conditions, as well as their prospective use. At last, we also discuss the progress of WFDC protein in clinical trials and put forward some research difficulties and the directions of follow-up research. Our review highlights the functional diversity and clinical significance of WFDC proteins family, while providing potential targets for drug development and innovative therapeutic strategies, this review lays the foundation and direction for future research on WFDC proteins.

Bioinformatics analysis of BTK expression in lung adenocarcinoma: implications for immune infiltration, prognostic biomarkers, and therapeutic targeting.

In recent years, as more and more lung-cancer patients have been treated with immunotherapeutic agents, their survival has been prolonged compared to before. It is well known that BTK (Bruton's tyrosine kinase) is predominantly found in cells of the hematopoietic system. However, there is a distinct lack of literature on BTK expression in lung adenocarcinoma (LUAD) patients and its effect on the immune microenvironment. Consequently, the main goal of this investigation was to analyze how BTK expression in lung adenocarcinoma affects its progression, along with its prognostic significance, through the utilization of bioinformatics online resources and publicly available databases. Data on the sequencing results and clinical records of lung adenocarcinoma patients were gathered from The Cancer Genome Atlas (TCGA) database. Based on the expression level of BKT, TCGA categorized lung adenocarcinoma patients into BTK high-expression and low-expression groups. We investigated the effects of BKT on clinicopathologic, genomic, and immunologic characteristics of lung adenocarcinoma patients. We analyzed BTK mRNA expression in tumors and normal tissues using two key resources: Tumor Immuno Estimation Resource 2.0 (TIMER 2.0) and Gene Expression Profiling Interactive Analysis 2 (GEPIA 2). We analyzed the prognosis of the patients using GEPIA2 and validated the results using univariate and multivariate analyses. In addition, we assessed BTK protein expression by Human Protein Atlas (HPA). We sought to elucidate the clinical prognostic significance of BTK in The TCGA using the online tool GEPIA 2. Furthermore, to clarify the biologic roles and pathways linked to BTK, we conducted a genomic enrichment analysis of the information. To predict the proportion of various immune cell infiltrations in the immune microenvironment of lung adenocarcinoma patients diagnosed in the TCGA database, we performed an analysis using the TIMER online tool. Using TIMER and CIBERSORT, the correlation between genes co-expressed with BTK and the corresponding tumor-infiltrating immune cells was explored; finally, the relationship between BTK expression and immune infiltration and immune checkpoints in the TMB group and the high and low groups was analyzed by R language analysis using the TCGA database. The expression of BTK provides some hints about the prognosis of the patients. The high expression of BTK is involved in immune response regulation signaling pathways, leukocyte-mediated immunity, leukocyte intercellular adhesion, graft rejection, and complement. Analysis of the GEPIA 2 database showed that BTK was co-expressed with the genes FGD2, SASH3, NCKAP1L, CD53, ARHGAP30 and LPXN. Increased expression of the above-mentioned genes resulted in increased proportions of CD8 + T cells, memory CD4 + T cells, B cells, macrophages, and dendritic cells, and decreased proportions of Treg cells and TH2 cells. In addition, our study revealed a strong positive correlation between various key immune checkpoints (e.g., PDCD1, CD274, PDCD1LG2, CTLA4, HAVCR2, LAG3, TIGIT, and SIGLEC15) and BTK expression. In conclusion, increased BTK expression in lung adenocarcinoma is closely associated with prolonged survival of lung-cancer patients. Moreover, the genes classified under the BTK high-expression group exhibit significant enrichment in immune-related pathways, suggesting a potential impact on the tumor microenvironment. We investigated the potential of BTK as a tumor suppressor gene in predicting prolonged patient survival. In addition, we further investigated the possibility that BTK further affects the immunotherapeutic response of patients by influencing the microenvironment of tumor immune infiltration, but the relevant mechanisms remain to be further studied.

Connexin 43 regulates pyroptosis by influencing intracellular calcium levels in X-ray induced vascular endothelial cell damage.

Our prior research has established that X-ray exposure induces pyroptosis in human umbilical vein endothelial cells (HUVECs), with Cx43 playing a regulatory role in this process. However, the precise mechanism by which Cx43 regulates pyroptosis remains unclear. The objective of this study is to assess the involvement of the calcium signaling pathway in Cx43-mediated regulation of X-ray-induced pyroptosis in HUVECs. HUVECs were exposed to 10 Gy X-ray radiation either alone or combined with Cx43 overexpression or knockdown. Calcium ions (Ca2+) were stained using Fluo-4/AM and analyzed via flow cytometry and confocal microscopy. Pyroptosis was assessed through flow cytometry by staining with FLICA (fluorescent-labeled inhibitor of caspase) and propidium iodide (PI). Calcium signaling was inhibited using BAPTA/AM, 2-APB, or nifedipine. Protein expression levels were assessed by western blotting. X-ray irradiation induced an increase in intracellular calcium levels in HUVECs in a dose- and time-dependent manner. The results demonstrated that regulating calcium release with BAPTA/AM, 2-APB, or nifedipine significantly reduced pyroptosis. Also, the overexpression of Cx43 significantly attenuated the increase in intracellular calcium. Conversely, Cx43 knockdown via siRNA significantly increased the intracellular calcium levels. Also, interfering with calcium signaling using BAPTA/AM, 2-APB, or nifedipine reduced the raised pyroptosis levels induced by Cx43 knockdown. Individual HUVECs exposed to high-dose X-ray irradiation exhibited an increase in intracellular calcium, leading to pyroptosis. Also, upregulating Cx43 expression reduced the pyroptosis levels by inhibiting intracellular calcium concentration. This study introduces new concepts for identifying targets for the prophylaxis and therapy of radiation-induced damage.

Global transcriptomic network analysis of the crosstalk between microbiota and cancer-related cells in the oral-gut-lung axis.

The diagnosis and treatment of lung, colon, and gastric cancer through the histologic characteristics and genomic biomarkers have not had a strong impact on the mortality rates of the top three global causes of death by cancer. Twenty-five transcriptomic analyses (10 lung cancer, 10 gastric cancer, and 5 colon cancer datasets) followed our own bioinformatic pipeline based on the utilization of specialized libraries from the R language and DAVID´s gene enrichment analyses to identify a regulatory metafirm network of transcription factors and target genes common in every type of cancer, with experimental evidence that supports its relationship with the unlocking of cell phenotypic plasticity for the acquisition of the hallmarks of cancer during the tumoral process. The network's regulatory functional and signaling pathways might depend on the constant crosstalk with the microbiome network established in the oral-gut-lung axis. The global transcriptomic network analysis highlighted the impact of transcription factors (SOX4, TCF3, TEAD4, ETV4, and FOXM1) that might be related to stem cell programming and cancer progression through the regulation of the expression of genes, such as cancer-cell membrane receptors, that interact with several microorganisms, including human T-cell leukemia virus 1 (HTLV-1), the human papilloma virus (HPV), the Epstein-Barr virus (EBV), and SARS-CoV-2. These interactions can trigger the MAPK, non-canonical WNT, and IFN signaling pathways, which regulate key transcription factor overexpression during the establishment and progression of lung, colon, and gastric cancer, respectively, along with the formation of the microbiome network. The global transcriptomic network analysis highlights the important interaction between key transcription factors in lung, colon, and gastric cancer, which regulates the expression of cancer-cell membrane receptors for the interaction with the microbiome network during the tumorigenic process.

Dysregulation of lipid metabolism in chronic kidney disease and the role of natural products.

Dysregulation of lipid metabolism plays a key role in the onset and progression of CKD, and a thorough understanding of its regulatory mechanisms is essential for the development of effective treatments. In recent years, an increasing number of studies have focused on the pharmacological activities of natural products and their application in the treatment of chronic diseases. Natural products, including plant extracts and bioactive compounds, have been shown to exert anti-inflammatory, antioxidant, antifibrosis, and anti-apoptotic effects through various signaling pathways in the treatment of CKD. Many natural products have been shown to target dysregulated lipid metabolism through various signaling pathways. This review summarizes the key regulatory factors and signaling pathways involved in the dysregulation of lipid metabolism in chronic kidney disease (CKD), highlighting their importance as potential therapeutic targets. Recently published research on the potential therapeutic benefits of natural products for the treatment of CKD was described. These studies have revealed the multi-target role of natural products in the regulation of lipid metabolism. Natural products show great potential in targeting lipid metabolism-related pathways, offering a novel research direction for the treatment of CKD while providing a scientific basis and experimental support for the development of new treatment strategies.

Bioinformatics Analysis of Differentially Expressed Genes in Carpal Tunnel Syndrome Using RNA Sequencing.

Carpal tunnel syndrome (CTS) is a common disease resulting from the median nerve entrapment at the wrist. Although CTS (prevalence=5%-10% in the general population) is the most common neuropathy, its molecular mechanisms need elucidation. We used bioinformatics to detect genes with differential expressions in CTS and introduce the molecular regulatory noncoding RNAs and signaling pathways involved. The raw files of the RNA sequencing of CTS patients and controls were obtained from GEO (accession: GSE108023), and the samples were analyzed. Differentially expressed genes were isolated using DESeq2 R. Functional analyses were conducted on the signaling pathways, biological processes, molecular functions, and cellular components of the differentially expressed genes. Additionally, interactions between the most differentially expressed genes and miRNAs and lncRNAs were investigated bioinformatically. Upregulation and downregulation were observed in 790 and 922 genes, respectively. The signaling pathway analysis identified the metabolism pathways of arachidonic acid, linoleic acid, and tyrosine as the most significant pathways in CTS. Moreover, PLA2G2D and PLA2G2A with upregulated expressions and PLA2G2F, PLA2G4F, PLA2G4D, PLA2G3, and PLA2G4E with downregulated expressions were genes from the phospholipase family playing significant roles in the pathways. Further analyses demonstrated that hsa-miR-3150b-3p targeted PLA2G2A and PLA2G4F, and RP11-573D15.8-018 lncRNA had regulatory interactions with the aforementioned genes. Molecular studies on CTS will clarify the involved signaling pathways and provide critical data for biomedical research, drug development, and clinical applications.

Potential use of HSP70 as an indicator of heat stress in dairy cows – a review

Heat stress (HS) poses significant challenges to the dairy industry, resulting in reduced milk production, impaired reproductive performance, and compromised animal welfare. Therefore, understanding the molecular mechanisms underlying cellular responses to HS is crucial for developing effective strategies to mitigate its adverse effects. Heat shock protein 70 (HSP70) has emerged as a potential player involved in cellular thermotolerance in dairy cows. This review provides a comprehensive overview of the role of HSP70 as a molecular chaperone in cellular thermotolerance in dairy cows under HS. HSP70 facilitates proper protein folding and prevents the aggregation of denatured proteins. By binding to misfolded proteins, it helps maintain protein homeostasis and prevents the accumulation of damaged proteins during HS. Additionally, HSP70 interacts with various regulatory proteins and signaling pathways, contributing to the cellular adaptive response to HS. The upregulation of HSP70 expression in response to HS is regulated by a complex network involving heat-shock factors (HSFs), heat-shock element-binding proteins, and HSF co-chaperones. Therefore, HSP70 holds the potential to be a useful indicator of tissue stress due to its role in maintaining cellular balance, and as it is released both inside and outside cells in response to stress. Traditional methods of measuring HSP70 in blood samples are labor-intensive, and with the process being potentially stressful for the animals and may subsequently affect the results. Therefore, measuring HSP expression in cow's milk has shown promise as an easy, non-invasive, and accurate way to detect HS in dairy cows. Monitoring HSP70 levels in milk can be applied as a supplementary approach to identify HS or HS resistance of individual cows, selection of suitable animals and to guide targeted management strategies. However, despite the potential advantages of using HSP70 as a biomarker for monitoring HS on dairy cows, challenges remain in standardizing measurement protocols, establishing species-specific reference ranges, addressing inter-individual variations, and determining the specificity of changes in HSP70 due to HS. Future research should focus on developing non-invasive techniques for HSP70 detection, with consideration of climatic conditions, and unravelling the molecular interactions and regulatory networks involving HSP70.

Glutathione hybrid poly (beta-amino ester)-plasmid nanoparticles for enhancing gene delivery and biosafety

IntroductionCRISPR/Cas9 gene editing technology has significantly advanced gene therapy, with gene vectors being one of the key factors for its success. Poly (beta-amino ester) (PBAE), a distinguished non-viral cationic gene vector, is known to elevate intracellular reactive oxygen species (ROS) levels, which may cause cytotoxicity and, consequently, impact gene transfection efficacy (T.E.). ObjectivesTo develop a simple but efficient strategy to improve the gene delivery ability and biosafety of PBAE both in vivo and in vitro. MethodsWe used glutathione (GSH), a clinically utilized drug with capability to modulating intracellular ROS level, to prepare a hybrid system with PBAE-plasmid nanoparticles (NPs). This system was characterized by flow cytometry, RNA-seq, Polymerase Chain Reaction (PCR) and Sanger sequencing in vitro, and its safety and efficacy in vivo was evaluated by imaging, PCR, Sanger sequencing and histology analysis. ResultsThe particle size of GSH-PBAE-plasmid NPs were 168.31 nm with a ζ-potential of 15.21 mV. An enhancement in T.E. and gene editing efficiency, ranging from 10 % to 100 %, was observed compared to GSH-free PBAE-plasmid NPs in various cell lines. In vitro results proved that GSH-PBAE-plasmid NPs reduced intracellular ROS levels by 25 %–40 %, decreased the total number of upregulated/downregulated genes from 4,952 to 789, and significantly avoided the disturbance in gene expression related to cellular oxidative stress-response and cell growth regulation signaling pathway compared to PBAE-plasmid NPs. They also demonstrated lower impact on the cell cycle, slighter hemolysis, and higher cell viability after gene transfection. Furthermore, GSH hybrid PBAE-plasmid NPs exhibited superior safety and improved tumor suppression ability in an Epstein–Barr virus (EBV)-infected murine tumor model, via targeting cleavage the EBV related oncogene by delivering CRISPR/Cas9 gene editing system and down-regulating the expression levels. This simple but effective strategy is expected to promote clinical applications of non-viral vector gene delivery.

Pyroptosis, ferroptosis, and autophagy in spinal cord injury: regulatory mechanisms and therapeutic targets.

Regulated cell death is a form of cell death that is actively controlled by biomolecules. Several studies have shown that regulated cell death plays a key role after spinal cord injury. Pyroptosis and ferroptosis are newly discovered types of regulated cell deaths that have been shown to exacerbate inflammation and lead to cell death in damaged spinal cords. Autophagy, a complex form of cell death that is interconnected with various regulated cell death mechanisms, has garnered significant attention in the study of spinal cord injury. This injury triggers not only cell death but also cellular survival responses. Multiple signaling pathways play pivotal roles in influencing the processes of both deterioration and repair in spinal cord injury by regulating pyroptosis, ferroptosis, and autophagy. Therefore, this review aims to comprehensively examine the mechanisms underlying regulated cell deaths, the signaling pathways that modulate these mechanisms, and the potential therapeutic targets for spinal cord injury. Our analysis suggests that targeting the common regulatory signaling pathways of different regulated cell deaths could be a promising strategy to promote cell survival and enhance the repair of spinal cord injury. Moreover, a holistic approach that incorporates multiple regulated cell deaths and their regulatory pathways presents a promising multi-target therapeutic strategy for the management of spinal cord injury.

Withaferin A protects against epilepsy by promoting LCN2-mediated astrocyte polarization to stopping neuronal ferroptosis

BackgroundEpilepsy is among the most frequent severe brain diseases, with few treatment options available. Neuronal ferroptosis is an important pathogenic mechanism in epilepsy. As a result, addressing ferroptosis appears to be a promising treatment approach for epilepsy. Withaferin A (WFA) is a C28 steroidal lactone that has a broad range of neuroprotective properties. Nonetheless, the antiepileptic action of WFA and the intrinsic mechanism by which it inhibits ferroptosis following epilepsy remain unknown. PurposeThis study aimed at investigating to the antiepileptic potential of WFA in epilepsy, as well as to propose a potential therapeutic approach for epilepsy therapy. MethodsWe conducted extensive research to examine the impacts of WFA on epilepsy and ferroptosis, using the kainic acid (KA)-treated primary astrocyte as an in vitro model and KA-induced temporal lobe epilepsy mice as an in vivo model. To analyze the neuroprotective effects of WFA on epileptic mice, electroencephalogram (EEG) recording, Nissl staining, and neurological function assessments such as the Morris water maze (MWM) test, Y-maze test, Elevated-plus maze (O-maze) test, and Open field test were used. Furthermore, the mechanism behind the neuroprotective effect of WFA in epilepsy was investigated using the transcriptomics analysis and verified on epileptic patient and epileptic mouse samples using Western blotting (WB) and immunofluorescence (IF) staining. In addition, WB, IF staining and specific antagonists/agonists were used to investigate astrocyte polarization and the regulatory signaling pathways involved. More critically, ferroptosis was assessed utilizing lipocalin-2 (LCN2) overexpression cell lines, siRNA knockdown, JC-1 staining, WB, IF staining, flow cytometry, electron microscopy (TEM), and ferroptosis-related GSH and MDA indicators. ResultsIn this study, we observed that WFA treatment reduced the number of recurrent seizures and time in seizure, and the loss of neurons in the hippocampal area in in epileptic mice, and even improved cognitive and anxiety impairment after epilepsy in a dose depend. Furthermore, WFA treatment was proven to enhance to the transformation of post-epileptic astrocytes from neurotoxic-type A1 to A2 astrocytes in both in vivo and in vitro experiments by inhibiting the phosphoinositide 3-kinase /AKT signaling pathway. At last, transcriptomics analysis in combination with functional experimental validation, it was discovered that WFA promoted astrocyte polarity transformation and then LCN2 in astrocytes, which inhibited neuronal ferroptosis to exert neuroprotective effects after epilepsy. In addition, we discovered significant astrocytic LCN2 expression in human TLE patient hippocampal samples. ConclusionsTaken together, for the first, our findings suggest that WFA has neuroprotective benefits in epilepsy by modulating astrocyte polarization, and that LCN2 may be a novel potential target for the prevention and treatment of ferroptosis after epilepsy.