Pan-cancer transcriptome analysis reveals widespread regulation through alternative tandem transcription initiation.

Abnormal transcription initiation from alternative first exon has been reported to promote tumorigenesis. However, the prevalence and impact of gene expression regulation mediated by alternative tandem transcription initiation were mostly unknown in cancer. Here, we developed a robust computational method to analyze alternative tandem transcription start site (TSS) usage from standard RNA sequencing data. Applying this method to pan-cancer RNA sequencing datasets, we observed widespread dysregulation of tandem TSS usage in tumors, many of which were independent of changes in overall expression level or alternative first exon usage. We showed that the dynamics of tandem TSS usage was associated with epigenomic modulation. We found that significant 5' untranslated region shortening of gene TIMM13 contributed to increased protein production, and up-regulation of TIMM13 by CRISPR-mediated transcriptional activation promoted proliferation and migration of lung cancer cells. Our findings suggest that dysregulated tandem TSS usage represents an addtional layer of cancer-associated transcriptome alterations.

H6N2 reassortant avian influenza virus isolate in wild birds in Jiangxi Province, China.

H6 avian influenza virus is widely prevalent in wild birds and poultry and has caused human infection in 2013 in Taiwan, China. During our active influenza surveillance program in wild waterfowl at Poyang Lake, Jiangxi Province, an H6N2 AIV was isolated and named A/bean goose/JiangXi/452-4/2013(H6N2). The isolate was characterized as a typical low pathogenic avian influenza virus (LPAIV) due to the presence of the amino acid sequence PQIETR↓GLFGAI at the cleavage site of the hemagglutinin (HA) protein. The genetic evolution analysis revealed that the NA gene of the isolate originated from North America and exhibited the highest nucleotide identity (99.29%) with a virus recovered from wild bird samples in North America, specifically A/bufflehead/California/4935/2012(H11N2). Additionally, while the HA and PB1 genes belonged to the Eurasian lineage, they displayed frequent genetic interactions with the North American lineage. The remaining genes showed close genetic relationships with Eurasian viruses. The H6N2 isolate possessed a complex genome, indicating it is a multi-gene recombinant virus with genetic material from both Eurasian and North American lineages.

Fatty acids promote M1 polarization of monocyte-derived macrophages in healthy or ketotic dairy cows and a bovine macrophage cell line via impairing mTOR-mediated autophagy.

Excessive concentrations of free fatty acids (FFA) are the main factors causing immune dysfunction and inflammation in dairy cows with ketosis. Polarization of macrophages (the process of macrophages freely switching from one phenotype to another) into M1 or M2 phenotypes is an important event during inflammation induced by environmental stimuli. In non-ruminants, mammalian target of rapamycin (mTOR)-mediated autophagy (a major waste degradation process) regulates macrophage polarization. Thus, the objective was to unravel the role of mTOR-mediated autophagy on macrophage polarization in ketotic dairy cows. Four experiments were performed as follows: (1) In vitro differentiated monocyte-derived macrophages from healthy dairy cows or dairy cows with clinical ketosis (CK) were treated with 100 ng/mL lipopolysaccharide (LPS) and 100 ng/mL interferon-γ (IFN-γ) or 10 ng/mL interleukin-4 (IL4) and 10 ng/mL interleukin-10 (IL10) for 24 h; (2) Immortalized bovine macrophages were treated with 0, 0.3, 0.6, 1.2 mM FFA and LPS and IFN-γ or IL4 and IL10 for 24 h; (3) Macrophages were pretreated with 2 µM 4,6-dimorpholino-N-(4-nitrophenyl)-1,3,5-triazin-2-amine (MHY1485) for 30 min before treatment with LPS and IFN-γ or IL4 and IL10; (4) Macrophages were pretreated with 100 nM rapamycin (RAPA) for 2 h before treatment with LPS and IFN-γ or IL4 and IL10. Compared with healthy cows, cows with CK had a greater mean fluorescence intensity (MFI) of CD86+, but lower MFI of CD206+ and lower number of autophagosomes and autolysosomes in macrophages. Exogenous FFA treatment upregulated protein abundance of inducible nitric oxide synthase (iNOS) and mean fluorescence intensity of CD86, whereas it downregulated the protein abundance of arginase 1 (ARG1) and mean fluorescence intensity of CD206. In addition, FFA increased the p-p65/p65 protein abundance and tumor necrosis factor α (TNFA), interleukin-1B (IL1B), and interleukin-6 (IL6) mRNA abundance, but decreased LC3-phosphatidylethanolamine conjugate (LC3-II) protein abundance and autophagosomes and autolysosomes number. Pretreatment with MHY1485 promoted macrophage M1 polarization and inhibited macrophage M2 polarization via decreased mTOR-mediated autophagy. Activation of mTOR-mediated autophagy by pretreatment with RAPA attenuated the upregulation of inflammation in M1 macrophages that was induced by FFA. These data revealed that high concentrations of FFA promote macrophage M1 polarization in ketotic dairy cows via impairing mTOR-mediated autophagy.

Role of diacylglycerol O-acyltransferase 1 (DGAT1) in lipolysis and autophagy of adipose tissue from ketotic dairy cows.

High-yielding dairy cows in early lactation often encounter difficulties in meeting the energy requirements essential for maintaining milk production. This is primarily attributed to insufficient dry matter intake, which consequently leads to sustained lipolysis of adipose tissue. Fatty acids released by lipolysis can disrupt metabolic homeostasis. Autophagy, an adaptive response to intracellular environmental changes, is considered a crucial mechanism for regulating lipid metabolism and maintaining a proper cellular energy status. Despite its close relationship with aberrant lipid metabolism and cytolipotoxicity in animal models of metabolic disorders, the precise function of diacylglycerol o-acyltransferase 1 (DGAT1) in bovine adipose tissue during periods of negative energy balance is not fully understood, particularly regarding its involvement in lipolysis and autophagy. The objective of the present study was to assess the effect of DGAT1 on both lipolysis and autophagy in bovine adipose tissue and isolated adipocytes. Adipose tissue and blood samples were collected from cows diagnosed as clinically ketotic (n = 15) or healthy (n = 15) following a veterinary evaluation based on clinical symptoms and serum concentrations of BHB, which were 3.19 mM (interquartile range = 0.20) and 0.50 mM (interquartile range = 0.06), respectively. Protein abundance of DGAT1 and phosphorylation levels of unc-51-like kinase 1 (ULK1), were greater in adipose tissue from cows with ketosis, whereas phosphorylation levels of phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR) were lower. Furthermore, when adipocytes isolated from the harvested adipose tissue of 15 healthy cows were transfected with DGAT1 overexpression adenovirus or DGAT1 small interfering RNA followed by exposure to epinephrine (EPI), it led to greater ratios and protein abundance of phosphorylated hormone-sensitive triglyceride lipase (LIPE) to total LIPE and adipose triglyceride lipase (ATGL), while inhibiting the protein phosphorylation levels of ULK1, PI3K, AKT, and mTOR. Overexpression of DGAT1 in EPI-treated adipocytes reduced lipolysis and autophagy, whereas silencing DGAT1 further exacerbated EPI-induced lipolysis and autophagy. Taken together, these findings indicate that upregulation of DGAT1 may function as an adaptive response to suppress adipocytes lipolysis, highlighting the significance of maintaining metabolic homeostasis in dairy cows during periods of negative energy balance.

The chromatin accessibility dynamics during cell fate specifications in zebrafish early embryogenesis.

Chromatin accessibility plays a critical role in the regulation of cell fate decisions. Although gene expression changes have been extensively profiled at the single-cell level during early embryogenesis, the dynamics of chromatin accessibility at cis-regulatory elements remain poorly studied. Here, we used a plate-based single-cell ATAC-seq method to profile the chromatin accessibility dynamics of over 10 000 nuclei from zebrafish embryos. We investigated several important time points immediately after zygotic genome activation (ZGA), covering key developmental stages up to dome. The results revealed key chromatin signatures in the first cell fate specifications when cells start to differentiate into enveloping layer (EVL) and yolk syncytial layer (YSL) cells. Finally, we uncovered many potential cell-type specific enhancers and transcription factor motifs that are important for the cell fate specifications.

Different Degrees of Sulfated Laminaria Polysaccharides Recovered Damaged HK-2 Cells and Inhibited Adhesion of Nano-COM and Nano-COD Crystals.

Purpose: The crystal adhesion caused by the damage of renal tubular epithelial cells (HK-2) is the key to the formation of kidney stones. However, no effective preventive drug has been found. This study aims to explore the recovery effects of four Laminaria polysaccharides (SLPs) with different sulfate (-OSO3-) contents on damaged HK-2 cells and the difference in the adhesion of damaged cells to nanometer calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) before and after recovery. Methods: Sodium oxalate (2.6 mmol/L) was used to damage HK-2 cells to establish a damaged model. SLPs (LP0, SLP1, SLP2, and SLP3) with -OSO3- contents of 0.73%, 15.1%, 22.8%, and 31.3%, respectively, were used to restore the damaged cells, and the effects of SLPs on the adhesion of COM and COD, with a size of about 100 nm before and after recovery, were measured. Results: The following results were observed after SLPs recovered the damaged HK-2 cells: increased cell viability, restored cell morphology, decreased reactive oxygen levels, increased mitochondrial membrane potential, decreased phosphatidylserine eversion ratio, increased cell migration ability, reduced expression of annexin A1, transmembrane protein, and heat shock protein 90 on the cell surface, and reduced adhesion amount of cells to COM and COD. Under the same conditions, the adhesion ability of cells to COD crystals was weaker than that to COM crystals. Conclusions: As the sulfate content in SLPs increases, the ability of SLPs to recover damaged HK-2 cells and inhibit crystal adhesion increases. SLP3 with high -OSO3- content may be a potential drug to prevent kidney stones.

Cronkhite‒Canada syndrome as inflammatory hamartomatous polyposis: new evidence from whole transcriptome sequencing of colonic polyps.

BACKGROUND: Cronkhite-Canada syndrome (CCS) is a rare, nonhereditary disease characterized by diffuse gastrointestinal polyposis and ectodermal abnormalities. Although it has been proposed to be a chronic inflammatory condition, direct evidence of its pathogenesis is lacking. This study aims to investigate the pathophysiology of CCS by analyzing transcriptomic changes in the colonic microenvironment. METHODS: Next-generation sequencing-based genome-wide transcriptional profiling was performed on colonic hamartomatous polyps from four CCS patients and normal colonic mucosa from four healthy volunteers. Analyses of differential expression and multiple enrichment analyses were conducted from the molecular level to the cellular level. Quantitative real-time PCR (qRT-PCR) was carried out to validate the sequencing accuracy in samples from six CCS patients and six healthy volunteers. RESULTS: A total of 543 differentially expressed genes were identified, including an abundance of CC- and CXC-chemokines. Innate immune response-related pathways and processes, such as leukocyte chemotaxis, cytokine production, IL-17, TNF, IL-1 and NF-kB signaling pathways, were prominently enhanced in CCS colonic polyps. Upregulation of wound healing, epithelial-mesenchymal transition, Wnt, and PI3K-Akt signaling pathways were also observed. Enrichment analyses at different levels identified extracellular structure disorganization, dysfunction of the gut mucosal barrier, and increased angiogenesis. Validation by qRT-PCR confirmed increased expression of the LCN2, IL1B, CXCL1, and CXCL3 genes in CCS colonic polyps. CONCLUSIONS: This case-control whole transcriptome analysis of active CCS colonic hamartomatous polyps revealed intricate molecular pathways, emphasizing the role of the innate immune response, extracellular matrix disorganization, inflammatory cell infiltration, increased angiogenesis, and potential epithelial to mesenchymal transition. These findings supports CCS as a chronic inflammatory condition and sheds light on potential therapeutic targets, paving the way for more effective and personalized management of CCS in the future.

How do earthworms affect the pathway of sludge bio-stabilization via vermicomposting?

The synergy effects between earthworms and microorganisms promote nitrogen mineralization and enhance stabilization of organic matters in a vermicomposting system. However, the stabilization pathways of vermicomposting in the system remain unknown. The aim of this study was to investigate the effect of earthworms on the stabilization pathway and associated microbial population of waste activated sludge recycled by vermicomposting. The treatment of sludge with and without earthworms was conducted at 20 °C for 60 days. The trends in organic matter (OM), dissolved organic carbon (DOC), NH4+-N, electrical conductivity (EC), microbial biomass carbon (MBC), and dehydrogenase activity (DHA) were similar in both systems over time. At the end of the treatment, OM and DOC were significantly lower (p < 0.05), and EC, NH4+-N, and NO3--N were significantly higher (p < 0.05) in the vermicomposting group than in the control. Based on the statistical results of principal component analysis (PCA), it was proposed that the stabilization pathway in both treatment systems required a sequence of reactions characterized by the degradation of organic matter, accumulation of dissolved organic carbon, ammonification, and nitrification. Vermicomposting led to greater abundance and diversity (Shannon index) of 16S rDNA microbial species, but more even distribution in microbial community composition (Simpson index) than the control. However, the opposite performance for 18S rDNA microbes was observed. Vermicomposting enhanced the abundance of microorganisms involved in organic matter degradation and nitrification, facilitating the conversion of organic matter and favoring the nitrification. In short, the pathway of sludge bio-stabilization is not altered regardless of the addition of earthworms or not, which enables us to better understand vermicomposting process of sludge.

Bioinformatics analysis of carotid vulnerable plaques associated with the SARS-CoV-2 pattern.

The rupture of carotid artery vulnerable plaque plays a critical role in ischemic stroke, and the widely spread new coronavirus in recent years plays a certain role in the development of human carotid artery vulnerable plaque, we screened out 27 differential expression genes (DEGs) of stable plaque and vulnerable plaque associated with the new coronavirus. Through the construction of the protein-protein interaction (PPI) network, the Cathepsin B (CTSB) and Niemann-Pick Disease Type 2 (NPC2) were identified as crucial expression genes, and further, we confirmed the validity of core gene expression in two validation sets. Additionally, we discovered a significant connection between CTSB, NPC2 and 28 different kinds of immune cells in carotid plaque tissue. We screened out 65 target interacting drugs based on 10 differentially expressed genes through online tools and finally verified the high expression of 2 core genes in fragile plaques through clinical sample experiments. These findings imply that two core genes may be novel targets for molecular diagnostics and immunotherapy of vulnerable plaques.

Free Fatty Acids Induce Apoptosis of Mammary Epithelial Cells of Ketotic Dairy Cows via the Mito-ROS/NLRP3 Signaling Pathway.

Early lactation increases metabolic stress in ketotic dairy cows, leading to mitochondrial damage, apoptosis, and inflammatory response in mammary epithelial cells. The pyrin domain 3 (NLRP3) pathway involving the mitochondrial reactive oxygen species (Mito-ROS)-induced nucleotide-binding oligomerization domain-like receptor has been recognized as a key mechanism in this inflammatory response and cell apoptosis. This study aimed to elucidate the underlying regulatory mechanism of Mito-ROS-NLRP3 pathway-mediated mammary epithelial cell apoptosis in dairy cows with ketosis. Mitochondrial damage and cellular apoptotic program and NLRP3 inflammasome activation were observed in the mammary gland of ketotic cows. Similar damage was detected in MAC-T cells treated with exogenous fatty acids (FFAs). However, NLRP3 inhibitor MCC950 pretreatment or Mito-ROS scavenging by MitoTEMPO attenuated apoptosis in FFA-induced MAC-T cells by inhibiting the NLRP3 inflammasome pathway. These findings reveal that the Mito-ROS-NLRP3 pathway activation is a potent mechanism underlying mammary epithelial cell apoptosis in response to metabolic stress in ketotic dairy cows, which further contributes to reduced milk yield.

GPX2 promotes EMT and metastasis in non-small cell lung cancer by activating PI3K/AKT/mTOR/Snail signaling axis.

Lung cancer, with non-small cell lung cancer (NSCLC) being the main subtype, is the leading cause of cancer death worldwide, which is mainly due to the cancer metastasis. Glutathione peroxidase 2 (GPX2), an antioxidant enzyme, is involved in tumor progression and metastasis. Nevertheless, the role of GPX2 in NSCLC metastasis has not been clarified. In this study, we found that GPX2 expression was elevated in NSCLC tissues and high GPX2 expression was correlated with poor prognosis in patients with NSCLC. In addtion, GPX2 expression was related to the patient's clinicopathological features, including lymph node metastasis, tumor size, and TNM stage. Overexpression of GPX2 promoted epithelial-mesenchymal transition (EMT), migration, and invasion of NSCLC cells in vitro. Knockdown of GPX2 showed the opposite effects in vitro and inhibited the metastasis of NSCLC cells in nude mice. Furthermore, GPX2 reduced reactive oxygen species (ROS) accumulation and activated the PI3K/AKT/mTOR/Snail signaling axis. Therefore, our results indicate that GPX2 promotes EMT and metastasis of NSCLC cells by activating the PI3K/AKT/mTOR/Snail signaling axis via the removal of ROS. GPX2 may be an effective diagnostic and prognostic biomarker for NSCLC.

Mitochondrial Calcium Uniporter Regulator 1 (MCUR1) Relieves Mitochondrial Damage Induced by Lipopolysaccharide by Mediating Mitochondrial Ca2+ Homeostasis in Bovine Mammary Epithelial Cells.

The metabolic stress triggered by negative energy balance after calving induces mitochondrial damage of bovine mammary epithelial cells. Mitochondrial calcium uniporter regulator 1 (MCUR1) is a key protein-coding gene that mediates mitochondrial calcium ion (Ca2+) uptake and plays an important role in mediating homeostasis of mitochondria. The aim of the present study was to elucidate the effects of MCUR1-mediated Ca2+ homeostasis on mitochondria of bovine mammary epithelial cells in response to an inflammatory challenge with lipopolysaccharide (LPS). Exogenous LPS resulted in upregulation of the MCUR1 mRNA and protein abundance, mitochondrial Ca2+ content, and mitochondrial reactive oxygen species (Mito-ROS) content while decreasing mitochondrial membrane potential, causing mitochondrial damage, and increasing the rate of apoptosis. Ryanodine pretreatment attenuated the upregulation of the mitochondrial Ca2+ content and Mito-ROS content induced by LPS. Overexpression of MCUR1 increased the mitochondrial Ca2+ content and Mito-ROS content, while it decreased mitochondrial membrane potential, damaged mitochondria, and induced cell apoptosis. In addition, knockdown of MCUR1 by small interfering RNA attenuated LPS-induced mitochondrial dysfunction by inhibiting mitochondrial Ca2+ uptake. Our results revealed that exogenous LPS induces MCUR1-mediated mitochondrial Ca2+ overload in bovine mammary epithelial cells, which leads to mitochondrial injury. Thus, MCUR1-mediated Ca2+ homeostasis may be a potential therapeutic target against mitochondrial damage induced by metabolic challenges in bovine mammary epithelial cells.

The landscape of cryptic antisense transcription in human cancers reveals an oncogenic noncoding RNA in lung cancer.

Cryptic transcription initiation has been previously linked to activation of oncogenic transcripts. However, the prevalence and impact of cryptic antisense transcription from the opposite strand of protein-coding genes were mostly unknown in cancer. Applying a robust computational pipeline to publicly available transcriptome and epigenome datasets, we identified hundreds of previously unannotated cryptic antisense polyadenylated transcripts (CAPTs) that were enriched in tumor samples. We showed that the activation of cryptic antisense transcription was associated with increased chromatin accessibility and active histone marks. Accordingly, we found that many of the antisense transcripts were inducible by treatment of epigenetic drugs. Moreover, CRISPR-mediated epigenetic editing assays revealed that transcription of a noncoding RNA LRRK1-CAPT promoted LUSC cell proliferation, suggesting its oncogenic role. Our findings largely expand our understanding of cancer-associated transcription events, which may facilitate the development of novel strategies for cancer diagnosis and treatment.

A Salt-Tolerant Strain of Trichoderma longibrachiatum HL167 Is Effective in Alleviating Salt Stress, Promoting Plant Growth, and Managing Fusarium Wilt Disease in Cowpea.

Salt stress is a constraint factor in agricultural production and restricts crops yield and quality. In this study, a salt-tolerant strain of Trichoderma longibrachiatum HL167 was obtained from 64 isolates showing significant salt tolerance and antagonistic activity to Fusarium oxysporum. T. longibrachiatum HL167 inhibited F. oxysporum at a rate of 68.08% in 200 mM NaCl, penetrated F. oxysporum under 200 mM NaCl, and eventually induced F. oxysporum hyphae breaking, according to electron microscope observations. In the pot experiment, pretreatment of cowpea seedlings with T. longibrachiatum HL167 reduced the accumulation level of ROS in tissues and the damage caused by salt stress. Furthermore, in the field experiment, it was discovered that treating cowpea with T. longibrachiatum HL167 before root inoculation with F. oxysporum can successfully prevent and control the development of cowpea Fusarium wilt, with the best control effect reaching 61.54%. Moreover, the application of HL 167 also improved the K+/Na+ ratio of cowpea, alleviated the ion toxicity of salt stress on cowpea, and HL167 was found to effectively colonize the cowpea roots. T. longibrachiatum HL167, which normally survives in saline-alkali environments and has the functions of disease prevention and plant growth promotion capabilities, has important research implications for improving the saline-alkali soil environment and for the sustainable development of green agriculture.

The use of Chinese herbal medicines throughout the pregnancy life course and their safety profiles: a population-based cohort study.

BACKGROUND: Chinese herbal medicines have been long used among pregnant populations in China. However, despite the high susceptibility of this population to drug exposure, it continued to remain unclear about how often they were used, to what extent they were used at different pregnancy stages, and whether their use was based on sound safety profiles, particularly when used in combination with pharmaceutical drugs. OBJECTIVE: This descriptive cohort study aimed to systematically investigate the use of Chinese herbal medicines throughout pregnancy and their safety profiles. STUDY DESIGN: A large medication use cohort was developed by linking a population-based pregnancy registry and a population-based pharmacy database, which documented all prescriptions at both outpatients and inpatients from conception to 7 days after delivery, including pharmaceutical drugs and processed Chinese herbal medicine formulas that were approved by the regulatory authority and prepared under the guidance of national quality standards. The prevalence of the use of Chinese herbal medicine formulas, prescription pattern, and combination use of pharmaceutical drugs throughout pregnancy were investigated. Multivariable log-binomial regression was performed to assess temporal trends and further explore the potential characteristics associated with the use of Chinese herbal medicines. Of note, 2 authors independently conducted a qualitative systematic review of patient package inserts of the top 100 Chinese herbal medicine formulas used to identify their safety profiles. RESULTS: This study included 199,710 pregnancies; of those pregnancies, 131,235 (65.71%) used Chinese herbal medicine formulas, including 26.13% during pregnancy (corresponding to 14.00%, 8.91%, and 8.26% in the first, second, and third trimesters of pregnancy) and 55.63% after delivery. The peak uses of Chinese herbal medicines occurred between 5 and 10 weeks of gestation. The use of Chinese herbal medicines significantly increased over the years (from 63.28% in 2014 to 69.59% in 2018; adjusted relative risk, 1.11; 95% confidence interval, 1.10-1.13), which was particularly great during pregnancy (from 18.47% in 2014 to 32.46% in 2018; adjusted relative risk, 1.84; 95% confidence interval, 1.77-1.90). Our study observed 291,836 prescriptions involving 469 Chinese herbal medicine formulas, and the top 100 most used Chinese herbal medicines accounted for 98.28% of the total prescriptions. Of those, a third (33.39%) were dispensed at outpatient visits; 6.79% were external use, and 0.29% were administered intravenously. However, Chinese herbal medicines were very often prescribed in combination with pharmaceutical drugs (94.96% overall), involving 1175 pharmaceutical drugs with 1,667,459 prescriptions. The median of pharmaceutical drugs prescribed in combination with Chinese herbal medicines per pregnancy was 10 (interquartile range, 5-18). The systematic review of drug patient package inserts found that the 100 most frequently prescribed Chinese herbal medicines contained a total of 240 herb constituents (median, 4.5); 7.00% were explicitly indicated for pregnancy or postpartum conditions; 43.00% were reported with efficacy or safety data from randomized controlled trials. Information was lacking about whether the medications had any reproductive toxicity, were excreted in human milk, or crossed the placenta. CONCLUSION: The use of Chinese herbal medicines was prevalent throughout pregnancy and increased over the years. The use of Chinese herbal medicines peaked in the first trimester of pregnancy and was very often used in combination with pharmaceutical drugs. However, their safety profiles were mostly unclear or incomplete, suggesting a strong need for postapproval surveillance for the use of Chinese herbal medicines during pregnancy.

Activation of PINK1-mediated mitophagy protects bovine mammary epithelial cells against lipopolysaccharide-induced mitochondrial and inflammatory damage in vitro.

Increased metabolic stress during early lactation results in damage of mitochondria and inflammatory responses in bovine mammary epithelial cells, both of which could be aggravated by inhibition of mitophagy. PTEN-induced putative kinase 1 (PINK1)-mediated mitophagy is essential in the removal of damaged mitochondria and the regulation of inflammatory responses. The aim of the present study was to elucidate the role of PINK1-mediated mitophagy on mitochondrial damage and inflammatory responses in bovine mammary epithelial cells challenged with lipopolysaccharide (LPS). Exogenous LPS activated mitophagy and led to lower protein abundance of oxidative phosphorylation (OXPHOS) complexes (COI-V) and lower oxygen consumption rate (OCR) along with increased mitochondrial reactive oxygen species (Mito-ROS) content. These effects were also associated with increased protein abundance of Nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) in a time-dependent manner. Pretreatment with 3-Methyladenine (3-MA) or knockdown of PINK1 aggravated the downregulation of COI-V protein abundance, the increase in Mito-ROS content, and the protein abundance of NLRP3, Cleaved-Caspase-1 and IL-1ß induced by LPS. Overexpression of PINK1 activated mitophagy and alleviated LPS-induced NLRP3 inflammasome activation by reducing Mito-ROS production. Overall, the data suggested that PINK1-mediated mitophagy is a crucial anti-inflammatory mechanism that removes damaged mitochondria in bovine mammary epithelial cells experiencing an increased inflammatory load.

Radiosensitization-Related Cuproptosis LncRNA Signature in Non-Small Cell Lung Cancer.

A new treatment modality targeting cuproptosis is gradually entering the public horizon. Cuproptosis is a new form of regulated cell death distinct from ferroptosis, apoptosis, autophagy, and necrosis. Previous studies have discovered that the copper level varies considerably in various cancers and that an increase in copper content is directly associated with the proliferation and metastasis of cancer cells. In non-small cell lung cancer (NSCLC) after radiation, the potential utility of cuproptosis-related long noncoding RNAs (lncRNAs) is still unclear. This research aimed to develop a prediction signature based on lncRNAs associated with cuproptosis to predict the prognosis of NSCLC patients following radiation. Methods: Expression data of primary tumors and adjacent solid tissues were downloaded from The Cancer Genome Atlas (TCGA) database, along with the corresponding clinical and mutational data. Univariate and multivariate COX analyses and LASSO regression analyses were performed to obtain a predictive signature of lncRNAs associated with cuproptosis. The data were randomly grouped into a training group used for model construction and a test group used for model validation. The model was validated by drawing a survival curve, risk curve, independent prognostic analysis, ROC curve PFS analysis, etc. Results: The lncRNA signature consisting of six cuproptosis-related lncRNAs (AC104088.1, PPP4R3B-DT, AC006042.3, LUCAT1, HHLA3-AS1, and LINC02029) was used to predict the prognosis of patients. Among them, there were three high-risk lncRNAs (LUCAT1, HHLA3-AS1, and LINC02029) with HR > 1 and three protective lncRNAs (AC104088.1, PPP4R3B-DT, and AC006042.3), with an HR < 1. Data analysis demonstrated that the cuproptosis-related lncRNA signatures could well predict the prognosis of NSCLC patients after radiation. Patients in the high-risk category receive a worse prognosis than those in the low-risk group. Cuproptosis-related risk prediction demonstrated better predictive qualities than age, gender, and pathological stage factors. Conclusion: The risk proposed model can independently predict the prognosis of NSCLC patients after radiotherapy, provide a foundation for the role of cuproptosis-related lncRNAs in NSCLC after radiotherapy, and provide a clinical strategy for radiotherapy combined with cuproptosis in NSCLC patients.

ß-hydroxybutyrate impairs monocyte function via the ROS-NLR family pyrin domain-containing three inflammasome (NLRP3) pathway in ketotic cows.

Cows with ketosis display severe metabolic stress and immune dysfunction which renders them more susceptible to infections. Monocytes, one of the major subtypes of white blood cells, play an important role in innate immune defense against infections. Thus, the aim of this study was to investigate alterations in immune function, reactive oxygen species (ROS) production and activity of the NLR family pyrin domain containing 3 (NLRP3) inflammasome pathway in monocytes (CD14+) of cows with clinical ketosis (CK). Twelve healthy multiparous Holstein cows [blood ß-hydroxybutyrate (BHB) concentration < 1.2 mM] and 12 cows with CK (BHB > 3.0 mM) at 3 to 14 days in milk were used for blood sample collection. To determine effects of BHB on phagocytosis, ROS and protein abundance of the NLRP3 inflammasome pathway in vitro, monocytes isolated from healthy cows were treated with 3.0 mM BHB for 0, 6, 12 or 24 h. Dry matter intake (22.7 vs. 19.0 kg) was lower in cows with CK. Serum concentrations of fatty acids (0.30 vs. 0.88 mM) and BHB (0.52 vs. 3.78 mM) were greater in cows with CK, whereas concentration of glucose was lower (4.09 vs. 2.23 mM). The adhesion, migration and phagocytosis of monocytes were lower in cows with CK, but apoptosis and ROS content were greater. Protein abundance of NLRP3, cysteinyl aspartate specific proteinase 1 (caspase 1) and interleukin-1B p17 (IL1B p17) were greater in monocytes of cows with CK, while abundance of NADPH oxidase isoform 2 (NOX2) was lower. Compared with 0 h BHB, ROS content and apoptosis were greater in the monocytes challenged for 6, 12 or 24 h BHB. Compared with 0 h BHB, protein abundance of NLRP3, caspase 1, IL1B p17 and concentration of IL1B in medium were greater in the monocytes challenged for 6, 12 or 24 h BHB. However, compared with 0 h BHB, protein abundance of NOX2 and phagocytosis of monocytes were lower in the monocytes challenged for 6, 12 or 24 h BHB. Overall, the data suggested that exogenous BHB activated the ROS-NLRP3 pathway, which might be partly responsible for immune dysfunction of dairy cows with CK.