Inter-species Transcriptomic Analysis Reveals a Constitutive Adaptation Against Oxidative Stress for the Highly Virulent Leptospira Species.

Transcriptomic analyses across large scales of evolutionary distance have great potential to shed light on regulatory evolution but are complicated by difficulties in establishing orthology and limited availability of accessible software. We introduce here a method and a graphical user interface wrapper, called Annotator-RNAtor, for performing interspecies transcriptomic analysis and studying intragenus evolution. The pipeline uses third-party software to infer homologous genes in various species and highlight differences in the expression of the core-genes. To illustrate the methodology and demonstrate its usefulness, we focus on the emergence of the highly virulent Leptospira subclade known as P1+, which includes the causative agents of leptospirosis. Here, we expand on the genomic study through the comparison of transcriptomes between species from P1+ and their related P1- counterparts (low-virulent pathogens). In doing so, we shed light on differentially expressed pathways and focused on describing a specific example of adaptation based on a differential expression of PerRA-controlled genes. We showed that P1+ species exhibit higher expression of the katE gene, a well-known virulence determinant in pathogenic Leptospira species correlated with greater tolerance to peroxide. Switching PerRA alleles between P1+ and P1- species demonstrated that the lower repression of katE and greater tolerance to peroxide in P1+ species was solely controlled by PerRA and partly caused by a PerRA amino-acid permutation. Overall, these results demonstrate the strategic fit of the methodology and its ability to decipher adaptive transcriptomic changes, not observable by comparative genome analysis, that may have been implicated in the emergence of these pathogens.

Identifying the predictive role and the related drugs of oxidative stress genes in the hepatocellular carcinoma.

BACKGROUND AND AIMS: Oncogenesis and tumor development have been related to oxidative stress (OS). The potential diagnostic utility of OS genes in hepatocellular carcinoma (HCC), however, remains uncertain. As a result, this work aimed to create a novel OS related-genes signature that could be used to predict the survival of HCC patients and to screen OS related-genes drugs that might be used for HCC treatment. METHODS: We used The Cancer Genome Atlas (TCGA) database and the Gene Expression Omnibus (GEO) database to acquire mRNA expression profiles and clinical data for this research and the GeneCards database to obtain OS related-genes. Following that, biological functions from Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed on differentially expressed OS-related genes (DEOSGs). Subsequently, the prognostic risk signature was constructed based on DEOSGs from the TCGA data that were screened by using univariate cox analysis, and the Least Absolute Shrinkage and Selection Operator (LASSO) regression, and multivariate cox analysis. At the same time, we developed a prognostic nomogram of HCC patients based on risk signature and clinical-pathological characteristics. The GEO data was used for validation. We used the receiver operating characteristic (ROC) curve, calibration curves, and Kaplan-Meier (KM) survival curves to examine the prediction value of the risk signature and nomogram. Finally, we screened the differentially expressed OS genes related drugs. RESULTS: We were able to recognize 9 OS genes linked to HCC prognosis. In addition, the KM curve revealed a statistically significant difference in overall survival (OS) between the high-risk and low-risk groups. The area under the curve (AUC) shows the independent prognostic value of the risk signature model. Meanwhile, the ROC curves and calibration curves show the strong prognostic power of the nomogram. The top three drugs with negative ratings were ZM-336372, lestaurtinib, and flunisolide, all of which inversely regulate different OS gene expressions. CONCLUSION: Our findings indicate that OS related-genes have a favorable prognostic value for HCC, which sheds new light on the relationship between oxidative stress and HCC, and suggests potential therapeutic strategies for HCC patients.

An Intricate Network Involving the Argonaute ALG-1 Modulates Organismal Resistance to Oxidative Stress.

Cellular response to redox imbalance is crucial for organismal health. microRNAs are implicated in stress responses. ALG-1, the C. elegans ortholog of human AGO2, plays an essential role in microRNA processing and function. Here we investigated the mechanisms governing ALG-1 expression in C. elegans and the players controlling lifespan and stress resistance downstream of ALG-1. We show that upregulation of ALG-1 is a shared feature in conditions linked to increased longevity (e.g., germline-deficient glp-1 mutants). ALG-1 knockdown reduces lifespan and oxidative stress resistance, while overexpression enhances survival against pro-oxidant agents but not heat or reductive stress. R02D3.7 represses alg-1 expression, impacting oxidative stress resistance at least in part via ALG-1. microRNAs upregulated in glp-1 mutants (miR-87-3p, miR-230-3p, and miR-235-3p) can target genes in the protein disulfide isomerase pathway and protect against oxidative stress. This study unveils a tightly regulated network involving transcription factors and microRNAs which controls organisms' ability to withstand oxidative stress.

Lipopolysaccharide binding protein resists hepatic oxidative stress by regulating lipid droplet homeostasis.

Oxidative stress-induced lipid accumulation is mediated by lipid droplets (LDs) homeostasis, which sequester vulnerable unsaturated triglycerides into LDs to prevent further peroxidation. Here we identify the upregulation of lipopolysaccharide-binding protein (LBP) and its trafficking through LDs as a mechanism for modulating LD homeostasis in response to oxidative stress. Our results suggest that LBP induces lipid accumulation by controlling lipid-redox homeostasis through its lipid-capture activity, sorting unsaturated triglycerides into LDs. N-acetyl-L-cysteine treatment reduces LBP-mediated triglycerides accumulation by phospholipid/triglycerides competition and Peroxiredoxin 4, a redox state sensor of LBP that regulates the shuttle of LBP from LDs. Furthermore, chronic stress upregulates LBP expression, leading to insulin resistance and obesity. Our findings contribute to the understanding of the role of LBP in regulating LD homeostasis and against cellular peroxidative injury. These insights could inform the development of redox-based therapies for alleviating oxidative stress-induced metabolic dysfunction.

Machine learning-based identification of novel hub genes associated with oxidative stress in lupus nephritis: implications for diagnosis and therapeutic targets.

BACKGROUND: Lupus nephritis (LN) is a complication of SLE characterised by immune dysfunction and oxidative stress (OS). Limited options exist for LN. We aimed to identify LN-related OS, highlighting the need for non-invasive diagnostic and therapeutic approaches. METHODS: LN-differentially expressed genes (DEGs) were extracted from Gene Expression Omnibus datasets (GSE32591, GSE112943 and GSE104948) and Molecular Signatures Database for OS-associated DEGs (OSEGs). Functional enrichment analysis was performed for OSEGs related to LN. Weighted gene co-expression network analysis identified hub genes related to OS-LN. These hub OSEGs were refined as biomarker candidates via least absolute shrinkage and selection operator. The predictive value was validated using receiver operating characteristic (ROC) curves and nomogram for LN prognosis. We evaluated LN immune cell infiltration using single-sample gene set enrichment analysis and CIBERSORT. Additionally, gene set enrichment analysis explored the functional enrichment of hub OSEGs in LN. RESULTS: The study identified four hub genes, namely STAT1, PRODH, TXN2 and SETX, associated with OS related to LN. These genes were validated for their diagnostic potential, and their involvement in LN pathogenesis was elucidated through ROC and nomogram. Additionally, alterations in immune cell composition in LN correlated with hub OSEG expression were observed. Immunohistochemical analysis reveals that the hub gene is most correlated with activated B cells and CD8 T cells. Finally, we uncovered that the enriched pathways of OSEGs were mainly involved in the PI3K-Akt pathway and the Janus kinase-signal transducer and activator of transcription pathway. CONCLUSION: These findings contribute to advancing our understanding of the complex interplay between OS, immune dysregulation and molecular pathways in LN, laying a foundation for the identification of potential diagnostic biomarkers and therapeutic targets.

Clinical mutations in the TERT and TERC genes coding for telomerase components induced oxidative stress, DNA damage at telomeres and cell apoptosis besides decreased telomerase activity.

Telomeres are nucleoprotein structures at the end of chromosomes that maintain their integrity. Mutations in genes coding for proteins involved in telomere protection and elongation produce diseases such as dyskeratosis congenita or idiopathic pulmonary fibrosis known as telomeropathies. These diseases are characterized by premature telomere shortening, increased DNA damage and oxidative stress. Genetic diagnosis of telomeropathy patients has identified mutations in the genes TERT and TERC coding for telomerase components but the functional consequences of many of these mutations still have to be experimentally demonstrated. The activity of twelve TERT and five TERC mutants, five of them identified in Spanish patients, has been analyzed. TERT and TERC mutants were expressed in VA-13 human cells that express low telomerase levels and the activity induced was analyzed. The production of reactive oxygen species, DNA oxidation and TRF2 association at telomeres, DNA damage response and cell apoptosis were determined. Most mutations presented decreased telomerase activity, as compared to wild-type TERT and TERC. In addition, the expression of several TERT and TERC mutants induced oxidative stress, DNA oxidation, DNA damage, decreased recruitment of the shelterin component TRF2 to telomeres and increased apoptosis. These observations might indicate that the increase in DNA damage and oxidative stress observed in cells from telomeropathy patients is dependent on their TERT or TERC mutations. Therefore, analysis of the effect of TERT and TERC mutations of unknown function on DNA damage and oxidative stress could be of great utility to determine the possible pathogenicity of these variants.

miR-214 aggravates oxidative stress in thalassemic erythroid cells by targeting ATF4.

Oxidative damage to erythroid cells plays a key role in the pathogenesis of thalassemia. The oxidative stress in thalassemia is potentiated by heme, nonheme iron, and free iron produced by the Fenton reaction, due to degradation of the unstable hemoglobin and iron overload. In addition, the levels of antioxidant enzymes and molecules are significantly decreased in erythrocytes in α- and ß-thalassemia. The control of oxidative stress in red blood cells (RBCs) is known to be mediated by microRNAs (miRNAs). In erythroid cells, microR-214 (miR-214) has been reported to respond to external oxidative stress. However, the molecular mechanisms underlying this phenomenon remain unclear, especially during thalassemic erythropoiesis. In the present study, to further understand how miR-214 aggravates oxidative stress in thalassemia erythroid cells, we investigated the molecular mechanism of miR-214 and its regulation of the oxidative status in thalassemia erythrocytes. We have reported a biphasic expression of miR-214 in ß- and α-thalassemia. In the present study the effect of miR-214 expression was investigated by using miR -inhibitor and -mimic transfection in erythroid cell lines induced by hemin. Our study showed a biphasic expression of miR-214 in ß- and α-thalassemia. Subsequently, we examined the effect of miR-214 on erythroid differentiation in thalassemia. Our study reveals the loss-of-function of miR-214 during translational activation of activating transcription factor 4 mRNA, leading to decreased reactive oxygen species levels and increased glutathione levels in thalassemia erythroid cell. Our results suggest that the expression of activating transcription factor 4 regulated by miR-214 is important for oxidative stress modulation in thalassemic erythroid cells. Our findings can help to better understand the molecular mechanism of miRNA and transcription factors in regulation of oxidative status in erythroid cells, particularly in thalassemia, and could be useful for managing and relieving severe anemia symptoms in patients in the future.

Chk2 Modulates Bmi1-Deficiency-Induced Renal Aging and Fibrosis via Oxidative Stress, DNA Damage, and p53/TGFß1-Induced Epithelial-Mesenchymal Transition.

Renal aging may lead to fibrosis and dysfunction, yet underlying mechanisms remain unclear. We explored whether deficiency of the Polycomb protein Bmi1 causes renal aging via DNA damage response (DDR) activation, inducing renal tubular epithelial cell (RTEC) senescence and epithelial-mesenchymal transition (EMT). Bmi1 knockout mice exhibited oxidative stress, DDR activation, RTEC senescence, senescence-associated secretory phenotype (SASP), and age-related fibrosis in kidneys. Bmi1 deficiency impaired renal structure and function, increasing serum creatinine/urea, reducing creatinine clearance, and decreasing cortical thickness and glomerular number. However, knockout of the serine-threonine kinase Chk2 alleviated these aging phenotypes. Transcriptomics identified transforming growth factor beta 1 (TGFß1) upregulation in Bmi1-deficient RTECs, but TGFß1 was downregulated upon Chk2 knockout. The tumor suppressor protein p53 transcriptionally activated TGFß1, promoting EMT in RTECs. Bmi1 knockout or oxidative stress (induced with H2O2) increased TGFß1 expression, and EMT in RTECs and was partly reversed by p53 inhibition. Together, Bmi1 deficiency causes oxidative stress and DDR-mediated RTEC senescence/SASP, thus activating p53 and TGFß1 to induce EMT and age-related fibrosis. However, blocking DDR (via Chk2 knockout) or p53 ameliorates these changes. Our study reveals mechanisms whereby Bmi1 preserves renal structure and function during aging by suppressing DDR and p53/TGFß1-mediated EMT. These pathways represent potential targets for detecting and attenuating age-related renal decline.

Dynamics of Mitochondrial DNA Copy Number and Membrane Potential in Mouse Pre-Implantation Embryos: Responses to Diverse Types of Oxidative Stress.

Mitochondria undergo a myriad of changes during pre-implantation embryo development, including shifts in activity levels and mitochondrial DNA (mtDNA) replication. However, how these distinct aspects of mitochondrial function are linked and their responsiveness to diverse stressors is not well understood. Here, we show that mtDNA content increased between 8-cell embryos and the blastocyst stage, with similar copy numbers per cell in the inner cell mass (ICM) and trophectoderm (TE). In contrast, mitochondrial membrane potential (MMP) was higher in TE than ICM. Culture in ambient oxygen (20% O2) altered both aspects of mitochondrial function: the mtDNA copy number was upregulated in ICM, while MMP was diminished in TE. Embryos cultured in 20% O2 also exhibited delayed development kinetics, impaired implantation, and reduced mtDNA levels in E18 fetal liver. A model of oocyte mitochondrial stress using rotenone showed only a modest effect on on-time development and did not alter the mtDNA copy number in ICM; however, following embryo transfer, mtDNA was higher in the fetal heart. Lastly, endogenous mitochondrial dysfunction, induced by maternal age and obesity, altered the blastocyst mtDNA copy number, but not within the ICM. These results demonstrate that mitochondrial activity and mtDNA content exhibit cell-specific changes and are differentially responsive to diverse types of oxidative stress during pre-implantation embryogenesis.

ER stress and ERO1: Potential therapeutic targets for inherited myopathies.

Selenoprotein N-related myopathy (SEPN1-RM) is a genetic disease that causes muscle weakness and respiratory failure. Germani et al.1 demonstrate that diaphragm weakness in SEPN1-RM is prevented by the inhibition of ER stress or ERO1 oxidoreductase regulated by transcription factor CHOP.

Association between the oxidative balance score and thyroid function: Results from the NHANES 2007-2012 and Mendelian randomization study.

BACKGROUND: Oxidative stress is a significant contributor to the development of various diseases, and the oxidative balance score (OBS) is a valuable tool for assessing the impact of dietary and lifestyle factors on oxidative stress in humans. Nevertheless, the precise relationship between OBS and thyroid function in adults remains elusive. METHODS: This cross-sectional study comprised 6222 adult participants drawn from the National Health and Nutrition Examination Survey (NHANES) conducted from 2007 to 2012. Employing weighted multivariable linear regression modeling, the study estimated the connection between OBS quartiles and thyroid functions. The causal relationship between OBS components and thyroid function was analyzed by Mendelian randomization (MR). RESULTS: We found a significant negative correlation between OBS and free thyroxine (FT4) and total thyroxine (TT4). Univariate and multivariate MR Analyses showed a causal relationship between BMI and FT4. Copper, smoking, and riboflavin showed a causal relationship with FT4 after moderation. CONCLUSION: We found that a lifestyle high in antioxidant exposure reduced FT4 and TT4 levels in the population. We suggest that BMI, Copper, and Riboflavin are important factors in the regulation of FT4 levels.

Fluorescent proteins generate a genetic color polymorphism and counteract oxidative stress in intertidal sea anemones.

Fluorescent proteins (FPs) are ubiquitous tools in research, yet their endogenous functions in nature are poorly understood. In this work, we describe a combination of functions for FPs in a clade of intertidal sea anemones whose FPs control a genetic color polymorphism together with the ability to combat oxidative stress. Focusing on the underlying genetics of a fluorescent green "Neon" color morph, we show that allelic differences in a single FP gene generate its strong and vibrant color, by increasing both molecular brightness and FP gene expression level. Natural variation in FP sequences also produces differences in antioxidant capacity. We demonstrate that these FPs are strong antioxidants that can protect live cells against oxidative stress. Finally, based on structural modeling of the responsible amino acids, we propose a model for FP antioxidant function that is driven by molecular surface charge. Together, our findings shed light on the multifaceted functions that can co-occur within a single FP and provide a framework for studying the evolution of fluorescence as it balances spectral and physiological functions in nature.

Construction of an oxidative stress-associated genes signature in breast cancer by machine learning algorithms.

OBJECTIVE: To construct a prognostic model of a breast cancer-related oxidative stress-related gene (OSRG) signature using machine learning algorithms. METHODS: The OSRGs of breast cancer were constructed by least absolute shrinkage and selection operator (LASSO) and multivariate Cox regression analysis. The Cancer Genome Atlas (TCGA) was used to analyse the gene expression and prognostic value. The Human Protein Atlas was used to analyse the protein expression of hub genes. Receiver operating characteristic analysis, calibration curve and decision curve analysis were used to predict the stability of this model. RESULTS: The area under the curve of 1-, 3- and 5-year overall survival were 0.751, 0.707 and 0.645 in the TCGA training dataset; and 0.692, 0.678 and 0.602 in the TCGA testing dataset, respectively. Calibration plot showed good agreement between predicted probabilities and observed outcomes. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA) pathway analysis indicated that multiple cancer-related pathways were highly enriched in the high-risk group. Immune infiltration analysis showed immune cells and their functions may play a key role in the development and mechanism of breast cancer. CONCLUSIONS: This new OSRG signature was associated with the immune infiltration and it might be useful in predicting the prognosis in patients with breast cancer.

Single-cell RNA combined with bulk RNA analysis to explore oxidative stress and energy metabolism factors and found a new prostate cancer oncogene MXRA8.

BACKGROUND: Prostate cancer is the most common malignancy among men worldwide, and its diagnosis and treatment are challenging due to its heterogeneity. METHODS: Integrating single-cell RNA sequencing (scRNA-seq) and bulk RNA-seq data, we identified two molecular subtypes of prostate cancer based on dysregulated genes involved in oxidative stress and energy metabolism. We constructed a risk score model (OMR) using common differentially expressed genes, which effectively evaluated prostate cancer prognosis. RESULTS: Our analysis demonstrated a significant correlation between the risk score model and various factors, including tumor immune microenvironment, genomic variations, chemotherapy resistance, and immune response. Notably, patients with low-risk scores exhibited increased sensitivity to chemotherapy and immunotherapy compared to those with high-risk scores, indicating the model's potential to predict patient response to treatment. Additionally, our investigation of MXRA8 in prostate cancer showed significant upregulation of this gene in the disease as confirmed by PCR and immunohistochemistry. Functional assays including CCK-8, transwell, plate cloning, and ROS generation assay demonstrated that depletion of MXRA8 reduced the proliferative, invasive, migratory capabilities of PC-3 cells, as well as their ROS generation capacity. CONCLUSIONS: Our study highlights the potential of oxidative stress and energy metabolism-related genes as prognostic markers and therapeutic targets in prostate cancer. The integration of scRNA-seq and bulk RNA-seq data enables a better understanding of prostate cancer heterogeneity and promotes personalized treatment development. Additionally, we identified a novel oncogene MXRA8 in prostate cancer.

Transcriptional analysis of oxidative and nitrosative stress on oral opportunistic Candida albicans.

The yeast Candida albicans is one of the most aggressive opportunistic pathogens in immunocompromised patients. The ability of the yeast to withstand stresses and radicals is of great concern. In the present study, four isolates of C. albicans were taken from patients with oral candidiasis and grown on RPMI for 24 hours at 37°C. Then, they were exposed to various concentrations of oxidative (H2O2) and nitrosative (HNO3) stress for two hours, and gene expression rates were measured through RT-PCR. After initial biofilm formation steps and growth validation, RNA extracted from the yeast and gene expression status were evaluated. Upon treatment with H2O2, the gene expression profile for ALS1, MLH1, and EXO1 showed approximately a fold increase in expression. While within HNO3 the yeast gene expression exhibited a dramatic increase in ALS1 up to 217 folds, while others such as MLH1, HWP1, and ERG11 showed a one-fold increase in the expression rate. The findings of this research indicate a considerable expression activity within the biofilm of Candida albicans, increased rate of DNA mismatch repair and break fixation may indicate the ability of the yeast to tolerate high concentrations of free radicals. It paves the way toward understanding the pathogenicity of the yeast and its survival capability inside macrophages. The study also revealed that the biofilm strategy of the yeast is more active within these stresses.

miR-107-5p ameliorates neurological damage, oxidative stress, and immune responses in mice with Alzheimer's disease by suppressing the Toll-like receptor 4 (TLR4)/nuclear factor-kappaB(NF-κB) pathway.

Alzheimer's disease (AD) is a progressively debilitating neurodegenerative condition primarily affecting the elderly. Emerging research suggests that microRNAs (miRNAs) play a role in the development of AD. This study investigates the impact of miR-107-5p on neurological damage, oxidative stress, and immune responses in AD. We utilized APP/PS1 mice as AD mouse models and C57BL/6 J mice as controls. AD mice received treatment with agomir miR-107-5p (to overexpress miR-107-5p) or BAY11-7082 (an NF-κB pathway inhibitor). We evaluated learning and memory abilities through the Morris water maze test. Histopathological changes, hippocampal neuron distribution, and apoptosis were assessed using hematoxylin-eosin, Nissl, and TUNEL staining. Reactive oxygen species (ROS) levels, amyloid-Aß (Aß1-40/42) contents, and inflammatory factors (TNF-α, IL-6, IL-1ß) in hippocampal tissues were measured using ROS kits and enzyme-linked immunosorbent assay (ELISA). Microglial activation in hippocampal tissues was observed under a fluorescence microscope. miR-107-5p's binding to TLR4 was predicted via the TargetScan database and confirmed through a dual-luciferase assay. miR-107-5p expression, along with TLR4, APOE, and TREM2 in hippocampal tissue homogenate, and NF-κB p65 protein expression in the nucleus and cytoplasm were assessed via RT-qPCR and Western blot. Overexpression of miR-107-5p ameliorated hippocampal neurological damage, oxidative stress, and immune responses. This was evidenced by improved enhanced learning/memory abilities, reduced Aß1-40 and Aß1-42 levels, diminished neuronal injuries, decreased ROS and TNF-α, IL-6, and IL-1ß levels, increased APOE and TREM2 levels, and suppressed microglial activation. miR-107-5p directly targeted and inhibited TLR4 expression, leading to reduced nuclear translocation of NF-κB p65 in the NF-κB pathway. Inhibition of the NF-κB pathway similarly improved neurological damage, oxidative stress, and immune response in AD mice. miR-107-5p exerts its beneficial effects by suppressing the TLR4/NF-κB pathway, ultimately ameliorating neurological damage, oxidative stress, and immune responses in AD mice.

MicroRNA-124 negatively regulates STAT3 to alleviate hypoxic-ischemic brain damage by inhibiting oxidative stress.

MicroRNA-124 (miR-124) is implicated in various neurological diseases; however, its significance in hypoxic-ischaemic brain damage (HIBD) remains unclear. This study aimed to elucidate the underlying pathophysiological mechanisms of miR-124 in HIBD. In our study performed on oxygen-glucose deprivation followed by reperfusion (OGD)/R-induced primary cortical neurons, a substantial reduction in miR-124 was observed. Furthermore, the upregulation of miR-124 significantly mitigated oxidative stress, apoptosis, and mitochondrial impairment. We demonstrated that miR-124 interacts with the signal transducer and activator of transcription 3 (STAT3) to exert its biological function using the dual-luciferase reporter gene assay. As the duration of OGD increased, miR-124 exhibited a negative correlation with STAT3. STAT3 overexpression notably attenuated the protective effects of miR-124 mimics, while knockdown of STAT3 reversed the adverse effects of the miR-124 inhibitor. Subsequently, we conducted an HIBD model in rats. In vivo experiments, miR-124 overexpression attenuated cerebral infarction volume, cerebral edema, apoptosis, oxidative stress, and improved neurological function recovery in HIBD rats. In summary, the neuroprotective effects of the miR-124/STAT3 axis were confirmed in the HIBD model. MiR-124 may serve as a potential biomarker with significant therapeutic implications for HIBD.

MicroRNA-320a enhances LRWD1 expression through the AGO2/FXR1-dependent pathway to affect cell behaviors and the oxidative stress response in human testicular embryonic carcinoma cells.

BACKGROUND: Testicular cancer is fairly rare but can affect fertility in adult males. Leucine-rich repeats- and WD repeat domain-containing protein 1 (LRWD1) is a sperm-specific marker that mainly affects sperm motility in reproduction. Our previous study demonstrated the impact of LRWD1 on testicular cancer development; however, the underlying mechanisms remain unclear. METHODS: In this study, various plasmids associated with LRWD1 and miR-320a manipulation were used to explore the roles and regulatory effects of these molecules in NT2D1 cellular processes. A Dual-Glo luciferin-luciferase system was used to investigate LRWD1 transcriptional activity, and qRT-PCR and western blotting were used to determine gene and protein expression. RESULTS: The results suggested that miR-320a positively regulated LRWD1 and positively correlated with NT2D1 cell proliferation but negatively correlated with cell migration and invasion ability. In addition, the miRNA-ribonucleoprotein complex AGO2/FXR1 was shown to be essential in the mechanism by which miR-320a regulates LRWD1 mRNA expression. As miR-320a was required to regulate LRWD1 expression through the AGO2 and FXR1 complex, eEF2 and eLF4E were also found to be involved in miR-320a increasing LRWD1 expression. Furthermore, miR-320a and LRWD1 were responsive to oxidative stress, and NRF2 was affected by the presence of miR-320a in response to ROS stimulation. CONCLUSIONS: This is the first study showing the role of miR-320a in upregulating the testicular cancer-specific regulator LRWD1 and the importance of the AGO2/FXR1 complex in miR-320a-mediated upregulation of LRWD1 during testicular cancer progression.

Sequestrase chaperones protect against oxidative stress-induced protein aggregation and [PSI+] prion formation.

Misfolded proteins are usually refolded to their functional conformations or degraded by quality control mechanisms. When misfolded proteins evade quality control, they can be sequestered to specific sites within cells to prevent the potential dysfunction and toxicity that arises from protein aggregation. Btn2 and Hsp42 are compartment-specific sequestrases that play key roles in the assembly of these deposition sites. Their exact intracellular functions and substrates are not well defined, particularly since heat stress sensitivity is not observed in deletion mutants. We show here that Btn2 and Hsp42 are required for tolerance to oxidative stress conditions induced by exposure to hydrogen peroxide. Btn2 and Hsp42 act to sequester oxidized proteins into defined PQC sites following ROS exposure and their absence leads to an accumulation of protein aggregates. The toxicity of protein aggregate accumulation causes oxidant sensitivity in btn2 hsp42 sequestrase mutants since overexpression of the Hsp104 disaggregase rescues oxidant tolerance. We have identified the Sup35 translation termination factor as an in vivo sequestrase substrate and show that Btn2 and Hsp42 act to suppress oxidant-induced formation of the yeast [PSI+] prion, which is the amyloid form of Sup35. [PSI+] prion formation in sequestrase mutants does not require IPOD (insoluble protein deposit) localization which is the site where amyloids are thought to undergo fragmentation and seeding to propagate their heritable prion form. Instead, both amorphous and amyloid Sup35 aggregates are increased in btn2 hsp42 mutants consistent with the idea that prion formation occurs at multiple intracellular sites during oxidative stress conditions in the absence of sequestrase activity. Taken together, our data identify protein sequestration as a key antioxidant defence mechanism that functions to mitigate the damaging consequences of protein oxidation-induced aggregation.

Methyltransferase METTL3-mediated maturation of miR-4654 facilitates high glucose-induced apoptosis and oxidative stress in lens epithelial cells via decreasing SOD2.

N6-methyladenosine (m6 A) modification has been reported to have roles in modulating the development of diabetic cataract (DC). Methyltransferase-like 3 (METTL3) is a critical m6 A methyltransferase involving in m6 A modification activation. Here, we aimed to explore the action and mechanism of METTL3-mediated maturation of miR-4654 in DC progression. Human lens epithelial cells (HLECs) were exposed to high glucose (HG) to imitate DC condition in vitro. Levels of genes and proteins were tested via qRT-PCR and western blotting assays. The proliferation and apoptosis of HLECs were evaluated by cell counting kit-8, 5-ethynyl-2'-deoxyuridine (EdU), and flow cytometry assays, respectively. Oxidative stress was analyzed by detecting the contents of reactive oxygen species (ROS), superoxide dismutase (SOD) and malondialdehyde (MDA). The binding of miR-4654 and SOD2 was confirmed by dual-luciferase reporter assay. The m6 A-RNA immunoprecipitation (MeRIP) assay detected the m6 A modification profile. Thereafter, we found that miR-4654 expression was elevated in DC samples and HG-induced HLECs. MiR-4654 knockdown reversed HG-mediated apoptosis and oxidative stress in HLECs. Mechanistically, miR-4654 directly targeted SOD2, silencing of SOD2 abolished the protective effects of miR-4654 knockdown on HLECs under HG condition. In addition, METTL3 induced miR-4654 maturation through promoting pri-miR-4654 m6 A modification, thereby increasing miR-4654 content in HLECs. METTL3 was highly expressed in DC samples and HG-induced HLECs, METTL3 deficiency protected HLECs against HG-mediated apoptotic and oxidative injury via down-regulating miR-4654. In all, METTL3 induced miR-4654 maturation in a m6 A-dependent manner, which was then reduced SOD2 expression, thus promoting apoptosis and oxidative stress in HLECs, suggesting a novel path for DC therapy.