Dynamic genomic changes in methotrexate-resistant human cancer cell lines beyond DHFR amplification suggest potential new targets for preventing drug resistance.

BACKGROUND: Although DHFR gene amplification has long been known as a major mechanism for methotrexate (MTX) resistance in cancer, the early changes and detailed development of the resistance are not yet fully understood. METHODS: We performed genomic, transcriptional and proteomic analyses of human colon cancer cells with sequentially increasing levels of MTX-resistance. RESULTS: The genomic amplification evolved in three phases (pre-amplification, homogenously staining region (HSR) and extrachromosomal DNA (ecDNA)). We confirm that genomic amplification and increased expression of DHFR, with formation of HSRs and especially ecDNAs, is the major driver of resistance. However, DHFR did not play a detectable role in the early phase. In the late phase (ecDNA), increase in FAM151B protein level may also have an important role by decreasing sensitivity to MTX. In addition, although MSH3 and ZFYVE16 may be subject to different posttranscriptional regulations and therefore protein expressions are decreased in ecDNA stages compared to HSR stages, they still play important roles in MTX resistance. CONCLUSION: The study provides a detailed evolutionary trajectory of MTX-resistance and identifies new targets, especially ecDNAs, which could help to prevent drug resistance. It also presents a proof-of-principal approach which could be applied to other cancer drug resistance studies.

Electroacupuncture protects the intestinal mucosal barrier in diarrhea-predominant Irritable Bowel Syndrome rats by regulating the MCs/Tryptase/PAR-2/MLCK pathway.

OBJECTIVE: The pathogenesis of diarrhea-predominant irritable bowel syndrome (IBS-D) is related to damage to the intestinal mucosal barrier function. Based on the Mast cell (MC)/Tryptase/Protease-activated receptor-2 (PAR-2)/Myosin light chain kinase (MLCK) pathway, this study explored the effect of electroacupuncture (EA) on IBS-D rats and its possible mechanism of protecting the intestinal mucosal barrier. METHODS: The IBS-D rat model was established by mother-offspring separation, acetic acid enema, and chronic restraint stress. The efficacy of EA on IBS-D rats was evaluated by observing the rate of loose stool (LSP) and the minimum volume threshold of abdominal withdrawal reflex (AWR) in rats. Mast cells and the ultrastructure of intestinal mucosa were observed by H&E staining, toluidine blue staining, and transmission electron microscopy. The expression levels of Tryptase, PAR-2, MLCK, zonula occludens-1 (ZO-1), and Occludin in rats were detected by ELISA, qRT-PCR, and western blot. RESULTS: After 7 days of intervention, compared to the IBS-D group, the loose stool rates of rats in IBS-D + EA group and IBS-D + ketotifen group were decreased (P < 0.01), the minimum volume thresholds of AWR were improved (P < 0.01), the inflammation of colon tissue decreased, the number of MCs were decreased (P < 0.01), the expression of Tryptase, PAR-2, and MLCK were lowered (P < 0.01, P < 0.05), and the expression of ZO-1 and Occludin were enhanced (P < 0.01, P < 0.05). Compared to the EA group, there was no significant difference in each index between the ketotifen groups (P > 0.05). CONCLUSION: EA has a good therapeutic effect on IBS-D rats. Regulating the MCs/Tryptase/PAR-2/MLCK pathway may be a mechanism to protect the intestinal mucosal barrier.

Combined SNPs sequencing and allele specific proteomics capture reveal functional causality underpinning the 2p25 prostate cancer susceptibility locus.

Genome wide association studies (GWASs) have identified numerous risk loci associated with prostate cancer, yet unraveling their functional significance remains elusive. Leveraging our high-throughput SNPs-seq method, we pinpointed rs4519489 within the multi-ancestry GWAS-discovered 2p25 locus as a potential functional SNP due to its significant allelic differences in protein binding. Here, we conduct a comprehensive analysis of rs4519489 and its associated gene, NOL10, employing diverse cohort data and experimental models. Clinical findings reveal a synergistic effect between rs4519489 genotype and NOL10 expression on prostate cancer prognosis and severity. Through unbiased proteomics screening, we reveal that the risk allele A of rs4519489 exhibits enhanced binding to USF1, a novel oncogenic transcription factor (TF) implicated in prostate cancer progression and prognosis, resulting in elevated NOL10 expression. Furthermore, we elucidate that NOL10 regulates cell cycle pathways, fostering prostate cancer progression. The concurrent expression of NOL10 and USF1 correlates with aggressive prostate cancer characteristics and poorer prognosis. Collectively, our study offers a robust strategy for functional SNP screening and TF identification through high-throughput SNPs-seq and unbiased proteomics, highlighting the rs4519489-USF1- NOL10 regulatory axis as a promising biomarker or therapeutic target for clinical diagnosis and treatment of prostate cancer.

Molecular insights into FucR transcription factor to control the metabolism of L-fucose in Bifidobacterium longum subsp. infantis.

Bifidobacterium longum subsp. infantis commonly colonizes the human gut and is capable of metabolizing L-fucose, which is abundant in the gut. Multiple studies have focused on the mechanisms of L-fucose utilization by B. longum subsp. infantis, but the regulatory pathways governing the expression of these catabolic processes are still unclear. In this study, we have conducted a structural and functional analysis of L-fucose metabolism transcription factor FucR derived from B. longum subsp. infantis Bi-26. Our results indicated that FucR is a L-fucose-sensitive repressor with more α-helices, fewer ß-sheets, and ß-turns. Transcriptional analysis revealed that FucR displays weak negative self-regulation, which is counteracted in the presence of L-fucose. Isothermal titration calorimetry indicated that FucR has a 2:1 stoichiometry with L-fucose. The key amino acid residues for FucR binding L-fucose are Asp280 and Arg331, with mutation of Asp280 to Ala resulting in a decrease in the affinity between FucR and L-fucose with the Kd value from 2.58 to 11.68 µM, and mutation of Arg331 to Ala abolishes the binding ability of FucR towards L-fucose. FucR specifically recognized and bound to a 20-bp incomplete palindrome sequence (5'-ACCCCAATTACGAAAATTTTT-3'), and the affinity of the L-fucose-loaded FucR for the DNA fragment was lower than apo-FucR. The results provided new insights into the regulating L-fucose metabolism by B. longum subsp. infantis.

Clinical efficacy and pathological outcomes of transanal endoscopic intersphincteric resection for low rectal cancer.

BACKGROUND: Transanal endoscopic intersphincteric resection (ISR) surgery currently lacks sufficient clinical research and reporting. AIM: To investigate the clinical effectiveness of transanal endoscopic ISR, in order to promote the clinical application and development of this technique. METHODS: This study utilized a retrospective case series design. Clinical and pathological data of patients with lower rectal cancer who underwent transanal endoscopic ISR at the First Affiliated Hospital of Xiamen University between May 2018 and May 2023 were included. All patients underwent transanal endoscopic ISR as the surgical approach. We conducted this study to determine the perioperative recovery status, postoperative complications, and pathological specimen characteristics of this group of patients. RESULTS: This study included 45 eligible patients, with no perioperative mortalities. The overall incidence of early complications was 22.22%, with a rate of 4.44% for Clavien-Dindo grade ≥ III events. Two patients (4.4%) developed anastomotic leakage after surgery, including one case of grade A and one case of grade B. Postoperative pathological examination confirmed negative circumferential resection margins and distal resection margins in all patients. The mean distance between the tumor lower margin and distal resection margin was found to be 2.30 ± 0.62 cm. The transanal endoscopic ISR procedure consistently yielded high quality pathological specimens. CONCLUSION: Transanal endoscopic ISR is safe, feasible, and provides a clear anatomical view. It is associated with a low incidence of postoperative complications and favorable pathological outcomes, making it worth further research and application.

Quality assessment of paediatric education and research in southwest China: A cross-sectional study.

Currently, there is a limited analysis of medical education research both domestically and internationally. To enhance, improve the quality of medical education, this study conducted a quantitative analysis of teaching project data from an affiliated hospital during the period 2016-2022. The results indicated that a total of 133 teaching projects were initiated during this period, with an average age of project leaders being 42.73±6.45 years. Regarding professional ranks and titles, municipal-level project leaders had a high concentration of seniors (48.15%), while at the university-level, most project leaders held the title of deputy seniors (58.82%). At the university-level, project leaders were mainly distributed between deputy senior titles (37.08%) and intermediate titles (38.20%). In terms of research content, nearly half of the studies (46.62%) focused on teaching methods and models. Further regression analysis revealed that professional ranks and titles were an independent factor influencing the project level (P<0. 05). These findings suggest the need for improvement in medical education research, including addressing the uneven distribution of research topics, enhancing the research capacity of junior and mid-career medical education teachers, and improving the dissemination of research results.

Brain tumor recognition by an optimized deep network utilizing ammended grasshopper optimization.

Brain tumors are abnormal cell masses that can get originated in the brain spread from other organs. They can be categorized as either malignant (cancerous) or benign (noncancerous), and their growth rates and locations can impact the functioning of the nerve system. The timely detection of brain tumors is crucial for effective treatment and prognosis. In this study, a new approach has been proposed for diagnosing brain tumors using deep learning and a meta-heuristic algorithm. The method involves three main steps: (1) extracting features from brain MRI images using AlexNet, (2) reducing the complexity of AlexNet by employing an Extreme Learning Machine (ELM) network as a classification layer, and (3) fine-tuning the parameters of the ELM network using an Amended Grasshopper Optimization Algorithm (AGOA). The performance of the method has been evaluated on a publicly available dataset consisting of 20 patients with newly diagnosed glioblastoma that is compared with several state-of-the-art techniques. Experimental results demonstrate that the method achieves the highest accuracy, precision, specificity, F1-score, sensitivity, and MCC with values of 0.96, 0.94, 0.96, 0.96, 0.94, and 0.90, respectively. Furthermore, the robustness and stability of the method have been illustrated when subjected to different levels of noise and image resolutions. The proposed approach offers a rapid, accurate, and dependable diagnosis of brain tumors and holds potential for application in other medical image analysis tasks.

Dental caries status and related factors among 5-year-old children in Shanghai.

BACKGROUND: Dental caries in young children is a difficult global oral health problem. In the last decade, China has put a great deal of effort into reducing the prevalence of dental caries. This study, which is part of the China Population Chronic Disease and Nutrition Surveillance 2021, aimed to investigate the prevalence of dental caries among children aged 5 in Shanghai, China, and its associated factors. METHODS: A total of 1281 children aged 5 years from 6 districts in Shanghai were selected by a stratified sampling method. The survey consisted of an oral health questionnaire and an oral health examination. The questionnaire included questions on oral health knowledge, attitudes, and behaviours. The oral health examination used WHO standards. After screening, the data were input and analysed. Chi-square tests and logistic regression analyses were used to study the relevant factors affecting dental caries. RESULTS: The prevalence of dental caries among 1281 children was 51.0%, the dmft index score was 2.46, the Significant Caries Index (SiC) score was 6.39, and the SiC10 score was 10.35. Dental caries experience was related to the frequency of sweet drink consumption, the age of starting tooth brushing, eating habits after brushing, whether the children had received an oral examination provided by the government (p < 0.05), and the mother's education level but was not related to sex, the use of fluoride toothpaste, the frequency of brushing, whether the parents assisted brushing, or the frequency of flossing (p > 0.05). Logistic regression analysis showed that the region of residence, eating after brushing and the age of starting brushing were associated with dental caries. CONCLUSIONS: Dental caries remained prevalent among 5-year-old children in Shanghai, China. Prevention strategies that target the associated factors including region of residence, eating after brushing, and the age of starting brushing should be considered.

Epicardial Adipose Tissue and Major Adverse Cardiovascular Events in Myocardial Infarction Patients with and without Diabetes.

BACKGROUND: Epicardial adipose tissue (EAT) accumulation plays a key role in the progression myocardial infarction (MI) and diabetes. Diabetic patients have elevated risk of major adverse cardiac events (MACEs) compared to non-diabetic patients. We aimed to investigate the prognostic value of EAT volume in MI patients with and without diabetes. METHODS: This study included 458 MI patients who underwent cardiac computed tomography (CT) imaging and received successful stent implantation. EAT volume was quantified with cardiac CT imaging. Sub-study stratification of patients by diabetes status was further analyzed. Cox proportional hazards regression models were applied to evaluate the association between EAT volume and MACEs. RESULTS: Diabetes was identified in 135 of the 458 patients (29.5%). EAT volume was significantly higher in diabetes than non-diabetes. During a median follow-up of 1154 days, MACEs occurred more frequently in patients with versus without diabetes. EAT volume was independent predictor of MACEs in all MI patients after adjustment for risk factors, and showed good predictive value in the evaluation of MACEs. Moreover, EAT volume was also significantly associated with MACEs after adjustment for risk factors in diabetes and non-diabetes in the subgroup analysis. CONCLUSION: MI patients with diabetes had higher EAT volume and experienced higher rate of MACEs compared to non-diabetes. EAT volume is an independent risk of prognosis of MI, regardless of the diabetes status.

The adaptability, distribution, ecological function and restoration application of biological soil crusts on metal tailings: A critical review.

A large amount of metal tailings causes many environmental issues. Thus, the techniques for their ecological restoration have garnered extensive attention. However, they are still in the exploratory stage. Biological soil crusts (BSCs) are a coherent layer comprising photoautotrophic organisms, heterotrophic organisms and soil particles. They are crucial in global terrestrial ecosystems and play an equal importance in metal tailings. We summarized the existing knowledge on BSCs growing on metal tailings. The main photosynthetic organisms (cyanobacteria, eukaryotic algae, lichens, and mosses) of BSCs exhibit a high heavy metal(loid) (HM) tolerance. BSCs also have a strong adaptability to other adverse conditions in tailings, such as poor structure, acidification, and infertility. The literature about tailing BSCs has been rapidly increasing, particularly after 2022. The extensive literature confirms that the BSCs distributed on metal tailings, including all major types of metal tailings in different climatic regisions, are common. BSCs perform various ecological functions in tailings, including HM stress reduction, soil structure improvement, soil nutrient increase, biogeochemical cycle enhancement, and microbial community restoration. They interact and accelerate revegetation of tailings (at least in the temperate zone) and soil formation. Restoring tailings by accelerating/inducing BSC formation (e.g., resource augmentation and inoculation) has also attracted attention and achieved small-scale on-site application. However, some knowledge gaps still exist. The potential areas for further research include the relation between BSCs and HMs, large-scale quantification of tailing BSCs, application of emerging biological techniques, controlled laboratory experiments, and other restoration applications.

Tertiary lymphoid structures in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide. Smoking, drinking, and human papillomavirus (HPV) infection are the main risk factors. Early-stage patients can benefit from radical surgery, chemotherapy, and radiotherapy, but the prognosis of locally advanced, recurrent, or metastatic patients is poor. Programmed cell death receptor 1 (PD-1) inhibitor significantly prolongs the survival of these patients, but only about 20 % of the population can benefit significantly. Exploring effective predictive indicators of immunotherapy efficacy and new therapeutic targets is necessary. Tertiary lymphoid structure (TLS) is an ectopic lymphoid organ formed in non-lymphoid tissues, which usually occurs in chronic inflammation including autoimmune diseases, infectious diseases, and tumors. The structure and function of TLS are similar to those of secondary lymphoid organs. The existence of TLS is closely related to the favorable prognosis and immune response of patients. This article will review the formation, prognosis, and predictive value of TLS as well as inducing TLS neogenesis in HNSCC.

The CBS/H2S signalling pathway regulated by the carbon repressor CreA promotes cellulose utilization in Ganoderma lucidum.

Cellulose is an important abundant renewable resource on Earth, and the microbial cellulose utilization mechanism has attracted extensive attention. Recently, some signalling molecules have been found to regulate cellulose utilization and the discovery of underlying signals has recently attracted extensive attention. In this paper, we found that the hydrogen sulfide (H2S) concentration under cellulose culture condition increased to approximately 2.3-fold compared with that under glucose culture condition in Ganoderma lucidum. Further evidence shown that cellulase activities of G. lucidum were improved by 18.2-27.6% through increasing H2S concentration. Then, we observed that the carbon repressor CreA inhibited H2S biosynthesis in G. lucidum by binding to the promoter of cbs, a key gene for H2S biosynthesis, at "CTGGGG". In our study, we reported for the first time that H2S increased the cellulose utilization in G. lucidum, and analyzed the mechanism of H2S biosynthesis induced by cellulose. This study not only enriches the understanding of the microbial cellulose utilization mechanism but also provides a reference for the analysis of the physiological function of H2S signals.

[Gene Profile and Clinical Significance of Concomitant Mutations in CN-AML Patients with CEBPA Mutation].

OBJECTIVE: To analyze the occurrence of concomitant gene mutations in cytogenetically normal acute myeloid leukemia (CN-AML) patients with CEBPA mutation and its impact on the clinical characteristics and prognosis of the patients. METHODS: 151 newly diagnosed patients with CN-AML in the Second Hospital of Shanxi Medical University from June 2013 to June 2020 were analyzed retrospectively. 34 common genetic mutations associated with hematologic malignancies were detected by next-generation sequencing technology. The occurrence of concomitant gene mutations in patients with CEBPA positive and negative groups was compared, and the correlation between concomitant mutations in different functional groups and the clinical characteristics and prognosis of CN-AML patients with CEBPA mutation was analyzed. RESULTS: In 151 patients with CN-AML, 55 (36.42%) were positive for CEBPA mutation (including 36 cases of CEBPAdm and 19 cases of CEBPAsm), of which 41 (74.55%) had co-mutations with other genes. The main mutated genes were GATA2 (25.45%, 14/55), TET2 (21.82%, 12/55), FLT3 (20.00%, 11/55), NRAS (12.73%, 7/55) and WT1 (9.09%, 9/55), etc. Some cases had two or more concomitant gene mutations. Grouping the mutant genes according to their functions showed that CEBPA+ group had lower mutation rates of histone methylation (P =0.002) and chromatin modification genes (P =0.002, P =0.033), and higher mutation rates of transcription factors (P =0.037) than CEBPA- group. In 55 patients with CEBPA+ CN-AML, the platelet count at diagnosis in signaling pathway gene mutation-positive group was lower than that in the mutation-negative group (P =0.005), the proportion of bone marrow blasts in transcription factor mutation-positive group was higher than that in the mutation-negative group (P =0.003), and the onset age in DNA methylation gene mutation-positive group and chromatin modifier mutation-positive group was older than that in the mutation-negative group, respectively (P =0.002, P =0.008). DFS of CEBPA+ CN-AML patients in signaling pathway gene mutation group was shorter than that in signaling pathway gene mutation-negative group (median DFS: 12 months vs not reached) (P =0.034). Compared with DNA methylation gene mutation-negative group, CEBPA+ CN-AML patients with DNA methylation gene mutation had lower CR rate (P =0.025) significantly shorter OS and DFS (median OS: 20 months vs not reached, P =0.006; median DFS: 15 months vs not reached, P =0.049). OS in patients with histone methylation gene mutation was significantly shorter than that in the histone methylation gene mutation-negative group (median OS: 12 months vs 40 months) (P =0.008). Multivariate analysis of prognostic factors showed that the proportion of bone marrow blasts (P =0.046), concomitant DNA methylation gene mutation (P =0.006) and histone methylation gene mutation (P =0.036) were independent risk factors affecting the prognosis. CONCLUSION: CN-AML patients with CEBPA mutation have specific concomitant gene profile, and the concomitant mutations of different functional genes have a certain impact on the clinical characteristics and prognosis of the patients.

Short-term responses of soil enzyme activities and stoichiometry to litter input in Castanopsis carlesii and Cunninghamia lanceolata plantations.

Litter input triggers the secretion of soil extracellular enzymes and facilitates the release of carbon (C), nitrogen (N), and phosphorus (P) from decomposing litter. However, how soil extracellular enzyme activities were controlled by litter input with various substrates is not fully understood. We examined the activities and stoichiometry of five enzymes including ß-1,4-glucosidase, ß-D-cellobiosidase, ß-1,4-N-acetyl-glucosaminidase, leucine aminopeptidase and acidic phosphatase (AP) with and without litter input in 10-year-old Castanopsis carlesii and Cunninghamia lanceolata plantations monthly during April to August, in October, and in December 2021 by using an in situ microcosm experiment. The results showed that: 1) There was no significant effect of short-term litter input on soil enzyme activity, stoichiometry, and vector properties in C. carlesii plantation. In contrast, short-term litter input significantly increased the AP activity by 1.7% in May and decreased the enzymatic C/N ratio by 3.8% in August, and decreased enzymatic C/P and N/P ratios by 11.7% and 10.3%, respectively, in October in C. lanceolata plantation. Meanwhile, litter input increased the soil enzymatic vector angle to 53.8° in October in C. lanceolata plantations, suggesting a significant P limitation for soil microorganisms. 2) Results from partial least squares regression analyses showed that soil dissolved organic matter and microbial biomass C and N were the primary factors in explaining the responses of soil enzymatic activity to short-term litter input in both plantations. Overall, input of low-quality (high C/N) litter stimulates the secretion of soil extracellular enzymes and accelerates litter decomposition. There is a P limitation for soil microorganisms in the study area.

Association between dietary inflammation index and inflammatory bowel disease in adults: Results from National Health and Nutrition Examination Survey 2009-2010.

Previous study has demonstrated that the Dietary Inflammation Index (DII) played a role in the risk of inflammatory bowel disease (IBD), however, the prevalence and risk factors for IBD are distinct across locations and groups, and therefore, the findings are debatable and warrant further investigation. A total of 4363 participants were calculated in the National Health and Nutrition Examination Survey (NHANES) 2009 to 2010, of whom 1.21% self-reported a history of IBD. DII values were performed as a good predictor of dietary inflammation based on data from two 24-h dietary reviews in the NHANES database. Comparing the multifarious effects along with variations of the whole population by grouping populations according to DII quartiles, dietary inflammation levels increased progressively from DII quartile 1(Q1) to quartile 4(Q4). The association between DII and IBD was tested with multi-variable logistic regression models, subgroup analyses and weighted generalized additive models. Participants in the Q4 group showed the highest levels of C-reactive protein and reduced haemoglobin and albumin levels. Logistic regression confirmed the odds ratios (95% confidence intervals) of IBD for DII were 0.99 (0.86, 1.15), 0.97 (0.84, 1.13) and 0.80 (0.66, 0.98) in models 1, 2 and 3, respectively. The negative correlation between DII and IBD among United States adults from the NHANES database became increasingly apparent as covariates were adjusted. Subgroup analyses and smoothed curve fitting confirmed the inverse results. The study revealed that DII was correlated with the overall physical well-being of participants. However, there was no significant association between DII and IBD.

Potential therapeutic targets of fibrosis in inflammatory rheumatic diseases.

Fibrosis is commonly associated with chronic rheumatic diseases, and causes substantial morbidity and mortality. Treatment of fibrosis is extremely challenging but is badly needed, as approved antifibrotic therapies fibrosis do not halt its progression, which will be discussed with a focus on pulmonary fibrosis. Findings from recent studies indicate several therapeutic targets for treating fibrosis. Interleukin-11 is emerging as a fibrogenic cytokine whose activity can be blocked with neutralizing monoclonal antibodies. Fibroblast activation protein (FAP) is highly expressed by activated fibroblasts in inflammatory and fibrotic tissues. Targeting FAP with different modalities has been extensively explored as adjunct treatment for cancer, which can also apply to treating fibrosis in rheumatic diseases.

Clown care in the clinical nursing of children: a meta-analysis and systematic review.

Background: Children treated in hospitals often experience high levels of anxiety and pain. The purpose of this meta-analysis was to analyze the effect of clown care in clinical nursing on children and to provide ideas for improving the clinical nursing care provided to children. Methods: Two authors searched PubMed, Embase, Clinical trials, Cochrane Library, Web of Science, CINAHL, Scopus, China National Knowledge Infrastructure (CNKI), Weipu, and Wanfang databases to identify randomized controlled trials (RCTs) related to clown care for children until 15 September 2023. The quality assessment of the included RCTs and the data extraction were performed by two researchers, and meta-analysis was carried out using RevMan5.4. Results: A total of 15 RCTs involving 2,252 children were finally included in this meta-analysis. The findings from this meta-analysis revealed that clown care was beneficial in reducing the pain [SMD = -0.96, 95% CI (-1.76, 0.16)], anxiety [SMD = -0.81, 95% CI (-1.16, -0.46)], and crying time [SMD = -1.09, 95% CI (-1.74, -0.44)] of children and the anxiety level of caregivers [SMD = -0.99, 95% CI (-1.95, -0.03)] (all P's < 0.05). No significant publication biases were detected in the synthesized outcomes (all P's > 0.05). Conclusions: Clown care is helpful in reducing the pain, anxiety, and crying time of children and the anxiety level of caregivers. However, additional high-quality studies with larger sample sizes are warranted to further analyze the role of clown care in clinical practice.

Sestrin2 and Sestrin3 protect spermatogenesis against heat-induced meiotic defects.

Heat stress (HS) induces testicular oxidative stress, impairs spermatogenesis, and increases the risk of male infertility. Recent studies have highlighted the antioxidative properties of the Sestrins family in reducing cellular oxidative damage. However, the role of Sestrins (Sestrin1, 2, and 3) in the testicular response to heat stress remains unclear. Here, we found that Sestrin 2 and 3 were highly expressed in the testis relative to Sestrin1. Then, the Sestrin2-/- and Sestrin3-/- mice were generated by CRISPR/Cas9 to investigate the role of them on spermatogenesis after HS. Our data showed that Sestrin2-/- and Sestrin3-/- mice testes exhibited more severe damage manifested by exacerbated loss of germ cells and higher levels of oxidative stress as compared to wild-type counterparts after HS. Notably, Sestrin2-/- and Sestrin3-/- mice underwent a remarkable increase in heat-induced spermatocyte apoptosis than that of controls. Mechanistically, the transcriptome landscape of spermatocytes and chromosome spreading showed that loss of Sestrin2 and Sestrin3 exacerbated meiotic failure by compromising DNA double-strand breaks (DSBs) repair after heat stress. Taken together, our work demonstrated a critical protective function of Sestrin2 and Sestrin3 in mitigating the impairments of spermatogenesis against heat stress.