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目的 本研究通过探讨N6-甲基腺苷（N6-methyladenosine，m6A）修饰及其调控蛋白在脑缺血小鼠中的表达变化，为脑缺血治疗的分子靶点研究提供参考。 方法 取60只雄性C57BL/6J小鼠，随机分为假手术组、脑缺血1 d组、脑缺血3 d组和脑缺血7 d组，每组各15只，采用线栓法制作右侧大脑中动脉梗死模型，于缺血1 h进行拔栓再灌注。通过RNA提取及斑点印迹实验检测小鼠缺血侧脑组织RNA m6A水平；使用荧光定量逆转录PCR检测小鼠缺血侧脑组织甲基转移酶3（methyltransferase 3，Mettl3）、甲基转移酶14（methyltransferase 14，Mettl14）、FTO α-酮戊二酸酯依赖性双加氧酶（FTO alpha-ketoglutarate dependent dioxygenase，Fto）、RNA去甲基化酶alkB同源基因5（alkB homolog 5，RNA demethylase；Alkbh5）、YTH N6-甲基腺苷RNA结合蛋白1（YTH N6-methyladenosine RNA binding protein 1，Ythdf1）、Ythdf2和Ythdf3的mRNA表达水平；利用免疫印迹实验检测缺血侧脑组织Mettl3、Mettl14、Fto、Alkbh5、Ythdf1、Ythdf2和Ythdf3蛋白的表达水平；借助免疫荧光染色观察缺血侧脑组织梗死周边区神经元中Mettl3、Fto、Ythdf1、Ythdf2和Ythdf3蛋白的表达变化情况。 结果 与假手术组相比，脑缺血3 d组脑组织RNA的m6A水平升高（1.620±0.339 vs. 1.000±0.192，P=0.0343）。①甲基转移酶表达情况：脑缺血7 d组Mettl3 mRNA水平降低（0.675±0.059 vs. 1.000±0.131，P=0.0331）；脑缺血1 d组Mettl3（0.548±0.107 vs. 1.000±0.056，P=0.0398）、Mettl14（0.534±0.218 vs. 1.000±0.018，P=0.0108）蛋白表达水平降低；脑缺血3 d组Mettl3（0.410±0.341 vs. 1.000±0.056，P=0.0084）、Mettl14（0.429±0.283 vs. 1.000±0.018，P=0.0026）蛋白表达水平也均下降；免疫荧光染色显示脑缺血3 d组梗死周边区神经元中Mettl3蛋白表达减少。②去甲基化酶表达情况：脑缺血1 d组（0.405±0.209 vs. 1.000±0.142，P=0.0108）、脑缺血3 d组（0.530±0.125 vs. 1.000±0.142，P=0.0412）Fto蛋白表达水平降低；免疫荧光染色显示脑缺血3 d组梗死周边区神经元中Fto表达减少。③m6A结合蛋白表达情况：脑缺血1 d组Ythdf1 mRNA水平降低（0.708±0.046 vs. 1.000±0.117，P=0.0331），Ythdf3 mRNA水平升高（1.473±0.093 vs. 1.000±0.142，P=0.0012）；脑缺血3 d组Ythdf1（0.593±0.240 vs. 1.000±0.117，P=0.0034）、Ythdf2（0.664±0.177 vs. 1.000±0.200，P=0.0100）mRNA水平降低，Ythdf3 mRNA水平升高（1.451±0.281 vs. 1.000±0.142，P=0.0018）；脑缺血1 d组Ythdf1（0.486±0.177 vs. 1.000±0.091，P=0.0197）、Ythdf3（0.536±0.107 vs. 1.000±0.125，P=0.0400）蛋白表达水平降低；脑缺血3 d组Ythdf1（0.404±0.299 vs. 1.000±0.091，P=0.0079）、Ythdf2（0.279±0.189 vs. 1.000±0.261，P=0.0136）、Ythdf3（0.450±0.220 vs. 1.000±0.125，P=0.0157）蛋白表达水平均降低；免疫荧光染色显示脑缺血3 d组梗死周边区神经元中m6A结合蛋白Ythdf1、Ythdf2、Ythdf3表达均减少。 结论 小鼠脑缺血后可能由Fto表达下调导致m6A水平升高，Ythdf1、Ythdf2、Ythdf3蛋白表达水平趋势基本一致，可能存在功能冗余。 Abstract: Objective To explore the expression changes of N6-methyladenosine (m6A) and its regulatory proteins in mouse models with cerebral ischemia and to provide a reference for finding potential molecular targets for cerebral ischemia therapy. Methods A total of 60 male C57BL/6J mice were randomly divided into 4 groups: sham-operated group, 1 day post-ischemia group, 3 days post-ischemia group, and 7 days post-ischemia group, with 15 mice in each group. The model of right middle cerebral artery occlusion was established in C57BL/6J mice by thread embolization method. Reperfusion was achieved after 1 hour of ischemia. RNA extraction and dot blotting were used to detect the levels of RNA m6A in the ischemic side of the mouse brain. Fluorescence quantitative reverse transcription PCR was used to detect the mRNA expression of methyltransferase 3 (Mettl3); methyltransferase 14 (Mettl14); FTO alpha-ketoglutarate dependent dioxygenase (Fto); alkB homolog 5, RNA demethylase (Alkbh5); YTH N6-methyladenosine RNA binding protein 1 (Ythdf1); Ythdf2; and Ythdf3 in the ischemic side of the mouse brain. Western blot was used to measure the protein expression levels of Mettl3, Mettl14, Fto, Alkbh5, Ythdf1, Ythdf2, and Ythdf3 in the ischemic side of the brain. Meanwhile, immunofluorescence staining was used to observe the expression changes of Mettl3, Fto, Ythdf1, Ythdf2, and Ythdf3 in the neurons of the ischemic side of the brain. Results Compared with the sham-operated group, the m6A level of the brain tissue RNA in 3 days post-ischemia group was increased (1.620±0.339 vs. 1.000±0.192, P=0.0343). ①Expression of methyltransferase: the mRNA level of Mettl3 decreased (0.675±0.059 vs. 1.000±0.131, P=0.0331) in 7 days post-ischemia group. The protein levels of Mettl3 (0.548±0.107 vs. 1.000±0.056, P=0.0398) and Mettl14 (0.534±0.218 vs. 1.000±0.018, P=0.0108) decreased in 1 day post-ischemia group. The protein levels of Mettl3 (0.410±0.341 vs. 1.000±0.056, P=0.0084) and Mettl14 (0.429±0.283 vs. 1.000±0.018, P=0.0026) decreased in 3 days post-ischemia group. Immunofluorescence staining revealed a reduction of Mettl3 expression in the neurons around the cerebral infarction area in 3 days post-ischemia group. ②Expression of demethylase: Fto protein levels were decreased in 1 day post-ischemia group (0.405±0.209 vs. 1.000±0.142, P=0.0108) and 3 days post-ischemia group (0.530±0.125 vs. 1.000±0.142, P=0.0412). Immunofluorescence staining showed that the expression of Fto in the neurons around the cerebral infarction area decreased after 3 days of cerebral ischemia. ③Expression of m6A binding protein: Ythdf1 mRNA level decreased in 1 day post-ischemia group (0.708±0.046 vs. 1.000±0.117, P=0.0331), while Ythdf3 mRNA level was increased (1.473±0.093 vs. 1.000±0.142, P=0.0012). The mRNA levels of Ythdf1 (0.593±0.240 vs. 1.000±0.117, P=0.0034) and Ythdf2 (0.664±0.177 vs. 1.000±0.200, P=0.0100) in 3 days post-ischemia group decreased, while the mRNA level of Ythdf3 was increased (1.451±0.281 vs. 1.000±0.142, P=0.0018). The protein levels of Ythdf1 (0.486±0.177 vs. 1.000±0.091, P=0.0197) and Ythdf3 (0.536±0.107 vs. 1.000±0.125, P=0.0400) in 1 day post-ischemia group were decreased. The protein levels of Ythdf1 (0.404±0.299 vs. 1.000±0.091, P=0.0079), Ythdf2 (0.279±0.189 vs. 1.000±0.261, P=0.0136), and Ythdf3 (0.450±0.220 vs. 1.000±0.125, P=0.0157) in 3 days post-ischemia group were decreased. Immunofluorescence staining further showed a decrease in the expression of m6A binding proteins Ythdf1, Ythdf2, and Ythdf3 in the neurons around the cerebral infarction area 3 days after ischemia. Conclusions Post-ischemic downregulation of Fto in mice may lead to an elevation of m6A levels.The expression trends of Ythdf1, Ythdf2, and Ythdf3 proteins are generally consistent, indicating the existence of functional redundancy.

Aging is a complex biological process leading to impaired functions, with a variety of hallmarks. In the testis of Drosophila, the terminal epithelium region is involved in spermatid release and maturation, while its functional diversity and regulatory mechanism remain poorly understood. In this study, we performed single‐cell RNA‐sequencing analysis (scRNA‐seq) to characterize the transcriptomes of terminal epithelium in Drosophila testes at 2‐, 10 and 40‐Days. Terminal epithelium populations were defined with Metallothionein A (MtnA) and subdivided into six novel sub‐cell clusters (EP0–EP5), and a series of marker genes were identified based on their expressions. The data revealed the functional characteristics of terminal epithelium populations, such as tight junction, focal adhesion, bacterial invasion, oxidative stress, mitochondrial function, proteasome, apoptosis and metabolism. Interestingly, we also found that disrupting genes for several relevant pathways in terminal epithelium led to male fertility disorders. Moreover, we also discovered a series of age‐biased genes and pseudotime trajectory mediated state‐biased genes during terminal epithelium aging. Differentially expressed genes during terminal epithelium aging were mainly participated in the regulation of several common signatures, e.g. mitochondria‐related events, protein synthesis and degradation, and metabolic processes. We further explored the Drosophila divergence and selection in the functional constraints of age‐biased genes during aging, revealing that age‐biased genes in epithelial cells of 2 Days group evolved rapidly and were endowed with greater evolutionary advantages. scRNA‐seq analysis revealed the diversity of testicular terminal epithelium populations, providing a gene target resource for further systematic research of their functions during aging. [ABSTRACT FROM AUTHOR]

Background: Evidence indicates that the risk of developing a secondary ovarian cancer (OC) is correlated with estrogen receptor (ER) status. However, the clinical significance of the relationship between ER-associated breast cancer (BC) and clear cell ovarian cancer (CCOC) remains elusive., Methods: Independent single nucleotide polymorphisms (SNPs) strongly correlated with exposure were extracted, and those associated with confounders and outcomes were removed using the PhenoScanner database. SNP effects were extracted from the outcome datasets with minor allele frequency > 0.01 as the filtration criterion. Next, valid instrumental variables (IVs) were obtained by harmonizing exposure and outcome effects and further filtered based on F-statistics (> 10). Mendelian randomization (MR) assessment of valid IVs was carried out using inverse variance weighted (IVW), MR Egger (ME), weighted median (WM), and multiplicative random effects-inverse variance weighted (MRE-IVW) methods. For sensitivity analysis and visualization of MR findings, a heterogeneity test, a pleiotropy test, a leave-one-out test, scatter plots, forest plots, and funnel plots were employed., Results: MR analyses with all four methods revealed that CCOC was not causally associated with ER-negative BC (IVW results: odds ratio (OR) = 0.89, 95% confidence interval (CI) = 0.66-1.20, P = 0.431) or ER-positive BC (IVW results: OR = 0.99, 95% CI = 0.88-1.12, P = 0.901). F-statistics were computed for each valid IV, all of which exceeded 10. The stability and reliability of the results were confirmed by sensitivity analysis., Conclusions: Our findings indicated that CCOC dids not have a causal association with ER-associated BC. The absence of a definitive causal link between ER-associated BC and CCOC suggested a minimal true causal influence of ER-associated BC exposure factors on CCOC. These results indicated that individuals afflicted by ER-associated BC could alleviate concerns regarding the developing of CCOC, thereby aiding in preserving their mental well-being stability and optimizing the efficacy of primary disease treatment., Competing Interests: None., (AJTR Copyright © 2024.)

The epididymis is a highly specialized tissue that plays vital roles in sperm maturation and storage. The spatio-temporal repertoire of epididymal cells and their gene expression in the epididymis remain less characterized. With the help of single-cell RNA sequencing (scRNA-seq), Shi et al., reveal a spatio- and segment-specific distribution pattern of mitochondria that adds another layer of complexity to our understanding of the epididymis. They unexpectedly find a higher abundance of mitochondria and mitochondrial transcription in the corpus and cauda compared to the caput of epididymis, which are believed to be responsible for providing the energy necessary for sperm maturation and motility., Competing Interests: None., (AJCEI Copyright © 2023.)

By the methods of sugar-inversion, plant community field survey and laboratory analysis, this paper measured the effective temperature of habitats, relative coverage of species, and aboveground biomass in subalpine meadow of Mt.Xiaowutai, and analyzed the relationship between species distribution and habitat effective temperature in landscape scale. The results showed that among 97 vascular plant species registered, the distribution patterns of about 36 % of total species were influenced by the effective temperature of the habitats, and the relative coverage of about 20% of them correlated significantly with the effective temperature. Eight species showed a negative correlation between their relative coverage and effective temperature, and eleven species had a positive correlation between their relative coverage or aboveground biomass and effective temperature. The eleven species with a positive correlation had certain drought resistance feature or a tendency distributing towards warmer habitats. Concerning the effectiveness of species feature applied in the analysis, relative coverage could be used to determine the relationship between species distribution and effective temperature efficiently, if the species frequency ranged from 90 % to 20 %. But, for the dominant species with their frequency over 90 %, aboveground biomass seemed to be more suitable for the analysis.