PURPOSE. X-linked retinitis pigmentosa (XLRP) is a severely blinding retinal disease, most of which are due to mutations in retinitis pigmentosa GTPase regulator (RPGR). The patients with RPGR mutations exhibit severe retinal pigment epithelium (RPE) atrophy and photoreceptor degeneration. Previous research mainly focused on the role of RPGR in the connecting cilia of photoreceptors. However, the mechanism underlying RPE deficiency in patients remains unclear. Moreover, the function of RPGR in RPE cells has not been investigated. METHODS. To investigate the mechanisms underlying RPE atrophy and the role of RPGR in RPE cells, the rpgra-/-zebrafish, human RPE cell line RPE-1, and ARPE-19 were utilized. Histological analysis, immunofluorescence, and lipid staining were used to investigate the morphology of photoreceptor and RPE cells, as well as the accumulation of lipid droplets (LDs) in RPE cells. FITC-labeled OS were used to evaluate the engulfment and degradation capabilities of RPE cells. RESULTS. The zebrafish homolog of human RPGRORF15, rpgra, is expressed in RPE cells. The rpgra-/- zebrafish exhibits RPE atrophy, followed by photoreceptor degeneration. Loss of rpgra impairs lysosome formation in RPE cells, leading to defective RPE phagocytosis. This triggers lipid metabolism disorders, ultimately causing RPE and retinal degeneration. CONCLUSIONS. RPGRORF15 is essential for maintaining lysosome function and lipid metabolism homeostasis in RPE cells. This finding elucidates the previously unrecognized role of RPGRORF15 in RPE cells. This study provides new insights into the mechanisms underlying RPGR-associated retinal diseases and offers potential therapeutic approaches.

Background: Alternative splicing is essential for the physiological and pathological development of the inner ear. Disruptions in this process can result in both syndromic and non-syndromic forms of hearing loss. DHX38, a DEAH box RNA helicase, is integral to pre-mRNA splicing regulation and plays critical roles in development, cell differentiation, and stem cell maintenance. However, its specific role in inner ear development remains undefined. Here, we utilized a dhx38 knockout zebrafish model to monitor the ear morphology and elucidate a crucial role for DHX38 in the development of the zebrafish inner ear. Methods: Bright-field morphological analysis and in situ hybridization were performed to observe ear morphology changes. Immunofluorescence and semi-quantitative RT-PCR were employed to test apoptotic cells and abnormal splicing. Results: The dhx38-/- mutant zebrafish showed significant inner ear impairments, including decrescent otocysts, absent semicircular canal protrusion, and smaller otoliths. These structural abnormalities were accompanied by substantial DNA damage and p53-dependent apoptosis within the inner ear cells. Alternative splicing analysis showed that genes related to DNA damage repair and inner ear morphogenesis are abnormal in dhx38 knockout mutants. In summary, we suggest that dhx38 promotes cell survival during the inner ear development of zebrafish by ensuring the correct splicing of genes related to DNA damage repair.

IntroductionInherited retinal diseases (IRDs) affect ∼4.5 million people worldwide. Elusive pathogenic variants in over 280 genes are associated with one or more clinical forms of IRDs. It is necessary to understand the complex interaction among retinal cell types and pathogenic genes by constructing a regulatory network. In this study, we attempt to establish a panoramic expression view of the cooperative work in retinal cells to understand the clinical manifestations and pathogenic bases underlying IRDs.MethodsSingle-cell RNA sequencing (scRNA-seq) data on the retinas from 35 retina samples of 3 species (human, mouse, and zebrafish) including 259,087 cells were adopted to perform a comparative analysis across species. Bioinformatic tools were used to conduct weighted gene co-expression network analysis (WGCNA), single-cell regulatory network analysis, cell–cell communication analysis, and trajectory inference analysis.ResultsThe cross-species comparison revealed shared or species-specific gene expression patterns at single-cell resolution, such as the stathmin family genes, which were highly expressed specifically in zebrafish Müller glias (MGs). Thirteen gene modules were identified, of which nine were associated with retinal cell types, and Gene Ontology (GO) enrichment of module genes was consistent with cell-specific highly expressed genes. Many IRD genes were identified as hub genes and cell-specific regulons. Most IRDs, especially the retinitis pigmentosa (RP) genes, were enriched in rod-specific regulons. Integrated expression and transcription regulatory network genes, such as congenital stationary night blindness (CSNB) genes GRK1, PDE6B, and TRPM1, showed cell-specific expression and transcription characteristics in either rods or bipolar cells (BCs). IRD genes showed evolutionary conservation (GNAT2, PDE6G, and SAG) and divergence (GNAT2, MT-ND4, and PDE6A) along the trajectory of photoreceptors (PRs) among species. In particular, the Leber congenital amaurosis (LCA) gene OTX2 showed high expression at the beginning of the trajectory of both PRs and BCs.ConclusionWe identified molecular pathways and cell types closely connected with IRDs, bridging the gap between gene expression, genetics, and pathogenesis. The IRD genes enriched in cell-specific modules and regulons suggest that these diseases share common etiological bases. Overall, mining of interspecies transcriptome data reveals conserved transcriptomic features of retinas across species and promising applications in both normal retina anatomy and retina pathology.

立德树人是高校教育事业的根本任务,课程思政建设是实现立德树人的重要途径.细胞生物学实验课程是生物学各专业的必修基础课程,该文结合课程的特点及授课对象,通过PBL、TBL和BOPPPS等多种教学方法,采用虚实结合、开放与半开放和线上线下结合的混合式教学模式;从思政育人目标、思政元素挖掘及其实现路径等方面进行了探索和实践,以期实现知识传授和育人目标的协同前行,也为其他实验课程的教学改革提供借鉴经验.

The pathogen Enterocytozoon hepatopenaei (EHP) has significantly affected the shrimp farming industry since the last decade. For effective prevention, accurate and reliable detection methods for EHP infection are important, especially the methods suitable for point-of-care testing (POCT) environment. By combining recombinase polymerase amplification (RPA) with the CRISPR-Cas12a system, this study developed a POCT-friendly detection method for EHP infection with one-pot procedure. The combined method utilized robust amplification capacity of RPA and high specificity of CRISPR, thus had better accuracy and reliability. The one-pot procedure was achieved by careful design and screening of the crRNA and systematic optimization of the reaction. The issues related to the compatibility of RPA and Cas12a cleavage have been successfully solved. The method was highly specific and the sensitivity reached 101 gene copies per reaction. The practical performance was validated with clinical shrimp samples. With the generous temperature requirement and convenient end-point reading of results, the one-pot method is highly suitable for POCT environment. It is a good example of one-pot RPA-CRISPR detection assay for POCT purpose, and has great application value in prevention of EHP infection in shrimp farming industry.

Regular high-intensity exercise can cause changes in athletes’ gut microbiota, and the extent and nature of these changes may be affected by the athletes’ exercise patterns. However, it is still unclear to what extent different types of athletes have distinct gut microbiome profiles and whether we can effectively monitor an athlete’s inflammatory risk based on their microbiota. To address these questions, we conducted a multi-cohort study of 543 fecal samples from athletes in three different sports: aerobics (n = 316), wrestling (n = 53), and rowing (n = 174). We sought to investigate how athletes’ gut microbiota was specialized for different types of sports, and its associations with inflammation, diet, anthropometrics, and anaerobic measurements. We established a microbiota catalog of multi-cohort athletes and found that athletes have specialized gut microbiota specific to the type of sport they engaged in. Using latent Dirichlet allocation, we identified 10 microbial subgroups of athletes’ gut microbiota, each of which had specific correlations with inflammation, diet, and anaerobic performance in different types of athletes. Notably, most inflammation indicators were associated with Prevotella-driven subgroup 7. Finally, we found that the effects of sport types and exercise intensity on the gut microbiota were sex-dependent. These findings shed light on the complex associations between physical factors, gut microbiota, and inflammation in athletes of different sports types and could have significant implications for monitoring potential inflammation risk and developing personalized exercise programs. IMPORTANCE This study is the first multi-cohort investigation of athletes across a range of sports, including aerobics, wrestling, and rowing, with the goal of establishing a multi-sport microbiota catalog. Our findings highlight that athletes’ gut microbiota is sport-specific, indicating that exercise patterns may play a significant role in shaping the microbiome. Additionally, we observed distinct associations between gut microbiota and markers of inflammation, diet, and anaerobic performance in athletes of different sports. Moreover, we expanded our analysis to include a non-athlete cohort and found that exercise intensity had varying effects on the gut microbiota of participants, depending on sex.

该文以信息技术与实验教学融合为背景,介绍了基于柔性设计虚拟仿真系统的智慧实验教学平台的总体框架与实施,重点阐述了柔性设计虚拟仿真系统的优势以及相关实验教学资源的建设范畴与要求,为实现高校智慧化实验教学探索出一条可供借鉴的方案.

Neural retina leucine zipper ( NRL ) is an essential gene for the fate determination and differentiation of the precursor cells into rod photoreceptors in mammals. Mutations in NRL are associated with the autosomal recessive enhanced S-cone syndrome and autosomal dominant retinitis pigmentosa. However, the exact role of Nrl in regulating the development and maintenance of photoreceptors in the zebrafish ( Danio rerio ), a popular animal model used for retinal degeneration and regeneration studies, has not been fully determined. In this study, we generated an nrl knockout zebrafish model via the CRISPR-Cas9 technology and observed a surprising phenotype characterized by a reduced number, but not the total loss, of rods and over-growth of green cones. We discovered two waves of rod genesis, nrl -dependent and -independent at the embryonic and post-embryonic stages, respectively, in zebrafish by monitoring the rod development. Through bulk and single-cell RNA sequencing, we characterized the gene expression profiles of the whole retina and each retinal cell type from the wild type and nrl knockout zebrafish. The over-growth of green cones and mis-expression of green-cone-specific genes in rods in nrl mutants suggested that there are rod/green-cone bipotent precursors, whose fate choice between rod versus green-cone is controlled by nrl . Besides, we identified the mafba gene as a novel regulator of the nrl -independent rod development, based on the cell-type-specific expression patterns and the retinal phenotype of nrl / mafba double-knockout zebrafish. Gene collinearity analysis revealed the evolutionary origin of mafba and suggested that the function of mafba in rod development is specific to modern fishes. Furthermore, the altered photoreceptor composition and abnormal gene expression in nrl mutants caused progressive retinal degeneration and subsequent regeneration. Accordingly, this study revealed a novel function of the mafba gene in rod development and established a working model for the developmental and regulatory mechanisms regarding the rod and green-cone photoreceptors in zebrafish.

Mutations that occur in RNA-splicing machinery may contribute to hematopoiesis-related diseases. How splicing factor mutations perturb hematopoiesis, especially in the differentiation of erythro-myeloid progenitors (EMPs), remains elusive. Dhx38 is a pre-mRNA splicing-related DEAH box RNA helicase, for which the physiological functions and splicing mechanisms during hematopoiesis currently remain unclear. Here, we report that Dhx38 exerts a broad effect on definitive EMPs as well as the differentiation and maintenance of hematopoietic stem and progenitor cells (HSPCs). In dhx38 knockout zebrafish, EMPs and HSPCs were found to be arrested in mitotic prometaphase, accompanied by a 'grape' karyotype, owing to the defects in chromosome alignment. Abnormal alternatively spliced genes related to chromosome segregation, the microtubule cytoskeleton, cell cycle kinases and DNA damage were present in the dhx38 mutants. Subsequently, EMPs and HSPCs in dhx38 mutants underwent P53-dependent apoptosis. This study provides novel insights into alternative splicing regulated by Dhx38, a process that plays a crucial role in the proliferation and differentiation of fetal EMPs and HSPCs.

Two years of the COVID-19 pandemic has caused 444,837,463 cases and 5,998,581 deaths globally (retrieved

The pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led to more than 117 million reported cases and 2.6 million deaths. Accurate diagnosis technologies are vital for controlling this pandemic. Reverse transcription (RT)-based nucleic acid detection assays have been developed, but the strict sample processing requirement of RT has posed obstacles on wider applications. This study established a ligation and recombinase polymerase amplification (L/RPA) combined assay for rapid detection of SARS-CoV−2 on genes N and ORF1ab targeting the specific biomarkers recommended by the China CDC. Ligase-based strategies usually have a low-efficiency problem on RNA templates. This study has addressed this problem by using a high concentration of the T4 DNA ligase and exploiting the high sensitivity of RPA. Through selection of the ligation probes and optimization of the RPA primers, the assay achieved a satisfactory sensitivity of 101 viral RNA copies per reaction, which was comparable to RT-quantitative polymerase chain reaction (RT-qPCR) and other nucleic acid detection assays for SARS-CoV−2. The assay could be finished in less than 30 min with a simple procedure, in which the requirement for sophisticated thermocycling equipment had been avoided. In addition, it avoided the RT procedure and could potentially ease the requirement for sample processing. Once validated with clinical samples, the L/RPA assay would increase the practical testing availability of SARS-CoV-2. Moreover, the principle of L/RPA has an application potential to the identification of concerned mutations of the virus.

当前高等教育正处深刻变革之中,对人才培养模式、教育质量提出更高要求.我们通过对教学内容重组、优化及新内容开发,设计构建了三类四层次的以显微镜为主体的实验教学体系,实验注重学科交叉、强化实践探索.这是落实卓越拔尖计划、适应教育信息化的重要举措,有助于激发学生学习情趣和培养主动探索的精神,培养学生实践动手能力和解决问题的综合能力.

培养拔尖创新人才需要一流专业,而一流专业建设离不开创新型实验教学体系建设.以华中科技大学生命科学与技术学院生物物理学大实验课程为例,针对该领域商用教学设备少、缺乏针对性和前瞻性、滞后学科发展等问题,先后自制了膜片钳系统、视网膜电图仪、随机扫描光刺激系统以及小动物行为记录装置与光片式显微成像系统等教学仪器;并在此基础上开设了一系列新的实验项目,推进了学科的发展,使学生触及前沿技术,拓宽了实验的广度与深度,起到很好的教学效果,促进了实验室建设和实验教学改革,提升了学科专业特色和影响力.

Vibrio cholerae and Vibrio vulnificus are two most reported foodborne Vibrio pathogens related to seafood. Due to global ocean warming and an increase in seafood consumption worldwide, foodborne illnesses related to infection of these two bacteria are growing, leading to food safety issues and economic consequences. Molecular detection methods targeting species-specific genes are effective tools in the fight against bacterial infections for food safety. In this study, a duplex detection biosensor based on isothermal recombinase polymerase amplification (RPA) and a three-segment lateral flow strip (LFS) has been established. The biosensor used lolB gene of Vibrio cholerae and empV gene of Vibrio vulnificus as the detection markers based on previous reports. A duplex RPA reaction for both targets were constructed, and two chemical labels, FITC and DIG, of the amplification products were carefully tested for effective and accurate visualization on the strip. The biosensor demonstrated good specificity and achieved a sensitivity of 101 copies per reaction or one colony forming unit (CFU)/10 g of spiked food for both bacteria. Validation with clinical samples showed results consistent with that of real-time polymerase chain reaction. The detection process was simple and fast with a 30-min reaction at 37 °C and visualization on the strip within 5 min. With little dependence on laboratory settings, this biosensor was suitable for on-site detection, and the duplex system enabled simultaneous detection of the two important foodborne bacteria. Moreover, the principle can be extended to healthcare and food safety applications for other pathogens.

视网膜电图可用于评判视觉系统的发育、功能及病变,已成为眼科必备检查项目之一.目前已开发商用系统仅用于人类眼科检查和大的动物研究,无法用于斑马鱼视网膜功能测量.利用玻璃电极、四维度微操、LED光刺激模块及电生理放大器,搭建了适合于幼小斑马鱼视网膜电图测量的系统,系统具有高信噪比、适应性强、方便操作、实验成功率高等优点,将在斑马鱼视觉系统发育、遗传学研究等方面发挥积极的作用.

Shrimp is a globally popular seafood. Shrimp farming has been challenged by various infectious diseases that lead to significant economic losses. The prevention of two important shrimp infectious diseases, the acute hepatopancreatic necrosis disease (AHPND) and the Enterocytozoon hepatopenaei (EHP) infection, is highly dependent on early and accurate diagnostic. On-site monitoring of the two diseases in shrimp farming facilities demands point-of-care-testing (POCT) type of diagnostic assays. This study established a duplex recombinase polymerase amplification (RPA) and lateral flow dipstick (LFD) combined assay that could simultaneously diagnose the two diseases. The optimized RPA-LFD assay could finish the diagnostic in 35 min with good specificity, and the sensitivity reached 101 and 102 gene copies per reaction for EHP and AHPND, respectively, which were at the same level as the currently available molecular diagnostic assays. Test results of clinical samples showed 100% agreement of this assay with the industrial standard nested polymerase chain reaction (PCR) assays, and samples with both diseases were simultaneously identified. Because of the isothermal 37℃ amplification and the visual reading of the signal on dipsticks, the dependence on equipment is minimal. This duplex RPA-LFD assay is well suited for simultaneous POCT diagnostic of the two important shrimp infectious diseases. Moreover, the principle can be applied to multiplex POCT diagnostic of other infectious diseases in aquaculture.

Previous reports revealed that mutation of mitochondrial inner-membrane located protein SFXN1 led to pleiotropic hematological and skeletal defects in mice, associated with the presence of hypochromic erythroid cell, iron overload in mitochondrion of erythroblast and the development of sideroblastic anemia (SA). However, the potential role of sfxn1 during erythrocyte differentiation and the development of anemia, especially the pathological molecular mechanism still remains elusive. In this study, the correlation between sfxn1 and erythroid cell development is explored through zebrafish in vivo coupled with human hematopoietic cells assay ex vivo. Both knockdown and knockout of sfxn1 result in hypochromic anemia phenotype in zebrafish. Further analyses demonstrate that the development of anemia attributes to the biosynthetic deficiency of hemoglobin, which is caused by the biosynthetic disorder of heme that associates with one‑carbon (1C) metabolism process of mitochondrial branch in erythrocyte. Sfxn1 is also involved in the differentiation and maturation of erythrocyte in inducible human umbilical cord blood stem cells. In addition, we found that functional disruption of sfxn1 causes hypochromic anemia that is distinct from SA. These findings reveal that sfxn1 is genetically conserved and essential for the maturation of erythrocyte via facilitating the production of hemoglobin, which may provide a possible guidance for the future clinical treatment of sfxn1 mutation associated hematological disorders.

Titanium dioxide nanoparticles (nano-TiO2), as a common nanomaterial, are widely used in water purification, paint, skincare and sunscreens. Its safety has always been a concern. Prior studies have shown that ultraviolet A (UVA) can exacerbate the toxicity of nano-TiO2, including inducing cell apoptosis, changing glycosylation levels, arresting cell cycle, inhibiting tumor cell and bacterial growth. However, whether the combination of UVA and nano-TiO2 cause cell necrosis and its mechanism are still rarely reported. In this study, we investigated the cytotoxicity and phototoxicity of mixture crystalline nano-TiO2 (25% rutile and 75% anatase, 21 nm) under UVA irradiation in HeLa cells. Our results showed that the abnormal membrane integrity and the ultrastructure of HeLa cells, together with the decreased viability induced by nano-TiO2 under UVA irradiation, were due to cell necrosis rather than caspase-dependent apoptosis. Furthermore, nano-TiO2 and UVA generated the reactive oxygen species (ROS) and caused the mitochondrial permeability transition pore (mPTP) of HeLa cells to abnormally open. Cell viability was significantly increased after adding vitamin C (VC) or cyclosporin A (CsA) individually to inhibit ROS and mPTP. Clearance of ROS could not only impede the opening of mPTP but also reduce the rate of cell necrosis. The results suggest the possible mechanism of HeLa cell necrosis caused by nano-TiO2 under UVA irradiation through the ROS-mPTP pathway.

Gut microbial communities of athletes differ from that of sedentary persons in both diversity and the presence of certain taxa. However, it is unclear to what degree elite athletes and non-elite athletes harbor different gut microbial community patterns and if we can effectively monitor the potential of athletes based on microbiota. A team of professional female rowing athletes in China was recruited and 306 fecal samples were collected from 19 individuals, which were separated into three cohorts: adult elite athlete's (AE), youth elite athlete's (YE), and youth non-elite athlete's (YN). The differences in gut microbiome among different cohorts were compared, and their associations with dietary factors, physical characteristics, and athletic performance were investigated. The microbial diversities of elite athletes were higher than those of youth non-elite athletes. The taxonomical, functional, and phenotypic compositions of AE, YE and YN were significantly different. Additionally, three enterotypes with clear separation were identified in athlete's fecal samples, with majority of elite athletes stratified into enterotype 3. And this enterotype-dependent gut microbiome is strongly associated with athlete performances. These differences in athlete gut microbiota lead to establishment of a random forest classifier based on taxonomical and functional biomarkers, capable of differentiating elite athletes and non-elite athletes with high accuracy. Finally, these versatilities of athlete microbial communities of athletes were found to be associated with dietary factors and physical characteristics, which can in concert explain 41% of the variability in gut microbiome.

Hematopoietic stem and progenitor cells (HSPCs) have the ability to self-renew and differentiate into various blood cells, thus playing an important role in maintenance of lifelong hematopoiesis. Brahma-related gene 1 (BRG1), which acts as the ATP subunit of mammalian SWI-SNF-related chromatin remodeling complexes, is involved in human acute myeloid leukemia and highly expresses in short-term HSPCs. But its role and regulatory mechanism for HSPC development have not yet been well established. Here, we generated abrg1knockout zebrafish model using TALEN technology. We found that inbrg1(-/-)embryo, the primitive hematopoiesis remained well, while definitive hematopoiesis formation was significantly impaired. The number of hemogenic endothelial cells was decreased, further affecting definitive hematopoiesis with reduced myeloid and lymphoid cells. During embryogenesis, the nitric oxide (NO) microenvironment inbrg1(-/-)embryo was seriously damaged and the reduction of HSPCs could be partially rescued by a NO donor. Chromatin immunoprecipitation (ChIP) assays showed that BRG1 could bind to the promoter ofKLF2and trigger its transcriptional activity of NO synthase. Our findings show that Brg1 promotesklf2aexpression in hemogenic endothelium and highlight a novel mechanism for HSPC formation and maintenance.