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Design of an automated robotic microinjection system for batch injection of zebrafish embryos and larvae

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Microsystems & Nanoengineering 2024年第1期10卷 89-103页

作者： Zhongyi Guo Nana Ai Wei Ge Qingsong Xu department of Electromechanical Engineering Faculty of Science and TechnologyUniversity of MacaoMacaoChina department of Biomedical Sciences and Centre of reproduction Development and Aging(CRDA)Faculty of Health SciencesUniversity of MacaoMacaoChina

This paper presents the design of a vision-based automated robotic microinjection system for batch injection of both zebrafish embryos and larvae.A novel visual recognition algorithm based on an automatic threshold and excessive dilatation is introduced to accurately identify the center of zebrafish embryos and larval yolks.A corresponding software system is developed using the producer-consumer model as the framework structure,and a friendly user interface is designed to allow operators to choose from a range of desired functions according to their different needs.In addition,a novel microstructural agarose device is designed and fabricated to simultaneously immobilize mixed batches of embryos and larvae.Moreover,a prototype microinjection system is fabricated by integrating hardware devices with visual algorithms.An experimental study is conducted to verify the performance of the robotic microinjection system.The results show that the reported system can accurately identify zebrafish embryos and larvae and efficiently complete batch microinjection tasks of the mixtures with an injection success rate of 92.05%in 13.88 s per sample.Compared with manual and existing microinjection systems,the proposed system demonstrates the merits of versatility,excellent efficiency,high success rate,high survival rate,and sufficient stability.

关键词： zebra automated hardware

RNA m6A dynamic modification mediated by nucleuslocalized FTO is involved in follicular reserve

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Zoological Research 2024年第2期45卷 415-428页

作者： Jiao-Na Zhang Rui-Ting Wang Francesca-Gioia Klinger Shun-Feng Cheng Wei Shen Xiao-Feng Sun College of Life Sciences Key Laboratory of Animal Reproduction and Germplasm Enhancement in Universities of ShandongQingdao Agricultural UniversityQingdaoShandong 266109China department of Biomedicine and Prevention University of Rome Tor VergataRome 00133Italy

In eukaryotic organisms,the most common internal modification of messenger RNA(m RNA)is N6-methyladenosine(m6A).This modification can be dynamically and reversibly controlled by specific enzymes known as m6A writers and erasers.The fat-mass and obesity-associated protein(FTO)catalyzes RNA demethylation and plays a critical role in various physiological and pathological processes.Our research identified dynamic alterations in both m6A and FTO during the assembly of primordial follicles,with an inverse relationship observed for m6A levels and nuclear-localized FTO expression.Application of Fto small interfering RNA(si RNA)altered the expression of genes related to cell proliferation,hormone regulation,and cell chemotaxis,and affected RNA alternative splicing.Overexpression of the full-length Fto gene led to changes in m6A levels,alternative splicing of Cdk5,cell proliferation,cell cycle progression,and proportion of primordial follicles.Conversely,overexpression of Fto lacking a nuclear localization signal(NLS)did not significantly alter m6A levels or primordial follicle assembly.These findings suggest that FTO,localized in the nucleus but not in the cytoplasm,regulates RNA m6A demethylation and plays a role in cell proliferation,cell cycle progression,and primordial follicle assembly.These results highlight the potential of m6A and its eraser FTO as possible biomarkers and therapeutic targets.

关键词： m6A FTO Ovary Primordial follicle assembly Alternative splicing

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Berberine alleviates ovarian tissue damage in mice with hepatolenticular degeneration by suppressing ferroptosis and endoplasmic reticulum stress

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Journal of Integrative Medicine 2024年第4期22卷 493-502页

作者： Qian-zhuo Liu Hui Han Xin-ru Fang Lu-yao Wang Dan Zhao Miao-zhu Yin Nian Zhang Peng-yu Jiang Zhi-hui Ji Li-min Wu department of Neurology the First Affiliated Hospital of Anhui University of Chinese Medicine Center for reproduction and Genetics the First Affiliated Hospital of USTC Division of Life Sciences and Medicine University of Science and Technology of China

Objective:Hepatolenticular degeneration (HLD) is an autosomal recessive disorder that manifests as multiorgan damage due to impaired copper (Cu) metabolism. Female patients with HLD often experience reproductive impairments. This study investigated the protective effect of berberine against ovarian damage in toxic-milk (TX) mice, a murine model for HLD.Methods:Mice were categorized into control group, HLD TX group (HLD group), penicillamine (Cu chelator)-treated TX group and berberine-treated TX group. Body weight, ovary weight and the number of ovulated eggs were recorded. Follicular morphology and cellular ultrastructure were examined. Total iron, ferrous iron (Fe2+) and trivalent iron (Fe3+) levels, as well as malondialdehyde (MDA), glutathione(GSH) and oxidized glutathione (GSSG), were measured in the ovaries. Western blot analysis was used to analyze the expression of proteins related to ferroptosis and endoplasmic reticulum (ER) stress.Results:Ovarian tissue damage was evident in the HLD group, with a significant increase in ferroptosis and ER stress compared to the control group. This damage was inhibited by treatment with penicillamine,a Cu chelator. Compared with the HLD group, berberine increased the number of ovulations, and improved ovarian morphology and ultrastructure. Further, we found that berberine reduced total iron,Fe2+, MDA and GSSG levels, elevated GSH levels, decreased the expression of the ferroptosis marker protein prostaglandin-endoperoxide synthase 2 (PTGS2), and increased glutathione peroxidase 4 (GPX4)expression. Furthermore, berberine inhibited the expression of ER stress-associated proteins mediated by the protein kinase RNA-like ER kinase (PERK) pathway.Conclusion:Ferroptosis and ER stress are involved in Cu-induced ovarian damage in TX mice. Berberine ameliorates ovarian damage in HLD TX mice by inhibiting ferroptosis and ER stress.

关键词： Berberine Copper deposition Endoplasmic reticulum stress Ferroptosis Hepatolenticular degeneration

Decoding the Cellular Trafficking of Prion-like Proteins in Neurodegenerative Diseases

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Neuroscience Bulletin 2024年第2期40卷 241-254页

作者： Chenjun Hu Yiqun Yan Yanhong Jin Jun Yang Yongmei Xi Zhen Zhong department of Neurology of the Second Afliated Hospital and department of Human Anatomy Histology and EmbryologyZhejiang University School of MedicineHangzhou 310058China department of Physiology and department of Cardiology of the Second Afliated Hospital Zhejiang University School of MedicineHangzhou 310058China Division of Human reproduction and Developmental Genetics Women’s Hospital and Institute of GeneticsZhejiang University School of MedicineHangzhou 310006China

The accumulation and spread of prion-like proteins is a key feature of neurodegenerative diseases(NDs)such as Alzheimer’s disease,Parkinson's disease,or Amyotrophic Lateral Sclerosis.In a process known as‘seeding’,prion-like proteins such as amyloid beta,microtubule-associated protein tau,α-synuclein,silence superoxide dismutase 1,or transactive response DNA-binding protein 43 kDa,propagate their misfolded conformations by transforming their respective soluble monomers into fibrils.Cellular and molecular evidence of prion-like propagation in NDs,the clinical relevance of their‘seeding’capacities,and their levels of contribution towards disease progression have been intensively studied over recent years.This review unpacks the cyclic prion-like propagation in cells including factors of aggregate internalization,endo-lysosomal leaking,aggregate degradation,and secretion.Debates on the importance of the role of prion-like protein aggregates in NDs,whether causal or consequent,are also discussed.Applications lead to a greater understanding of ND pathogenesis and increased potential for therapeutic strategies.

关键词： Neurodegenerative diseases Prion-like propagation Seeding Endocytosis Endolyosomal leaking Degradation

Therapeutic advances in atrial fibrillation based on animal models

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Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2024年第2期25卷 135-152页

作者： Qian GONG Xuan LE Pengcheng YU Lenan ZHUANG Institute of Genetics and reproduction College of Animal SciencesZhejiang UniversityHangzhou310058China Key Laboratory of Cardiovascular Intervention and Regenerative Medicine of Zhejiang Province Department of CardiologySir Run Run Shaw HospitalSchool of MedicineZhejiang UniversityHangzhou310016China

Atrial fibrillation(AF)is the most prevalent sustained cardiac arrhythmia among humans,with its incidence increasing significantly with age.Despite the high frequency of AF in clinical practice,its etiology and management remain elusive.To develop effective treatment strategies,it is imperative to comprehend the underlying mechanisms of AF;therefore,the establishment of animal models of AF is vital to explore its pathogenesis.While spontaneous AF is rare in most animal species,several large animal models,particularly those of pigs,dogs,and horses,have proven as invaluable in recent years in advancing our knowledge of AF pathogenesis and developing novel therapeutic options.This review aims to provide a comprehensive discussion of various animal models of AF,with an emphasis on the unique features of each model and its utility in AF research and treatment.The data summarized in this review provide valuable insights into the mechanisms of AF and can be used to evaluate the efficacy and safety of novel therapeutic interventions.

关键词： Atrial fibrillation Animal model Therapeutic

A mutation in TBXT causes congenital vertebral malformations in humans and mice

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Journal of Genetics and Genomics 2024年第4期51卷 433-442页

作者： Shuxia Chen Yunping Lei Yajun Yang Chennan Liu Lele Kuang Li Jin Richard HFinnell Xueyan Yang Hongyan Wang Obstetrics and Gynecology Hospital State Key Laboratory of Genetic Engineering at School of Life SciencesKey Laboratory of Reproduction Regulation of NPFPCInstitute of Reproduction and DevelopmentFudan UniversityShanghai 200438China Shanghai Key Laboratory of Metabolic Remodeling and Health Institute of Metabolism and Integrative BiologyFudan UniversityShanghai 200438China Center for Precision Environmental Health Department of Molecular and Cellular BiologyBaylor College of MedicineHoustonTX 77030USA MOE Key Laboratory of Contemporary Anthropology School of Life SciencesFudan UniversityShanghai 200438China department of Assisted reproduction Xinhua HospitalSchool of MedicineShanghai Jiao Tong UniversityShanghai 200092China Children’s Hospital Fudan University399 Wanyuan RoadShanghai 201102China

T-box transcription factor T(TBXT;T)is required for mesodermal formation and axial skeletal development.Although it has been extensively studied in various model organisms,human congenital vertebral malformations(CVMs)involving T are not well established.Here,we report a family with 15 CVM patients distributed across 4 generations.All affected individuals carry a heterozygous mutation,T c.596A>G(p.Q199R),which is not found in unaffected family members,indicating co-segregation of the genotype and phenotype.In vitro assays show that T p.Q199R increases the nucleocytoplasmic ratio and enhances its DNA-binding affinity,but reduces its transcriptional activity compared to the wild-type.To determine the pathogenicity of this mutation in vivo,we generated a Q199R knock-in mouse model that recapitulates the human CVM phenotype.Most heterozygous Q199R mice show subtle kinked or shortened tails,while homozygous mice exhibit tail filaments and severe vertebral deformities.Overall,we show that the Q199R mutation in T causes CVM in humans and mice,providing previously unreported evidence supporting the function of T in the genetic etiology of human CVM.

关键词： Congenital vertebral malformation TBXT T gene Loss-of-function mutation

Polyoxidovanadates a new therapeutic alternative for neurodegenerative and aging diseases

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Neural Regeneration Research 2024年第3期19卷 571-577页

作者： Sonia Irais Gonzalez-Cano Gonzalo Flores Jorge Guevara Julio Cesar Morales-Medina Samuel Treviño Alfonso Diaz Faculty of Chemical Sciences Benemerita Autonomous University of PueblaPueblaMexico Institute of Physiology Benemerita Autonomous University of PueblaPueblaMexico department of Biochemistry Faculty of MedicineNational Autonomous University of MexicoMexico CityMexico Animal reproduction Research Center CINVESTAV-Autonomous University of TlaxcalaTlaxcalaMexico

Aging is a natural phenomenon characterized by a progressive decline in physiological integrity,leading to a deterioration of cognitive function and increasing the risk of suffering from chronic-degenerative diseases,including cardiovascular diseases,osteoporosis,cancer,diabetes,and neurodegeneration.Aging is considered the major risk factor for Parkinson’s and Alzheimer’s disease develops.Likewise,diabetes and insulin resistance constitute additional risk factors for developing neurodegenerative disorders.Currently,no treatment can effectively reverse these neurodegenerative pathologies.However,some antidiabetic drugs have opened the possibility of being used against neurodegenerative processes.In the previous framework,Vanadium species have demonstrated a notable antidiabetic effect.Our research group evaluated polyoxidovanadates such as decavanadate and metforminium-decavanadate with preventive and corrective activity on neurodegeneration in brain-specific areas from rats with metabolic syndrome.The results suggest that these polyoxidovanadates induce neuronal and cognitive restoration mechanisms.This review aims to describe the therapeutic potential of polyoxidovanadates as insulin-enhancer agents in the brain,constituting a therapeutic alternative for aging and neurodegenerative diseases.

关键词： Alzheimer’s disease antidiabetic brain cognition diabetes insulin neurodegeneration neuroinflammation oxidative stress Vanadium species

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Chromatin condensation but not DNA integrity of pig sperm is greater in the sperm-rich fraction

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Journal of Animal Science and Biotechnology 2024年第1期15卷 171-181页

作者： Estel Viñolas-Vergés Jordi Ribas-Maynou Isabel Barranco Camila Peres Rubio Sergi Bonet Jordi Roca Marc Yeste Unit of Cell Biology Department of BiologyFaculty of SciencesUniversity of GironaGironaSpain Biotechnology of Animal and Human reproduction(TechnoSperm) Institute of Food and Agricultural TechnologyUniversity of GironaGironaSpain department of Medicine and Animal Surgery Faculty of Veterinary ScienceUniversity of MurciaMurciaSpain Catalan Institution for Research and Advanced Studies(ICREA) S08010 BarcelonaSpain

Background Protamination and condensation of sperm chromatin as well as DNA integrity play an essential role during fertilization and embryo development.In some mammals,like pigs,ejaculates are emitted in three separate fractions:pre-sperm,sperm-rich(SRF)and post sperm-rich(PSRF).These fractions are known to vary in volume,sperm concentration and quality,as well as in the origin and composition of seminal plasma(SP),with differences being also observed within the SRF one.Yet,whether disparities in the DNA integrity and chromatin condensation and pro-tamination of their sperm exist has not been interrogated.Results This study determined chromatin protamination(Chromomycin A3 test,CMA_(3)),condensation(Dibromobi-mane test,DBB),and DNA integrity(Comet assay)in the pig sperm contained in the first 10 m L of the SRF(SRF-P1),the remaining portion of the sperm-rich fraction(SRF-P2),and the post sperm-rich fraction(PSRF).While chromatin protamination was found to be similar between the different ejaculate fractions(P>0.05),chromatin condensation was seen to be greater in SRF-P1 and SRF-P2 than in the PSRF(P=0.018 and P=0.004,respectively).Regarding DNA integrity,no differences between fractions were observed(P>0.05).As the SRF-P1 has the highest sperm concentra-tion and ejaculate fractions are known to differ in antioxidant composition,the oxidative stress index(OSi)in SP,calcu-lated as total oxidant activity divided by total antioxidant capacity,was tested and confirmed to be higher in the SRF-P1 than in SRF-P2 and PSRF(0.42±0.06 vs.0.23±0.09 and 0.08±0.00,respectively;P<0.01);this index,in addition,was observed to be correlated to the sperm concentration of each fraction(Rs=0.973;P<0.001).Conclusion While sperm DNA integrity was not found to differ between ejaculate fractions,SRF-P1 and SRF-P2 were observed to exhibit greater chromatin condensation than the PSRF.This could be related to the OSi of each fraction.

关键词： Chromatin Condensation DNA integrity Ejaculate fractions Pig Protamination Sperm

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Biallelic variants in RBM42 cause a multisystem disorder with neurological,facial,cardiac,and musculoskeletal involvement

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Protein & Cell 2024年第1期15卷 52-68页

作者： Yiyao Chen Bingxin Yang Xiaoyu Merlin Zhang Songchang Chen Minhui Wang Liya Hu Nina Pan Shuyuan Li Weihui Shi Zhenhua Yang Li Wang Yajing Tan Jian Wang Yanlin Wang Qinghe Xing Zhonghua Ma Jinsong Li He-Feng Huang Jinglan Zhang Chenming Xu Institutes of Biomedical Sciences Fudan UniversityShanghai 200032China bstetrics and Gynecology Hospital Institute of Reproduction and DevelopmentFudan UniversityShanghai 200011China Intermational Peace Matermity and Child Health Hospital School of MedicineShanghai Jiao Tong UniversityShanghai 200030China Shanghai Key Laboratory of embryo Original Diseases Shanghai 200030China State Key Laboratory of Cell Biology Shanghai Key Laboratory of Molecular AndrologyShanghai Institute of Biochemistry and Cell BiologyCAS Center for Excellence in Molecular Cell ScienceUniversity of Chinese Academy of SciencesChinese Academy of SciencesShanghai 200031China State Key Laboratory of Rice Biology the Key Laboratory of Molecular Biology of Crop Pathogens and InsectsInstitute of BiotechnologyZhejiang UniversityHangzhou 310058China Verna and Marrs McLean department of Biochemistry and Molecular Biology Baylor College of MedicineHoustonTX 77030USA Children's hospital of Fudan University Shanghai 201102China School of Life Science and Technology Shanghai Tech UniversityShanghai 201210China School of Life Science Hangzhou Institute for Advanced StudyUniversity of Chinese Academy of SciencesHangzhou 310024ZhejiangChina Research Units of embryo Original Diseases Chinese Academy of Medical Sciences(No.2019RU056)Shanghai 200011China

Here,we report a previously unrecognized syndromic neurodevelopmental disorder associated with biallelic loss-of-function variants in the RBM42 gene.The patient is a 2-year-old female with severe central nervous system(CNs)abnormalities,hypotonia,hearing loss,congenital heart defects,and dysmorphic facial features.Familial whole-exome sequencing(WEs)reveals that the patient has two compound heterozygous variants,c.304C>T(p.R102*)and c.1312G>A(p.A438T),in the RBM42 gene which encodes an integral component of splicing complex in the RNA-binding motif protein family.The p.A438T variant is in the RRM domain which impairs RBM42 pro-tein stability in vivo.Additionally,p.A438T disrupts the interaction of RBM42 with hnRNP K,which is the causa-tive gene for Au-Kline syndrome with overlapping disease characteristics seen in the index patient.The human R102*or A438T mutant protein failed to fully rescue the growth defects of RBM42 ortholog knockout△FgRbp1 in Fusarium while it was rescued by the wild-type(WT)human RBM42.A mouse model carying Rbm42 compound heterozygous variants,c.280C>T(p.Q94*)and c.1306_1308delinsACA(p.A436T),demonstrated gross fetal develop-mental defects and most of the double mutant animals died by E13.5.RNA-seq data confirmed that Rbm42 was involved in neurological and myocardial functions with an essential role in alternative splicing(As).Overall,we present clinical,genetic,and functional data to demonstrate that defects in RBM42 constitute the underlying etiology of a new neurodevelopmental disease which links the dysregulation of global AS to abnormal embryonic development.

关键词： RBM42 gene RNA-binding protein neurodevelopmental disorder Au-Kline syndrome alternative splicing