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Objective:To optimize the existing protocols for the detection of sperm nuclear DNA integrity and to explore their application value in assisted reproductive technology.Methods:194 couples intending to undergo in vitro fertilization-embryo transfer(IVF-ET)treatment at the Affiliated Reproductive Hospital of Jiangxi University of Traditional Chinese Medicine from January 1,2021,to December 8,2022,were selected as the study subjects.The sperm samples from the male partners were collected as the control group,and the same semen,processed after optimization using a double-layer density gradient centrifugation method,was used as the observation group.According to the DNA fragmentation index(DFI)results,the control group and the observation group were divided into three subgroups,control group A and observation group A:DFI＜15％;control group B and observation group B:DFI 15％～30％;control group C and observation group C:DFI ≥30％.Then the DFI values of the observation group and control group were compared.The conditions of assisted pregnancy and pregnancy were analyzed among the subgroups.Results:①The sperm DNA fragmentation index(DFI)of the observation group was signifi-cantly lower than that of the control group[(13.55±10.17)％vs.(18.56±11.54)％,P＜0.05].②There was no significant difference in fertilization rate,cleavage rate and high-quality embryo rate among the six subgroups(P＞0.05).③There were significant differences in pregnancy rate and implantation rate among the six subgroups(P＜0.05);The clinical pregnancy rate(all above 65.00％)and implantation rate(all above 50.00％)were com-pared among four groups:control group A,control group B,observation group A and observation group B.There was no significant difference among the four groups(P＞0.05),but they were all higher than those of control group C(43.24％,31.67％)and observation group C(13.64％,8.82％)(P＜0.05).The clinical pregnancy rate and implantation rate in the control group were significantly higher than those in the observation group C(P＜0.05).Conclusions:The DNA integrity of sperm nucleus can be improved obviously after the sperm was opti-mized.Both of the two methods have good application value in assisted reproductive technology,but the DFI≥30％of semen after optimal treatment has a better predictive value for adverse pregnancy outcomes in ART.
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Objective To study the effect of mitochondrial reactive oxygen species(ROS)on male fertility and its application value in assisted reproduction so as to provide a new method for fertility assessment and etiological treatment of male infertility.Methods The relationship between sperm ROS and male infertility,sperm nuclear DNA integrity and sperm membrane function was analyzed.Then the relationships of the ROS of sperms for fertiliza-tion with fertilization rate,cleavage rate,embryo rate and pregnancy outcome were analyzed.Results(1)The percentage of the high ROS in sperm membrane of the male infertility group was significantly higher than that of the normal fertility group(P<0.01),but there was no statistical difference in the percentage of the high ROS in the sperm mitochondria between the two groups.(2)The normal rate of sperm membrane function in the normal group was significantly higher than that in the abnormal group(P<0.01),but there was no significant difference in the sperm DNA fragmentation index(DFI)between the groups.(3)There was significant difference in the fertilization rate between the IVF control group and IVF observation group,but no significant difference was seen in the cleavage rate,excellent embryo rate and clinical pregnancy rate between the groups.Conclusion Sperm ROS is related to male fertility to some extent.Abnormal ROS may lead to impaired sperm membrane function and affect sperm fertilization ability,thus affecting male fertility.The detection of ROS in sperm can be used as a new method to evaluate male fertility and provide a basis for diagnosis or treatment of male infertility.For patients with ART or infertility,the appropriate time to conceive can be selected according to the sperm ROS level.
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Cardiac manifestations are commonly observed in COVID-19 patients and prominently contributed to overall mortality. Human myocardium could be infected by SARS-CoV-2, and human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are susceptible to SARS-CoV-2 infection. However, molecular mechanisms of SARS-CoV-2 gene-induced injury and dysfunction of human CMs remain elusive. Here, we find overexpression of three SARS-CoV-2 coding genes, Nsp6, Nsp8 and M, could globally compromise transcriptome of hPSC-CMs. Integrated transcriptomic analyses of hPSC-CMs infected by SARS-CoV-2 with hPSC-CMs of Nsp6, Nsp8 or M overexpression identified concordantly activated genes enriched into apoptosis and immune/inflammation responses, whereas reduced genes related to heart contraction and functions. Further, Nsp6, Nsp8 or M overexpression induce prominent apoptosis and electrical dysfunctions of hPSC-CMs. Global interactome analysis find Nsp6, Nsp8 and M all interact with ATPase subunits, leading to significantly reduced cellular ATP level of hPSC-CMs. Finally, we find two FDA-approved drugs, ivermectin and meclizine, could enhance the ATP level, and ameliorate cell death and dysfunctions of hPSC-CMs overexpressing Nsp6, Nsp8 or M. Overall, we uncover the global detrimental impacts of SARS-CoV-2 genes Nsp6, Nsp8 and M on the whole transcriptome and interactome of hPSC-CMs, define the crucial role of ATP level reduced by SARS-CoV-2 genes in CM death and functional abnormalities, and explore the potentially pharmaceutical approaches to ameliorate SARS-CoV-2 genes-induced CM injury and abnormalities.
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Since its outbreak in December 2019, COVID-19 has caused 100,5844,555 cases and 2,167,313 deaths as of Jan 27, 2021. Comparing our previous study of SARS-CoV-2 single nucleotide variants (SNVs) before June 2020, we found out that the SNV clustering had changed considerably since June 2020. Apart from that the group SNVs represented by two non-synonymous mutations A23403G (S: D614G) and C14408T (ORF1ab: P4715L) became dominant and carried by over 95% genomes, a few emerging groups of SNVs were recognized with sharply increased monthly occurrence ratios up to 70% in November 2020. Further investigation revealed that several SNVs were strongly associated with the mortality, but they presented distinct distribution in specific countries, e.g., Brazil, USA, Saudi Arabia, India, and Italy. SNVs including G25088T, T25A, G29861T and G29864A were adopted in a regularized logistic regression model to predict the mortality status in Brazil with the AUC of 0.84. Protein structure analysis showed that the emerging subgroups of non-synonymous SNVs and those mortality-related ones in Brazil were located on protein surface area. The clashes in protein structure introduced by these mutations might in turn affect virus pathogenesis through conformation changes, leading to the difference in transmission and virulence. Particularly, we found that SNVs tended to occur in intrinsic disordered regions (IDRs) of Spike (S) and ORF1ab, suggesting a critical role of SNVs in protein IDRs to determine protein folding and immune evasion.
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Four signature groups of frequently occurred single-nucleotide variants (SNVs) were identified in over twenty-eight thousand high-quality and high-coverage SARS-CoV-2 complete genome sequences, representing different viral strains. Some SNVs predominated but were mutually exclusively presented in patients from different countries and areas. These major SNV signatures exhibited distinguishable evolution patterns over time. A few hundred patients were detected with multiple viral strain-representing mutations simultaneously, which may stand for possible co-infection or potential homogenous recombination of SARS-CoV-2 in environment or within the viral host. Interestingly nucleotide substitutions among SARS-CoV-2 genomes tended to switch between bat RaTG13 coronavirus sequence and Wuhan-Hu-1 genome, indicating the higher genetic instability or tolerance of mutations on those sites or suggesting that major viral strains might exist between Wuhan-Hu-1 and RaTG13 coronavirus.
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Objective Apoptosis or programmed cell death(PCD) has been studied extensively in multicellular organisms,however,very little is known about the molecular mechanisms by which apoptosis occurs in unicellular protozoan parasites.The aim of this study is to characterize the apoptosis or PCD of Trichomonas vaginalis induced by metronidazole (MTZ).Methods T. Vaginalis strain cultures were treated with various concentrations of MTZ and the number of viable cells were determined at different time intervals.The genomic DNA of MTZ treated T. Vaginalis was extracted and DNA fragmentation was analyzed.TUNEL assay was carried out to detect the endonuclease activity in T. Vaginalis after MTZ treatment.Flow cytometric analysis was used to analyse the phosphatidylserine (PS) exposure of T. Vaginalis.Results Metronidazole (MTZ) induced an apoptotic-like cell death in T. Vaginalis.This apoptotic-like cell death was demonstrated by cell shrinkage,phosphatidylserine exposure,and nuclear chromatin condensation.However, no oligonucleosmal DNA laddering was detected.Conclusion The regulatory pathway of apoptotic cell death in T. Vaginalis may be different from multicellular organisms.The determination of protozoan apoptotic pathways leading to cell death might ultimately allow the identification of new therapeutic targets.
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Objective To clone and characterize a RRas-like gene from Trichomonas vaginalis for studying cellular signal transduction pathways in the organism. Methods A cDNA clone, which showed homology with RRas proteins of human being, was isolated and sequenced from a cDNA expression library of T. vaginalis. The genomic DNA corresponding to the cDNA sequence was amplified using PCR technique and sequenced. Sequence analysis was per-formed using BLASTP, RPS-BLAST and ClustalW programs. Phylogenetic tree was constructed and bootstrapped with 1 050 replicates using the software MEGA3. Results The cDNA sequence showed a length of 705 bp with an open reading frame of 615 bp. The deduced amino acid sequence from the open reading frame possesses 205 residuals. Sequencing of the PCR product of genomic DNA revealed that the genomic DNA sequence encompassing the putative 5′-ATG and 3′-stop codons was identical to the cDNA sequence. Sequence analysis demonstrated that this gene was most homologous to the RRas members of Homo sapiens and Mus musculus (both having 51% identity and 70% similarity), and the amino acid sequence contains highly conserved GTP-binding domains and a fully conserved effector domain of human RRas member. Phylogenetic analysis showed that TvRRas clustered with RAS oncoprotein branch and RRAS branch of human. Conclusion The encoding protein probably belongs to a RRas family of T. vaginalis.