Applications of single-cell RNA sequencing in spermatogenesis and molecular evolution

Spermatogenic cell heterogeneity is determined by the complex process of spermatogenesis differentiation.However,effectively revealing the regulatory mechanisms underlying mammalian spermatogenic cell development and differentiation via traditional methods is difficult.Advances in technology have led to the emergence of many single-cell transcriptome sequencing protocols,which have partially addressed these challenges.In this review,we detail the principles of 10x Genomics technology and summarize the methods for downstream analysis of single-cell transcriptome sequencing data.Furthermore,we explore the role of single-cell transcriptome sequencing in revealing the heterogeneity of testicular ecological niche cells,delineating the establishment and disruption of testicular immune homeostasis during human spermatogenesis,investigating abnormal spermatogenesis in humans,and,ultimately,elucidating the molecular evolution of mammalian spermatogenesis.

Molecular Evolution of Visual Opsin Genes during the Behavioral Shifts between Different Photic Environments in Geckos

Reptiles are the most morphologically and physiologically diverse tetrapods,with the squamates having the most diverse habitats.Lizard is an important model system for understanding the role of visual ecology,phylogeny and behavior on the structure of visual systems.In this study,we compared three opsin genes(RH2,LWS and SWS1)among 49 reptile species to detect positively selected genes as well as amino acid sites.Our results indicated that visual opsin genes have undergone divergent selection pressures in all lizards and RH2 and LWS suffered stronger positive selection than SWS1.Twelve positively selected sites were picked out for RH2 and LWS.Moreover,many diagnostic sites were found between geckos and non-gecko lizards,most of which were located near the positively selected sites and some of them have already been reported to be responsible for significant shifts of the wavelength of maximum absorption(λ_(max)).The results indicated that the gecko lineage accelerated the evolution of these genes to adapt to the dim-light environment or nocturnality as well as the switch between nocturnality and diurnality.

Molecular evolution,virology and spatial distribution of HCV genotypes in Pakistan:A meta-analysis

Background:Hepatitis C,caused by the Hepatitis C Virus(HCV),is the second most common form of viral hepatitis.The geographical distribution of HCV genotypes can be quite complex,making it challenging to ascertain the most prevalent genotype in a specific area.Methods:To address this,a review was conducted to determine the prevalence of HCV genotypes across various provinces and as a whole in Pakistan.The scientific literature regarding the prevalence,distribution,genotyping,and epidemiology of HCV was gathered from published articles spanning the years 1996-2020.Results:Genotype 1 accounted for 5.1%of the patients,with its predominant subtype being 1a at 4.38%.The frequencies of its other subtypes,1b and 1c,were observed to be 1.0%and 0.31%respectively.Genotype 2 had a frequency of 2.66%,with the most widely distributed subtype being 2a at 2.11%of the patients.Its other subtypes,2b and 2c,had frequencies of 0.17%and 0.36%respectively.The most prevalent genotype among all isolates was 3(65.35%),with the most frequent subtype being 3a(55.15%),followed by 3b(7.18%).The prevalence of genotypes 4,5,and 6 were scarce in Pakistan,with frequencies of 0.97%,0.08%,and 0.32%respectively.The prevalence of untypeable and mixed genotypes was 21.34%and 3.53%respectively.Estimating genotypes proves to be a productive method in assisting with the duration and selection of antiviral treatment.Different HCV genotypes can exhibit variations in their response to specific antiviral treatments.Different genotypes may have distinct natural histories,including variations in disease progression and severity.Some genotypes may lead to more rapid liver damage,while others progress more slowly.Conclusions:This information can guide screening and testing strategies,helping to identify individuals at higher risk of developing severe complications.Studying the distribution of HCV genotypes in a population can provide valuable insights into the transmission dynamics of the virus.

Molecular evolution of Na^(+) channels in teleost fishes

Voltage-dependent sodium channels are critical for electrical excitability.Invertebrates possess a single sodium channel gene;two rounds of genome duplication early in vertebrates increased the number to four.Since the teleost-tetrapod split,independent gene duplications in each lineage have further increased the number of sodium channel genes to 10 in tetrapods and 8 in teleosts.Here we review how the occurrence of multiple sodium channel paralogs has influenced the evolutionary history of three groups of fishes:pufferfish,gymnotiform and mor-myriform electric fish.Pufferfish(tetraodontidae)produce a neurotoxin,tetrodotoxin,that binds to and blocks the pore of sodium channels.Pufferfish evolved resistance to their own toxins by amino acid substitutions in the pore of their sodium channels.These substitutions had to occur in parallel across multiple paralogs for organismal re-sistance to evolve.Gymnotiform and mormyriform fishes independently evolved electric organs to generate elec-tricity for communication and object localization.Two sodium channel genes are expressed in muscle in most fishes.In both groups of weakly electric fishes,one gene lost its expression in muscle and became compartmen-talized in the evolutionary novel electric organ,which is a muscle derivative.This gene then evolved at elevated rates,whereas the gene that is still expressed in muscle does not show elevated rates of evolution.In the electric organ-expressing gene,amino acid substitutions occur in parts of the channel involved in determining how long the channel will be open or closed.The enhanced rate of sequence evolution of this gene likely underlies the spe-cies-level variations in the electric signal.

Molecular evolution and species-specific expansion of the NAP members in plants

he NAP（NAC-Like, Activated by AP3/PI） subfamily is one of the important plant-specific transcription factors, and controls many vital biological processes in plants. In the current study, 197 NAP proteins were identified from 31 vascular plants,but no NAP members were found in eight non-vascular plants.All NAP proteins were phylogenetically classified into two groups（NAP I and NAP II）, and the origin time of the NAP I group might be relatively later than that of the NAP II group.Furthermore, species-specific gene duplications, caused by segmental duplication events, resulted in the expansion of the NAP subfamily after species-divergence. Different groups have different expansion rates, and the NAP group preference was found during the expansion in plants. Moreover, the expansion of NAP proteins may be related to the gain and loss of introns.Besides, functional divergence was limited after the gene duplication. Abscisic acid（ABA） might play an important role in leaf senescence, which is regulated by NAP subfamily. These results could lay an important foundation for expansion and evolutionary analysis of NAP subfamily in plants.

Molecular Evolution of the Substrate Specificity of Chloroplastic Aldolases/Rubisco Lysine Methyltransferases in Plants

Rubisco and fructose-1,6-bisphosphate aldolases （FBAs） are involved in CO2 fixation in chloroplasts. Both enzymes are trimethylated at a specific lysine residue by the chloroplastic protein methyltransferase LSMT. Genes coding LSMT are present in all plant genomes but the methylation status of the substrates varies in a species-specific manner. For example, chloroplastic FBAs are naturally trimethylated in both Pisum sati- vum and Arabidopsis thaliana, whereas the Rubisco large subunit is trimethylated only in the former spe- cies. The in vivo methylation status of aldolases and Rubisco matches the catalytic properties of AtLSMT and PsLSMT, which are able to trimethylate FBAs or FBAs and Rubisco, respectively. Here, we created chimera and site-directed mutants of monofunctional AtLSMT and bifunctional PsLSMT to identify the molecular determinants responsible for substrate specificity. Our results indicate that the His-Ala/Pro- Trp triad located in the central part of LSMT enzymes is the key motif to confer the capacity to trimethylate Rubisco. Two of the critical residues are located on a surface loop outside the methyltransferase catalytic site. We observed a strict correlation between the presence of the triad motif and the in vivo methylation status of Rubisco. The distribution of the motif into a phylogenetic tree further suggests that the ancestral function of LSMT was FBA trimethylation. In a recent event during higher plant evolution, this function evolved in ancestors of Fabaceae, Cucurbitaceae, and Rosaceae to include Rubisco as an additional substrate to the archetypal enzyme. Our study provides insight into mechanisms by which SET-domain protein methyltransferases evolve new substrate specificity.

Molecular evolution of methanogens based on their metabolic facets

The information provided by completely sequenced genomes of methanogens can yield insights into a deeper molecular understanding of evolutionary mechanisms.This review describes the advantages of using metabolic pathways to clarify evolutionary correlation of methanogens with archaea and prokaryotes.Metabolic trees can be used to highlight similarities in metabolic networks related to the biology of methanogens.Metabolic genes are among the most modular in the cell and their genes are expected to travel laterally,even in recent evolution.Phylogenetic analysis of protein superfamilies provides a perspective on the evolutionary history of some key metabolic modules of methanogens.Phage-related genes from distantly related organisms typically invade methanogens by horizontal gene transfer.Metabolic modules in methanogenesis are phylogenetically aligned in closely related methanogens.Reverse order reactions of methanogenesis are achieved in methylotrophic methanogens using metabolic and structural modules of key enzymes.A significant evolutionary process is thought to couple the utilization of heavy metal ions with energetic metabolism in methanogens.Over 30 of methanogens genomes have been sequenced to date,and a variety of databases are being developed that will provide for genome annotation and phylogenomic analysis of methanogens.Into the context of the evolutionary hypothesis,the integration of metabolomic and proteomic data into large-scale mathematical models holds promise for fostering rational strategies for strain improvement.

Random Penetrance of Mutations Among Individuals:A New Type of Genetic Drift in Molecular Evolution

The determinative view of mutation penetrance is a fundamental assumption for the building of molecular evolutionary theory:individuals in the population with the same genotype have the same fitness effect.Since this view has been constantly challenged by experimental evidence,it is desirable to examine to what extent violation of this view could affect our under-standing of molecular evolution.To this end,the author formulated a new theory of molecular evolution under a random model of penetrance:for any individual with the same mutational genotype,the coefficient of selection is a random variable.It follows that,in addition to the conventional Ne-genetic drift(Ne is the effective population size),the variance of penetrance among individuals(ε^(2))represents a new type of genetic drift,coined by theε^(2)-genetic drift.It has been demonstrated that these two genetic drifts together provided new insights on the nearly neutral evolution:the evolutionary rate is inversely related to the log-of-Ne when theε^(2)-genetic drift is nontrivial.This log-of-Ne feature ofε^(2)-genetic drift did explain well why the dN/dS ratio(the nonsynonymous rate to the synonymous rate)in humans is only as twofold as that in mice,while the effective population size(Ne)of mice is about two-magnitude larger than that of humans.It was estimated that,for the first time,the variance of random penetrance in mammalian genes was approximatelyε^(2)≈5.89×10^(-3).

Massive Molecular Parallel Evolution of the HSP90AA1 Gene between High-elevation Anurans

HSP90 AA1 is part of the heat shock protein 90 gene family and has important functions against heat stress. We report a case of molecular level parallel evolution of the HSP90 AA1 gene in high elevation amphibians. HSP90 AA1 gene sequences of four high-elevation anurans, Bufo gargarizans, Nanorana parkeri, Rana kukunoris, and Scutiger boulengeri, were compared along with five of their low-elevation relatives. A total of 16 amino-acid sites were identified as parallel evolution between N. parkeri and R. kukunoris. We generated both model based（Zhang and Kumar＇s test） and empirical data based（parallel/divergence plotting） null distributions for non-parallel evolution, and both methods clearly determined that the observed number of parallel substitutions were significantly more than the null expectation. Furthermore, on the HSP90 AA1 gene tree, N. parkeri and R. kukunoris formed a strongly supported clade that was away from their respective relatives. This study provides a clear case of molecular parallel evolution, which may have significant implications in understanding the genetic mechanisms of high-elevation adaptation.

Fluctuation theorem for the mutation process in in vitro evolution

A proposition based on the fluctuation theorem in thermodynamics is formulated to quantitatively describe molecular evolution processes in biology. Although we cannot give full proof of its generality, we demonstrate via computer simulation its applicability in an example of DNA in vitro evolution. According to this theorem, the evolution process is a series of exponentially rare fluctuations fixed by the force of natural selection

20世纪古生物学的重大进展及21世纪战略重点

20世纪人类积累了大量的生物学、分类学和生物地层数据 ,这些数据的积累使得进化论和门德尔遗传学结合起来 ,如遗传学和物种起源、分类学和物种起源、演化的速率和模式、分支系统学等的运用使得古生物学在研究方法上有了新的进展。并提出了 2 1世纪古生物学的战略重点。

Identification of a Newly Isolated Getah Virus in the China-Laos Border,China

In this study, we isolated a virus strain （YN12031） from specimens of Armigeres subalbatus collected in the China-Laos border. BHK-21 cells infected with YN12031 exhibited an evident cytopathic effect （CPE） 32 h post-infection. The virus particles were spherical, 70 nm in diameter, and enveloped; they also featured surface fibers.

Advances in research on Zika virus

Zika virus(ZIKV) is rapidly spreading across the America and its devastating outcomes for pregnant women and infants have driven this previously ignored pathogen into the limelight.Clinical manifestations are fever,joint pain or rash and conjunctivitis.Emergence of ZIKV started with a first outbrcak in the Pacific arca in 2007.a second large outbreak occurred in the Pacific in 2013/2014 and subsequently the virus spread in other Pacific islands.Threat of explosive global pandemic and severe clinical complications linked with the more immediate and recurrent epidemics necessitate the development of an effective vaccine.Several vaccine platforms such as DNA vaccine,recombinant subunit vaccine.ZIKV purified inactivated vaccine,and chimeric vaccines have shown potent efficacy in ritro and in rim trials.Moreover,number of drugs such as Sofosbuvir.BCX4450.NITD008 and 7-DMA are ready to enter phase I clinical trial because of proven anti-ZIKV activity.Monoclonal based antibodies offer promise as an intervention effective for use in pregnant women.In this review,we describe the advances in research on ZIKV such as research strategies for the development of antiviral drugs & vaccines,molecular evolution,epidemiology emergence,neurological complications and other teratogenic outcomes as well as pathogenesis.

Molecular Phylogenetics and Functional Evolution of Major RNA Recognition Domains of Recently Cloned and Characterized Autoimmune RNA-Binding Particle

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are spliceosomal macromole-cular assemblages and thus actively participate in pre-mRNA metabolism. They are composed of evolutionary conserved and tandemly repeated motifs, where both RNA-binding and protein-protein recognition occur to achieve cellular activities. By yet unknown mechanisms, these ribonucleoprotein (RNP) particles are targeted by autoantibodies and hence play significant role in a variety of human systemic autoimmune diseases. This feature makes them important prognostic markers in terms of molecular epidemiology and pathogenesis of autoimmunity. Since RNP domain is one of the most conserved and widespread scaffolds, evolutionary analyses of these RNA-binding domains can provide further clues on disease-specific epitope formation. The study presented herein represents a sequence comparison of RNA-recognition regions of recently cloned and characterized human hnRNP A3 with those of other relevant hnRNP A/B-type proteins. Their implications in human autoimmunity are particularly emphasized.

Sequencing of p35 Gene from Heliothis armigera Polyhedrosis Virus and Its Homologous Comparison

Using p35 gene primers of AcNPV, about 1 kb fragment was obtained by PCR from HaNPV DNA and was sequenced thereafter. It has a full reading frame encode 299 amino acids. Sharing identity of 94% in nucleotides and 84% in predict amino acids with AcNPV, a apoptosis inhibiting gene was found. According to comparison of p35 genes in five kinds of baculovirus, it is found that AcNPV, TnNPV and BmNPV share the most intimate blood relation. HaNPV is near the above three species. LsNPV is little remote from them. This result reconfirmed those we have done with gp37 genes and vp39 genes. It is more accurate to use conserved gene for species division than that of the serological identification.

Evolutionary genomics analysis reveals gene expansion and functional diversity of arylalkylamine N-acetyltransferases in the Culicinae subfamily of mosquitoes

Arylalkylamine N-acetyltransferase(aaNAT),considered a potential new insecticide target,catalyzes the acetylation of arylalkylamine substrates such as serotonin and dopamine and,hence,mediates diverse functions in insects.However,the origin of insect aaNATs(iaaNATs)and the evolutionary process that generates multiple aaNATs in mosquitoes remain largely unknown.Here,we have analyzed the genomes of 33 species to explore and expand our understanding of the molecular evolution of this gene family in detail.We show that aaNAT orthologs are present in Bacteria,Cephalochordata,Chondrichthyes,Cnidaria,Crustacea,Mammalia,Placozoa,and Teleoste,as well as those from a number of insects,but are absent in some species of Annelida,Echinozoa,and Mollusca as well as Arachnida.Particularly,more than 10 aaNATs were detected in the Culicinae subfamily of mosquitoes.Molecular evolutionary analysis of aaNAT/aaNAT-like genes in mosquitoes reveals that tandem duplication events led to gene expansion in the Culicinae subfamily of mosquitoes more than 190 million years ago.Further selection analysis demonstrates that mosquito aaNATs evolved under strongly positive pressures that generated functional diversity following gene duplication events.Overall,this study may provide novel insights into the molecular evolution of the aaNAT family in mosquitoes.

当代生物进化论研究新进展

分子进化学说着眼于分子层次的进化机制 ,自然选择学说着眼于群体水平的进化机制 ,两者结合把生物进化理论提高到一个新的水平。另外 ,基因调控下的细胞分化、分子驱动机制均从不同侧面揭示了生物进化原理。

Effects of Strain Rate,Temperature and Grain Size on the Mechanical Properties and Microstructure Evolutions of Polycrystalline Nickel Nanowires:A Molecular Dynamics Simulation

Through molecular dynamics（MD） simulation, the dependencies of temperature, grain size and strain rate on the mechanical properties were studied. The simulation results demonstrated that the strain rate from 0.05 to 2 ns–1 affected the Young＇s modulus of nickel nanowires slightly, whereas the yield stress increased. The Young＇s modulus decreased approximately linearly; however, the yield stress firstly increased and subsequently dropped as the temperature increased. The Young＇s modulus and yield stress increased as the mean grain size increased from 2.66 to 6.72 nm. Moreover, certain efforts have been made in the microstructure evolution with mechanical properties association under uniaxial tension. Certain phenomena such as the formation of twin structures, which were found in nanowires with larger grain size at higher strain rate and lower temperature, as well as the movement of grain boundaries and dislocation, were detected and discussed in detail. The results demonstrated that the plastic deformation was mainly accommodated by the motion of grain boundaries for smaller grain size. However, for larger grain size, the formations of stacking faults and twins were the main mechanisms of plastic deformation in the polycrystalline nickel nanowire.

Evolutionary History of the Non-Specific Lipid Transfer Proteins

The non-specific lipid transfer proteins （nsLTPs） are small, basic proteins characterized by a tunnel-like hydrophobic cavity, capable of transferring various lipid molecules between lipid bilayers. Most nsLTPs are synthesized with an N-terminal signal peptide that localizes the protein to the apoplastic space. The nsLTPs have only been identified in seed plants, where they are encoded by large gene families. We have initiated an analysis of the evolutionary history of the nsLTP family using genomic and EST information from non-seed land plants and green algae to determine： （1） when the nsLTP family arose, （2） how often new nsLTP subfamilies have been created, and （3） how subfamilies differ in their patterns of expansion and loss in different plant lineages. In this study, we searched sequence databases and found that genes and transcripts encoding nsLTPs are abundant in liverworts, mosses, and all other investigated land plants, but not present in any algae. The tertiary structures of representative liverwort and moss nsLTPs were further studied with homology modeling. The results indicate that the nsLTP family has evolved after plants conquered land. Only two of the four major subfamilies of nsLTPs found in flowering plants are present in mosses and liverworts. The additional subfamilies have arisen later, during land plant evolution. In this report, we also introduce a modified nsLTP classification system.

Highlight the significance of genetic evolution of H5N1 avian flu

Agrowing concern has focused on the recent ,identification of influenza A H5N1 virus in Asia. Previously thought to infect only wild birds and poultry, H5N1 has now infected humans, cats, pigs, and other mammals in an ongoing outbreak, often with fatal results. According to a report from the World Health Organization （WHO）, 217 human H5N1 cases have been confirmed and 123 of them have been fatal as of May 19, 2006.1 But many questions remain unanswered, for example how the H5N1 virus could cross species barriers and acquire the ability to infect humans;