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.

Evolution of molecular structure of TATB under shock loading from transient Raman spectroscopic technique

By combination of the transient Raman spectroscopic measurement and the density functional theoretical calculations,the structural evolution and stability of TATB under shock compression was investigated.Due to the improvement in synchronization control between two-stage light gas gun and the transient Raman spectra acquisition,as well as the sample preparation,the Raman peak of the N-O mode of TATB was firstly observed under shock pressure up to 13.6 GPa,noticeably higher than the upper limit of 8.5 GPa reported in available literatures.By taking into account of the continuous shift of the main peak and other observed Raman peaks,we did not distinguish any structural transition or any new species.Moreover,both the present Raman spectra and the time-resolved radiation of TATB during shock loading showed that TATB exhibits higher chemical stability than previous declaration.To reveal the detailed structural response and evolution of TATB under compression,the density functional theoretical calculations were conducted,and it was found that the pressure make N-O bond lengths shorter,nitro bond angles larger,and intermolecular and intra-molecular hydrogen bond interactions enhanced.The observed red shift of Raman peak was ascribed to the abnormal enhancement of H-bound effect on the scissor vibration mode of the nitro group.

Sequence Variation and Molecular Evolution of Hormone-Sensitive Lipase Genes in Species of Bovidae

The partial sequences of exon Ⅰ of hormone-sensitive lipase （HSL） genes in yak （Bos grunniens）, cattle （Bos taurus）, zebu （Bos indicus）, and buffalo （Bubalus bubalis） were analyzed. Comparisons of these sequences and the deduced amino acid sequences with the homologous HSL gene and protein sequences in other mammalian species including pig （Sus scrofa）, human （Homo sapiens）, mouse （Mus musculus）, and rat （Rattus sp.） retrieved from the GenBank were carried out and finally a phylogenetic tree was constructed using the partial DNA sequences of the HSL genes in all species. The results showed that the homologies of the partial exon Ⅰ sequences of the HSL genes between yak and cattle, zebu, buffalo, pig, human, mouse, and rat were as high as 99.8%, 99.6%, 97.4%, 90.6%, 88.4%, 83.5%, and 82.3%, respectively. This was accompanied by highly homologous amino acid sequences of the HSLs： 100%, 100%, 98.2%, 94.0%, 92.2%, 89.8%, and 89.8% identity, respectively. There are more transitions, less transversions, and no insertion or deletion in variable nucleotides of the HSL genes between the yak and other species. The majority of the variable mutations was synonymous and was found most frequently at the third codon, followed by the first and second codons, a finding that was in accordance with the neutralism hypothesis for molecular evolution. In the phylogenetic tree, the cattle and zebu were clustered together first, followed by the yak, buffalo, pig, human, mouse, and rat. This was in agreement with taxonomy suggesting that the partial sequences of exon Ⅰ of the HSL genes were useful in constructing the phylogenetic tree of mammalian species. Among the four species of Bovidae, genetic differentiation in the HSL genes between yak and buffalo is equivalent to that between buffalo and cattle and between buffalo and zebu. Furthermore, the genetic distances in the HSL genes are much smaller between yak, cattle, and zebu than those between each of the three species and the buffalo. Therefore, it is reasonable to consider yak as an independent species of the genus Bos.

Sequence and Molecular Evolution Analysis of Ubiquitin Proteins Encoded by Baculoviruses

[Objective] The aim of this study was to analyze the sequence characteristics and molecular evolution of ubiquitins encoded by baculoviruses.[Methods]Clustal W software was used for multiple sequence alignment analysis,and neighbor-joining method（NJ）and maximum parsimony method（MP）were used for the construction of phylogenetic tree.[Results]The baculoviral ubiquitins showed 73%-86% sequence identity to eukaryotic ubiquitin.Two heterogeneous regions of baculoviral ubiquitins were observed：one was the residues from 15-32,the other was located from residues 53 to 60.The else parts were conserved,where many functional amino acids were also observed.Phylogenetic analysis indicated that baculoviral ubiquitins could be divided into three sub-families,including sub-family GV,sub-family I and sub-family II.The molecular evolution of baculoviral ubiquitins might be under negative selection to maintain their functional and structural stability.[Conclusion]The analysis had provided reference for the researches on functional characterization of baculoviral ubiquitins.

Hepatitis C virus molecular evolution:Transmission,disease progression and antiviral therapy

Hepatitis C virus(HCV)infection represents an important public health problem worldwide.Reduction of HCV morbidity and mortality is a current challenge owned to several viral and host factors.Virus molecular evolution plays an important role in HCV transmission,disease progression and therapy outcome.The high degree of genetic heterogeneity characteristic of HCV is a key element for the rapid adaptation of the intrahost viral population to different selection pressures(e.g.,host immune responses and antiviral therapy).HCV molecular evolution is shaped by different mechanisms including a high mutation rate,genetic bottlenecks,genetic drift,recombination,temporal variations and compartmentalization.These evolutionary processes constantly rearrange the composition of the HCV intrahost population in a staging manner.Remarkable advances in the understanding of the molecular mechanism controlling HCV replication have facilitated the development of a plethora of direct-acting antiviral agents against HCV.As a result,superior sustained viral responses have been attained.The rapidly evolving field of anti-HCV therapy is expected to broad its landscape even further with newer,more potent antivirals,bringing us one step closer to the interferon-free era.

O-O bond formation mechanisms during the oxygen evolution reaction over synthetic molecular catalysts

Water oxidation is one of the most important reactions in natural and artificial energy conversion schemes.In nature,solar energy is converted to chemical energy via water oxidation at the oxygen-evolving center of photosystem II to generate dioxygen,protons,and electrons.In artificial energy schemes,water oxidation is one of the half reactions of water splitting,which is an appealing strategy for energy conversion via photocatalytic,electrocatalytic,or photoelectrocatalytic processes.Because it is thermodynamically unfavorable and kinetically slow,water oxidation is the bottleneck for achieving large-scale water splitting.Thus,developing highly efficient water oxidation catalysts has attracted the interests of researchers in the past decades.The formation of O-O bonds is typically the rate-determining step of the water oxidation catalytic cycle.Therefore,better understanding this key step is critical for the rational design of more efficient catalysts.This review focuses on elucidating the evolution of metal-oxygen species during transition metal-catalyzed water oxidation,and more importantly,on discussing the feasible O-O bond formation mechanisms during the oxygen evolution reaction over synthetic molecular catalysts.

Multiscale evolution mechanism of sandstone under wet-dry cycles of deionized water:From molecular scale to macroscopic scale

Water is the most abundant molecule found on the earth’s surface and is a key factor in multiscale rock destruction.However,given the fine-grained nature of rock and the complexity of its internal structure,the microstructural evolution of rock under the action of water has not yet been elucidated in detail,and little is understood about the relationship between the rock structure and solideliquid unit.A variety of techniques were used in this study to track the mechanical properties,pore and crack characteristics,and mineral structure degradation characteristics of sandstone at different stages under the action of deionized water,and the evolution mechanisms of the microstructure were analyzed at the molecular scale.The results showed that during the watererock interaction process,water was adsorbed onto the surface of dolomite minerals and the hydrophilic surface of clay minerals,forming a high-density hydrogen bond network.However,different mineral surface structures had different water adsorption structures,resulting in the strain of the dense clay mineral aggregates under expansion action.Stress concentrated at crack tips under the capillary force of dolomite minerals(very weak dolomite dissolution).These effects resulted in a substantial increase in the number of small pores and enhancements in poreecrack connectivity,and the rock strength exhibited varying degrees of decline at different stages of wet-dry cycles.In general,the results of this paper will help to further elucidate the internal connections between molecular-scale and macroscale processes in rock science.

Directed Molecular Evolution of Nitrite Oxido-reductase by DNA-shuffling

Objective To devellop directly molecular evolution Of nitrite oxido-reductase using DNA-shuffling technique because nitrobacteria grow extremelly slow and are unable to nitrify effectively inorganic nitrogen in wastewater treatmem. Methods The norB gene coding the ntitrite oxido-reductase in nitrobacteria was cloned and sequenced. Then, directed molecular evolution of nitrite oxido-reductase was developed by DNA-shuffling of 15 norB genes from different nitrobacteria. Results After DNA-shuffling with sexual PeR and staggered extension process PCR, the sequence was differem from its parental DNA fragmems and the homology ranged from 98% to 99%. The maximum nitrification rate of the modified bacterium of X16 by DNA-shuffling was up to 42.9 mg/L.d, which was almost 10 times higher than that of its parental bacteria. Furthermore, the modified bacterium had the same characteristics of its parental bacteria of E. coli and could grow rapidly in normal cultures. Conclusion DNA-shuffling was successfully used to engineer E. coli, which had norB gene and could degrade inorganic nitrogen effectively.

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.

Detecting Variation in the Rate of Molecular Evolution in Different Lineages of Mammals

A major research topic within molecular evolution studies is to understand more about the causes of the molecular evolutionary patterns that are recorded within and between taxa. The amount of germ cell divisions in females and males causes the majority of mutations, during DNA replication, that impact molecular evolution. In an XX female and an XY male system of diploid animals, the autosomes come in duplicates, with one copy from the male and female parent. Because of this, the idea that evolution is driven by male mutations has become increasingly more likely. This paper looks at the different male-mutation rates and determines that the male-mutation rate is much higher than female-mutation rates. Ry/a to be approximately 2.2, which means that Ry is approximately -24.2. From software analysis, x was approximated to be about 0.5. And since x and Ry are known, Ry/x was determined to be -49. The results for this paper show the calculated Rx/a and Ry/a are similar to the results of another study, but they are unique in that they produced a relatively high negative number for the Ry/a, which was about -49. This provides evidence that the male-mutation rate is higher than the female-mutation rate. This is interesting because this suggests that, from the data, the mutation rate in males is the defining force in molecular evolution. And because the rate goes beyond the prescribed model, future models of molecular systems will need to consider the rate of male mutations, as well as clarifying this male-mutation rate and calculating the rate of mutation in other sex-determinant systems.

The role of polysaccharides in the molecular evolution of biopolymers

The empirical (biochemical, the PCR method) and computational (quantum-chemical, PM3) methods have shown the ability of polysaccharides to catalyze the polymerization of amino acids and nucleotides within the physiologically acceptable temperature range. The possibility of nucleotide aminoacylation in the presence of polysaccharides has been established. The suggestion has been made that abiogenic aminoacylation of nucleotides by polysaccharides served as the prototype of the original aminoacylpre-tRNA-synthetase activity and subsequently determined the formation of the modern mechanism of genetic information transfer via three biopolymer types—nucleic acids, proteins and polysaccharides.

Taxonomy and molecular evolution of secondary metabolism pathway of isoflavones biosynthesis

We compared the cDNA sequences and amino acid sequences of Chalcone synthase （CHS）, Chalcone isomerase （CH1）, and lsoflavone synthase （IFS） in isoflavone biosynthesis pathway, found that their similarity were not always consistent; then we compared the amino acid sequences of CHS, CHI, and IFS, found that the clustering relationship of CHS and CHI were different in 13 kind of species; CHS and IFS were the same or similar in leguminous plants Medicago sativa, Medicago truncatula, Pisum sativum, Pueraria Montana, and Glycine max however disaccording with CHI. We prefer the amino acid sequence for cDNA sequence for evolution analysis, and think that the secondary metabolism mostly attributing to environmental stresses, and the functional genes on secondary metabolism pathway are unsuitable for taxonomy analysis.

Cloning and molecular evolution research of porcine GAD65 gene

Glutamate decarboxylase （GAD） has been found in animal and higher plant tissues as well as in yeasts and microorganisms. In animals the enzyme plays an important role in central nervous system activity because the enzyme substrate glutamic acid is a mediator of excitation process and the product, gamma-aminobutyric acid, is the most important mediator of inhibition process in the central nervous system. GAD65 is one form of the glutamate decarboxylases （GAD）, GAD65 has been identified as a major autoantigen in type 1 diabetes, so the GAD65 gene of porcine was cloned by RT-PCR method to construct phylogenetic tree, the homology of 13 glutamate decarboxylases （GAD） of different origin was analyzed by multiple alignment.

Cloning and molecular evolution of 9-cis-epoxycarotenoid dioxygenase gene (NCED3) in six species of Glycyrrhiza L

Objective:To understand the evolutionary relationship of NCED3 in Glycyrrhiza L.and find genetic evidence of its local adaptation.Meanwhile,lay the foundation for relieving the demand and subsequent starvation of Glycyrrhiza resources.Methods:NCED3 were isolated from six species of Glycyrrhiza L.by rapid amplification of cDNA ends and a homology cloning strategy.The basic properties and evolutionary relationships of NCED3 were analyzed by bioinformatics.Results:Six NCED3 genes formed two clusters;they were under purifying selection and were highly conserved in evolution (Ka/Ks≤).There was very little functional divergence among the NCED3s (Alpha value 0.071).The contents of ABA in the six species were divided into two groups.Conclusion:This study provides the first analysis of the evolution of NCED3s in six species of Glycyrrhiza.Six NCED3 genes were slow evolutionary rates in six NCED3s.Different genetic relationships to Glycyrrhiza adapted to various environments.The changes in ABA content were consistent with the evolution of NCED3.Whether there are direct relationships between NCED3 and environmental adaptation,the answer is uncertain,and we hope to show more positive results in the further study of NCED3 in Glycyrrhiza L.

Evolutions of CHCN abundance in molecular clumps

To investigate the effects of massive star evolution on surrounding molecules,we select nine massive clumps previously observed with the Atacama Pathfinder Experiment(APEX) telescope and the Submillimeter Array(SMA) telescope.Based on the observations of APEX,we obtain luminosity to mass ratios Lclump/Mclump that range from 10 to 154 L_(⊙)/M_(⊙),where some of them embedded ultra compact(UC) H Ⅱ region.Using the SMA,CH3CN(12K-11K) transitions were observed toward nine massive starforming regions.We derive the CH3CN rotational temperature and column density using the XCLASS program,and calculate its fractional abundance.We find that CH3CN temperature seems to increase with the increase of Lclump/Mclump when the ratio is between 10 to 40 L_(⊙)/M_(⊙),then decrease when L_(clump)/M_(clump)≥ 40 L_(⊙)/M_(⊙).Assuming that the CH3CN gas is heated by radiation from the central star,the effective distance of CH3CN relative to the central star is estimated.The distance ranges from - 0.003 to - 0.083 pc,which accounts for - 1/100 to - 1/1000 of clump size.The effective distance increases slightly as Lclump/Mclump increases(Reff-(L_(clump)/M_(clump))0.5±0.2).Overall,the CH3 CN abundance is found to decrease as the clumps evolve,e.g.,XCH3CN -(L_(clump)/M_(clump))-1.0±0.7.The steady decline of CH3CN abundance as the clumps evolution can be interpreted as a result of photodissociation.

The molecular evolution of spermatogenesis across mammals

The testis produces gametes through spermatogenesis and evolves rapidly at both the morphological and molecular level in mammals1-6,probably owing to the evolutionary pressure on males to be reproductively successful7.However,the molecular evolution of individual spermatogenic cell types across mammals remains largely uncharacterized.

Evolutionary history and phylogeography of Scots pine (Pinus sylvestris L.) in Europe based on molecular markers

In this review we summarized recent historical records and molecular studies on evolutionary history and phylogeography of Scots pine with focus on the European highly fragmented distribution area of the species. Fossilized pollen, plant micro- and macrofossil records provided evidences on the large-scale species’ range shifts and demographic changes during the Quaternary. Populations of Scots pine were documented both in the glacial (incl. full glaciation) and interglacial periods. Recolonization of Europe after the glaciation originated from the (Sub) Mediterranean areas like the Balkan Peninsula but also from around the Eastern Alps and the surroundings of the Danube plain. Fennoscandia and northern European Baltic regions were most probably colonized from two main directions, from Western Europe and from the Russian Plain. Modern history of Scots pine was hardly affected by anthropogenic activities that started to strengthen in the Bronze and Iron Age. Along with the fossil records, molecular genetic tools were used to infer the origin and putative history including migration, differentiation and demography of the species. In this paper we compiled the major publications (30) of molecular genetic studies of the past 20 years derived from distinctly inherited organelle genomes (mitochondrial, chloroplast, nuclear) revealed by different marker systems (mtDNA-cox1, -nad1, -nad3, -nad7, ISSR, cpSSR, nSSR, B-SAP, SNP). It is important to consider that different phylogeographic patterns can be drawn by the analysis of different DNA marker types. Accordingly the use of more than one marker simultaneously outlines the most sophisticated phylogeographical pattern on the genetic lineages and can reveal high differentiation of the European distribution. Combined marker systems and markers derived from coding sequences have also been used to detect species’ phylogeographic patterns, but these were rarely applied to Scots pine. Although new molecular techniques can provide higher resolution data for populations, the reviewed results can shape the direction of further studies.

MORPHOLOGY EVOLUTION IN PTFE AS A FUNCTION OF MELT TIME AND TEMPERATURE——Ⅰ.HIGH MOLECULAR WEIGHT SINGLE-AND MULTI-MOLECULE FOLDED CHAIN SINGLE CRYSTALS AND BAND STRUCTURES

The effect of sintering dispersed dispersion and nano-emulsion particles of high molecular weightpolytetrafluoroethylene(PTFE)on a substrate as a function of“melt”time and temperature is described.Folded chain singlecrystals parallel to the substrate and as ribbons on-edge(with double striations),as well as bands,are produced for longersintering times;particle merger and diffusion of individual molecules,crystallizing as folded chain,single(or few)molecule,single crystals when“trapped”on the substrate by cooling occur for shorter sintering times.It is suggested the observedstructures develop with sintering time,in a mesomorphic melt.The structure of the nascent particles is also discussed.

Evolutionary biogeography on Ophiocordyceps sinensis:An indicator of molecular phylogeny to geochronological and ecological exchanges

The fungus Ophiocordyceps sinensis is endemic to the vast region of the Qinghai-Tibetan plateau(QTP).The unique and complex geographical environmental conditions have led to the "sky island" distribution structure of O.sinensis.Due to limited and unbalanced sample collections,the previous data on O.sinensis regarding its genetic diversity and spatial structure have been deemed insufficient.In this study,we analyzed the diversity and phylogeographic structures of O.sinensis using internally transcribed spacer region(ITS) and 5-locus datasets by a large-scale sampling.A total of 111 haplotypes of ITS sequences were identified from 948 samples data of the fungus O.sinensis,with representing high genetic diversity,and 8 phylogenetic clades were recognized in O.sinensis.Both the southeastern Tibet and the northwestern Yunnan were the centers of genetic diversity and genetic differentiation of the fungus,and they were inferred as the glacial refugia in the Quaternary.Three distribution patterns were identified to correspond to the 8 clades,including but not limited to the coexistence of widely and specific local distributive structures.It also revealed that the differentiation pattern of O.sinensis did not fit for the isolation-by-distance model.The differentiation into the 8 clades occurred between 1.56 Myr and6.62 Myr.The ancestor of O.sinensis most likely originated in the late Miocene(6.62 Myr) in the northwestern Yunnan,and the Scene A-C of the Qinghai-Tibetan movements may have played an important role in the differentiation of O.sinensis during the late Miocene-Pliocene periods.Our current results provide a much clearer and detailed understanding of the genetic diversity and geographical spatial distribution of the endemic alpine fungus O.sinensis.It also revealed that the geochronology resulting from paleogeology could be cross-examined with biomolecular clock at a finer scale.

The Analysis of Thermal-Induced Phase Transformation and Microstructural Evolution in Ni-Ti Based Shape Memory Alloys By Molecular Dynamics

Shape memory alloys has been widely applied on actuators and medical devices.The transformation temperature and microstructural evolution play two crucial factors and dominate the behavior of shape memory alloys.In order to understand the influence of the composition of the Ni-Ti alloys on the two factors,molecular dynamics was adopted to simulate the temperature-induced phase transformation.The results were post-processed by the martensite variant identification method.The method allows to reveal the detailed microstructural evolution of variants/phases in each case of the composition of Ni-Ti.Many features were found and having good agreement with those reported in the literature,such as the well-known Rank-2 herringbone structures;the X-interface;Ni-rich alloys have lower transformation temperature than Ti-rich alloys.In addition,some new features were also discovered.For example,the Ti-rich alloys enabled an easier martensitic transformation;the nucleated martensite pattern determined the microstructural evolution path,which also changed the atomic volume and temperature curves.The results generated in the current study are expected to provide the design guidelines for the applications of shape memory alloys.