Safety assessment of a novel marine multi-stress-tolerant yeast Meyerozyma guilliermondii GXDK6 according to phenotype and whole genome-sequencing analysis

The application of microorganisms as probiotics is limited due to lack of safety evaluation.Here,a novel multi-stress-tolerant yeast Meyerozyma guilliermondii GXDK6 with aroma-producing properties was identified from marine mangrove microorganisms.Its safety and probiotic properties were assessed in accordance with phenotype and whole-genome sequencing analysis.Results showed that the genes and phenotypic expression of related virulence,antibiotic resistance and retroelement were rarely found.Hyphal morphogenesis genes(SIT4,HOG1,SPA2,ERK1,ICL1,CST20,HSP104,TPS1,and RHO1)and phospholipase secretion gene(VPS4)were annotated.True hyphae and phospholipase were absent.Only one retroelement(Tad1-65_BG)was found.Major biogenic amines(BAs)encoding genes were absent,except for spermidine synthase(JA9_002594),spermine synthase(JA9_004690),and tyrosine decarboxylase(inx).The production of single BAs and total BAs was far below the food-defined thresholds.GXDK6 had no resistance to common antifungal drugs.Virulence enzymes,such as gelatinase,DNase,hemolytic,lecithinase,and thrombin were absent.Acute toxicity test with mice demonstrated that GXDK6 is safe.GXDK6 has a good reproduction ability in the simulation gastrointestinal tract.GXDK6 also has a strong antioxidant ability,β-glucosidase,and inulinase activity.To sum up,GXDK6 is considered as a safe probiotic for human consumption and food fermentation.

Development and parentage analysis of SNP markers for Chestnut-vented Nuthatch(Sitta nagaensis)based on ddRAD-seq data

Extra-pair paternity(EPP)is commonly found in socially monogamous birds,especially in small passerine birds,and there are interspecific and intraspecific variations in the extent of EPP.The Chestnut-vented Nuthatch(Sitta nagaensis)is a socially monogamous passerine bird,and verifying whether this species has EPP relies on parentage testing-S.nagaensis is not known to have EPP.In this study,we developed SNP markers of this species that are informative for parentage analysis from double digest restriction site-associated DNA sequencing(ddRAD-seq)data.A panel consisting of 50 SNP markers,with a mean heterozygosity of 0.343,was used to resolve 95% of nestlings to fathers.The combined exclusion probabilities for the first parent and second parent were 0.991 and 0.9999,respectively.This panel of SNP markers is a powerful tool for parentage assignments in S.nagaensis.In addition,we found that three offspring(7.9%)from three nests(23.1%)were the result of extra-pair fertilization out of 38 offspring in 13 nests.Our study provided information on parentage analysis that has not been reported before in S.nagaensis.It also supplemented the understudied EPP behavior of birds in Asia,contributing to a general understanding of the EPP behaviors of birds.

Screening biomarkers for spinal cord injury using weighted gene co-expression network analysis and machine learning

Immune changes and inflammatory responses have been identified as central events in the pathological process of spinal co rd injury.They can greatly affect nerve regeneration and functional recovery.However,there is still limited understanding of the peripheral immune inflammato ry response in spinal cord inju ry.In this study.we obtained microRNA expression profiles from the peripheral blood of patients with spinal co rd injury using high-throughput sequencing.We also obtained the mRNA expression profile of spinal cord injury patients from the Gene Expression Omnibus(GEO)database(GSE151371).We identified 54 differentially expressed microRNAs and 1656 diffe rentially expressed genes using bioinformatics approaches.Functional enrichment analysis revealed that various common immune and inflammation-related signaling pathways,such as neutrophil extracellular trap formation pathway,T cell receptor signaling pathway,and nuclear factor-κB signal pathway,we re abnormally activated or inhibited in spinal cord inju ry patient samples.We applied an integrated strategy that combines weighted gene co-expression network analysis,LASSO logistic regression,and SVM-RFE algorithm and identified three biomarke rs associated with spinal cord injury:ANO10,BST1,and ZFP36L2.We verified the expression levels and diagnostic perfo rmance of these three genes in the original training dataset and clinical samples through the receiver operating characteristic curve.Quantitative polymerase chain reaction results showed that ANO20 and BST1 mRNA levels were increased and ZFP36L2 mRNA was decreased in the peripheral blood of spinal cord injury patients.We also constructed a small RNA-mRNA interaction network using Cytoscape.Additionally,we evaluated the proportion of 22 types of immune cells in the peripheral blood of spinal co rd injury patients using the CIBERSORT tool.The proportions of naive B cells,plasma cells,monocytes,and neutrophils were increased while the proportions of memory B cells,CD8^(+)T cells,resting natural killer cells,resting dendritic cells,and eosinophils were markedly decreased in spinal cord injury patients increased compared with healthy subjects,and ANO10,BST1 and ZFP26L2we re closely related to the proportion of certain immune cell types.The findings from this study provide new directions for the development of treatment strategies related to immune inflammation in spinal co rd inju ry and suggest that ANO10,BST2,and ZFP36L2 are potential biomarkers for spinal cord injury.The study was registe red in the Chinese Clinical Trial Registry(registration No.ChiCTR2200066985,December 12,2022).

Transcriptomic and bioinformatics analysis of the mechanism by which erythropoietin promotes recovery from traumatic brain injury in mice

Recent studies have found that erythropoietin promotes the recovery of neurological function after traumatic brain injury.However,the precise mechanism of action remains unclea r.In this study,we induced moderate traumatic brain injury in mice by intrape ritoneal injection of erythro poietin for 3 consecutive days.RNA sequencing detected a total of 4065 differentially expressed RNAs,including 1059 mRNAs,92 microRNAs,799 long non-coding RNAs,and 2115circular RNAs.Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analyses revealed that the coding and non-coding RNAs that were differentially expressed after traumatic brain injury and treatment with erythropoietin play roles in the axon guidance pathway,Wnt pathway,and MAPK pathway.Constructing competing endogenous RNA networks showed that regulatory relationship between the differentially expressed non-coding RNAs and mRNAs.Because the axon guidance pathway was repeatedly enriched,the expression of Wnt5a and Ephb6,key factors in the axonal guidance pathway,was assessed.Ephb6 expression decreased and Wnt5a expression increased after traumatic brain injury,and these effects were reversed by treatment with erythro poietin.These findings suggest that erythro poietin can promote recove ry of nerve function after traumatic brain injury through the axon guidance pathway.

Isolation and Analysis of α-Gliadin Gene Coding Sequences from Triticum durum

Three coding sequences of gliadins genes, designed as Gli2_Dul, Gli2_Du2 and Gli2_Du3, were isolated from the genomic DNA of Triticum durum accessions CItr5083. Gli2_Dul and Gli2_Du2 contain 945 and 864 bp, encoding the mature proteins with 314 and 287 amino acid residues, respectively. Gli2_Du3 is recognized as a pseudogene due to the stop codon occurring in the coding region. The pseudogenes, commonly occurring in gliadins family, are attributed to the single base change C→T. The amino acid sequences deduced from these gene sequences were characterized with the typical structure of α-gliadin proteins, including the toxic sequences （PSQQQP）. The peptide fraction PF（Y）PP（Q）is thought to be an extra unit of repetitive domain, slightly diverging from the previous report. Six cysteine residues were observed within two unique domains. Phylogenetic analysis showed Gli2_Du2 and Gli2_Du3 were closely related to the genes on chromosome 6A, whereas Gli2_Dul seems to be more homologous with the genes on chromosome 6B.

A new Multilocus Sequence Analysis Scheme for Mycobacterium tuberculosis

Objective Tuberculosis remains one of the most serious infectious diseases in the world. In this study, a scheme of Mycobacterium tuberculosis （M. tuberculosis） multilocus sequence analysis （MLSA） was established for the phylogenetic and epidemiology analysis. Methods To establish the scheme of M. tuberculosis MLSA, the genome of H37Rv, CCDC5079 and CCDC5180 were compared, and some variable genes were chosen to be the MLSA typing scheme. 44 M. tuberculosis clinical isolates were typed by MLSA, IS6110-RFLP, and soligotyping, to evaluate the MLSA methods. Results After comparison of the genome, seven high discrimination gene loci （recX, rpsL, rmlC, rpmG1, mprA, gcvH, ideR） were chosen to be the MLSA typing scheme finally. 11 variable SNP sites of those seven genes were found among the 44 M. tuberculosis isolate strains and 11 sequence types （STs） were identified. Based on the Hunter-Gaston Index （HGI）, MLSA typing was not as good for discrimination at the strain level as IS6110-RFLP, but the HGI was much better than that of spoligotyping. In addition, the MEGA analysis result of MLSA data was similar to spoligotyping/PGG lineage, showing a strong phylogenetic signal in the modern strains of M. tuberculosis. The MLSA data analysis by eBURST revealed that 4 sequence types （ST） came into a main cluster, showing the major clonal complexes in those 44 strains. Conclusion MLSA genotyping not only can be used for molecular typing, but also is an ideal method for the phylogenetic analysis for M. tuberculosis.

Isolation and Sequence Analysis of HMW Glutenin Subunit 1Dy10.1 Ecoding Gene from Xinjiang Wheat (Triticum petropavlovskyi Udacz. et Migusch)

A novel HMW glutenin subunit gene 1Dy10. 1 was isolated and characterized from Xinjiang wheat （Triticum petropavlovskyi. Udacz. et Migusch） accession Daomai 2. The complete open reading frame （ORF） of 1Dy10. 1 was 1 965 bp, encoding 655 amino acids. The numbers and distribution of cysteines in 1Dy 10.1 were similar to those of 1Dy10 and other y-type subunits. In the N-terminal of 1Dy 10.1, an amino acid was changed from L （leucine） to P （proline） at position 55. The repetitive domain of 1Dy10.1 differed from those of known HMW subunits by substitutions, insertions or/and deletions involving single or more amino acid residues. In the repetitive domain of subunit 1Dyl 0.1, the deletion of tripeptide GQQ in the consensus unit PGQGQQ resulted in the appearance of the motif PGQ that have not been observed in other known y-type HMW subunits. In comparison with the subunit 1Dy 12, a deletion of dipeptide GQ, which occurred in subunit IDy10, was also observed in subunit 1Dy10.1. The cloned IDylO. 1 gene had been successfully expressed in Escherichia coli, and the expressed protein had the identical mobility with the endogenous subunit IDy10.1 from seed.

Vitis amuerensis CBF3 Gene Isolation,Sequence Analysis and Expression

The full length cDNA sequence of CBF3 （CRT/DRE-binding factor） was cloned from Vitis amurensis by reverse transcription polymerase chain reaction （RT-PCR） using the primers designed based on CBF genes available in GenBank. Sequence analysis showed that the gene had 854 bp long and its coding sequence contained 720 bp, encoding a protein with 239 amino acids and an AP2 structural domain. The molecular mass of CBF3 was predicted to be 25.9 kDa and its theoretical isoelectric point was 7.02 and aliphatic index was 59.29. The average hydropathicity of the protein was -0.551. The tertiary structures of CBF3 were also analyzed. The prokaryotic expression vector pGEX-4T-CBF3 containing CBF3 gene was constructed and CBF3 fusion protein （52 kDa） was produced in Escherichia coli after induction with 1 mmol L-1 IPTG. Further studies are needed to evaluate its potential application for improving plant resistance to cold and other stress condition such as drought and salinity.

Cloning and Sequence Analysis of a Novel Cold-Adapted Lipase Gene from Strain lip35 (Pseudomonas sp.)

A combination method of the usual-PCR and reverse-PCR for the cloning of a novel lipase gene directly from the total genomic DNA of strain lip35 （Pseudomonas sp.） is described, whereby a lipase gene （lip） was cloned directly from genomic DNA. The sequence data have been deposited in the GenBank and EMBL data bank with the accession number EU414288. The nucleotide sequence showed a major open reading frame encoding a 59-kDa protein of 566 amino acid residues, which contained a lipase consensus sequence GXSXG. The lipase lip had 74 and 70% homologies with the lipases of an uncultured bacterium and P. fluorescens PfO-1, respectively, but it did not show any overall homology with lipases from other origins. The functional lipase was obtained when the lip gene was expressed in Pichia pastoris GS115.

Cloning and Sequence Analysis of Expansin Genes from Litchi chinensis Fruit

Using PCR degenerate primers, designed with reference to the sequences of the conserved amino acids of known expansins, to amplify cDNA fragments in litchi fruit by RT-PCR, two different cDNA fragments , named as Lc-Exp1 and Lc-Exp2 , were cloned. Lc-Exp1 and Lc-Exp2 was respectively composed of 531 bp encoding 177 amino acids and 537 bp encoding 179 amino acids. Eight cysteine residues and three tryp-tophan residues, which is supposed to be the characteristics of expansins, are conserved in both Lc-Exp1 and c-Exp2. In addition, the homology between the two expansins is 71. 6% at nucleotide acid sequences and 76.3% at amino acid sequences. The homology of Lc-Exp1 with Fa-Exp2 or Pp-Exp1 was 92.7% or 92.1%, but that of Lc-Exp2 with Fa-Exp2 or Pp-Exp1 was only 77. 4% or 76.3% at amino acid sequences.

Cloning and sequence analysis of the partial sequence of the rbcL from Bryopsis hypnoides

The partial sequence of the rbcL from Bryopsis hypnoides, including the sequences of the upstream, extron and partial intron, was amplified by PCR and their sequences were determined. With Spinacia oleracea as the outgroup, neighbor-joining method and maximum parsimony method were used respectively to build phylogenetic trees according to the rbcL exon sequence among 13 species that were the typical species of six phyla. Two kinds of trees showed clearly that there were two groups among those species, the green lineage and the non-green lineage. And the relationships of algae in the green lineage were similar in the two trees but those in the non-green lineage were not consistent. Analysis of codon preference indicated that the codon preference of the rbcL exon of Bryopsis hypnoides distinctly differed from that of the relevant sequence of photosynthetic bacteria.

Cloning and Expression Analysis of Downy Mildew Resistance-Related cDNA Sequences in Melon

Melon downy mildew caused by Pseudoperonospora cubensis leads to significant losses in melon yields worldwide. Reverse-transcription Polymerase Chain Reaction （RT-PCR） was performed using cDNAs as templates from melon-Huangdanzi induced with fungus Pseudoperonospora cubensis, and degenerate primers designed based on the conserved amino acid sequences of known plant disease-resistance genes. A polymorphic cDNA fragment which we named rap-19 was cloned and sequenced. The Open Reading Frame （ORF） of this product comprised of 510 base pairs which encodes DNA or RNA-binding protein with 170 amino acids. The putative amino acid sequence of mp-19 appeared highly homologous with those of NBS-type resistant-genes isolated from other plants. Southern blot indicated that the melon genome contained more than 3 copies of rap-19. The obvious expression differences detected by semi-quantitative RT- PCR could be observed between resistant-line Huangdanzi and susceptible-line Jiashi after Pseudoperonospora cubensis infection, which implied that mp-19 gene may be related to the resistance of downy mildew in melon.

Complete genome sequence and proteomic analysis of a thermophilic bacteriophage BV1

Viruses of thermophiles are of great interest due to their roles in gene transfer, global geochemical cycle and evolution of life on earth. However, the thermophilic bacteriophages have not been studied extensively. In this investigation, a typical bacteriophage BV1 was obtained from a thermophilic bacterium Geobacillus sp. 6k512, which was isolated from an inshore hot spring in Xiamen of China. The BV1 contained a double-stranded linear DNA of 35 055 bp, which encodes 54 open reading frames （ORFs）. Interestingly, eight of the 54 BV1 ORFs shared sequence similarities to genes from human disease-relevant bacteria. Seven proteins of the purified BV1 virions were identified by proteomic analysis. Determination of BV1 functional genomics would facilitate the better understanding of the mechanism for virus-thermophile interaction.

Genetic Diversity among Parents of Hybrid Rice Based on Cluster Analysis of Morphological Traits and Simple Sequence Repeat Markers

The genetic diversity of 41 parental lines popularized in commercial hybrid rice production in China was studied by using cluster analysis of morphological traits and simple sequence repeat （SSR） markers. Forty-one entries were assigned into two clusters （i.e. early or medium-maturing cluster; medium or late-maturing cluster） and further assigned into six sub-clusters based on morphological trait cluster analysis, The early or medium-maturing cluster was composed of 15 maintainer lines, four early-maturing restorer lines and two thermo-sensitive genic male sterile lines, and the medium or late-maturing cluster included 16 restorer lines and 4 medium or late-maturing maintainer lines. Moreover, the SSR cluster analysis classified 41 entries into two groups （i.e, maintainer line group and restorer line group） and seven sub-groups. The maintainer line group consisted of all 19 maintainer lines, two thermo-sensitive genic male sterile lines, while the restorer line group was composed of all 20 restorer lines. The SSR analysis fitted better with the pedigree information. From the views on hybrid rice breeding, the results suggested that SSR analysis might be a better method to study the diversity of parental lines in indica hybrid rice.

The structural analysis of protein sequences based on the quasi-amino acids code

Proteomics is the study of proteins and their interactions in a cell. With the successful completion of the Human Cenome Project, it comes the postgenome era when the proteomics technology is emerging. This paper studies protein molecule from the algebraic point of view. The algebraic system （∑, ＋, ＊） is introduced, where ∑ is the set of 64 codons. According to the characteristics of （∑, ＋, ＊）, a novel quasi-amino acids code classification method is introduced and the corresponding algebraic operation table over the set ZU of the 16 kinds of quasi-amino acids is established. The internal relation is revealed about quasi-amino acids. The results show that there exist some very close correlations between the properties of the quasi-amino acids and the codon. All these correlation relationships may play an important part in establishing the logic relationship between codons and the quasi-amino acids during the course of life origination. According to Ma F et al （2003 J. Anhui Agricultural University 30 439）, the corresponding relation and the excellent properties about amino acids code are very difficult to observe. The present paper shows that （ZU, ＋,×） is a field. Furthermore, the operational results display that the eodon tga has different property from other stop codons. In fact, in the mitochondrion from human and ox genomic codon, tga is just tryptophane, is not the stop codon like in other genetic code, it is the case of the Chen W C et al （2002 Acta Biophysiea Siniea 18（1） 87）. The present theory avoids some inexplicable events of the 20 kinds of amino acids code, in other words it solves the problem of ＇the 64 codon assignments of mRNA to amino acids is probably completely wrong＇ proposed by Yang （2006 Progress in Modern Biomedicine 6 3）.

Aequorea taiwanensis n. sp. (Hydrozoa,Leptomedusae) and mtCOI sequence analysis for the genus Aequorea

Nine species of Prionospio complex are recorded from China＇s waters,including one new species and six newly recorded species.Prionospio（Prionospio） pacifica sp.nov.,is characterized by having first and forth pairs of branchiae pinnate,second and third pairs of apinnate,ventral crest on Setiger 9 and dorsal crests on Setigers 10-25.Apoprionospio kirrae（Wilson,1990）,Prionospio（Aquilaspio） convexa Imajima,1990,Prionospio（Minuspio） multibranchiata Berkeley,1927,Prionospio（Prionospio） bocki Sderstrm,1920,Prionospio（Prionospio） dubia Maciolek,1985 and Prionospio（Prionospio） paradisea Imajima,1990 are recorded for the first time from China＇s waters.

Molecular Cloning and cDNA Sequence Analysis of Two New Lepidopteran OR83b Orthologue Chemoreceptors

The aim of this study was to isolate the full-length cDNA sequences of an unusually highly conserved olfactory receptor （orthologue to the Drosophila melanogaster DOR83b） from the antennae of Spodoptera exigua and S. litura by using reverse transcription-polymerase chain reaction （RT-PCR） and rapid amplification of cDNA ends （RACE） methods. Bioinformatics methods were used to further analyze the cDNA sequences and putative amino acid sequences. The two full-length cDNA sequences from olfactory receptor （OR） of male S. exigua and S. litura were named as SexiOR2 and SlitOR2, respectively. SexiOR2 and SlitOR2 consisted of nucleotide sequence of 1 906 and 2 483 bp, respectively, and both with deduced amino acid sequences of 473 residues. The sequence analysis indicated that the deduced amino acid sequences of the cDNA shared the high homologies with OR83b orthologue chemoreceptor sequences from previously reported moths, implying that the cDNA sequences were of OR83b orthologue chemoreceptor genes.

Stability analysis of backflling in subsiding area and optimization of the stoping sequence

In underground mining by sublevel caving method, the deformation and damage of the surface induced by subsidence are the major challenging issues. The dynamic and soft backflling body increases the safety risks in the subsiding area. In this paper, taking Zhangfushan iron mine as an example, the ore body and the general layout are focused on the safety of backflling of mined-out area. Then, we use the ANSYS software to construct a three-dimensional(3D) model for the mining area in the Zhangfushan iron mine. According to the simulation results of the initial mining stages, the ore body is stoped step by step as suggested in the design. The stability of the backflling is back analyzed based on the monitored displacements, considering the stress distribution to optimize the stoping sequence. The simulations show that a reasonable stoping sequence can minimize the concentration of high compressive stress and ensure the safety of stoping of the ore body.

Isolation and Sequence Analysis of NSP2, ORF3 and ORF5 of PRRSV XH-GD Strain from Guangdong Province

The diseased samples collected from Guangdong Province were inoculated to Marc-145 cells, PRRSV-specific cytopathogenic effects （CPE） such as cell aggregation, cell shrinkage and cell exfoliation were observed and a PRRSV strain XH-GD was obtained. By using primers designed in accordance with the pub- lished gene se^lences of PRRSV in GenBank, NSP2, ORF3 and ORF5 fragments were amplified by reverse transcription polymerase chain reaction and analyzed by sequence aligment. The results demonstrated that PRRSV strain XH-GD belonged to the North American genotype and the NSP2 gene contained discontinuous de- letions of 30 amino acids. NSP2, ORF3 and ORF5 genes of XH-GD shared 83.3% -98.9%, 88.6% -99.2% and 88.1% -99.2% nucleotide homology, and 76.8% -98.3%, 83.7% -98.8% and 88.1% -99.2% amino acid homology with HUB1, NX06, BJsy06, VR-2332, HB-1（sh）2002, HB-2（sh）2002, CH-1a and RespPRRS MLV, respectively; however, NSP2, ORb3 and ORF5 genes of XH-GD shared only 52.9%, 65.0% and 64. 3% nucleotide homology, and 31.3%, 57.5% and 58.9% amino acid homology with LV, respectively. Phylogenetic analysis revealed that XH-GD had a closer relationship with HUB1, NX06, BJsy06, VR-2332, HB-1 （sh） 2002, HB-2 （sh） 2002, CH-la and RespPRRS MLV strains compared with LV strain.

Sequence Analysis of Polypeptide by Mass Spectrometry

The method described in this work provides a sensitive and fast technique for investigating the primary structure of peptides with molecular weight up to 3340 amu. Usually, the metastable ion kinetic energy spectra (MIKES) and collisional activated decomposition (CAD) spectra provide complementary information for the FAB mass spectra, the MIKES and CAD spectra generally contain high-mass sequence ions.