Profiles and differences of submucosal microbial in peri-implantitis and health implants: A cross-sectional study / 北京大学学报(医学版)

OBJECTIVE@#To describe the submucosal microbial profiles of peri-implantitis and healthy implants, and to explore bacteria that might be correlated with clinical parameters.@*METHODS@#In the present cross-sectional study, 49 patients were recruited. Each patient contributed with one implant, submucosal biofilms were collected from 20 healthy implants and 29 implants with peri-implantitis. DNA was extracted and bacterial 16S ribosomal RNA (16S rRNA) genes were amplified. Submucosal biofilms were analyzed using 16S rRNA sequencing at Illumina MiSeq platform. Differences between the groups were determined by analyzing α diversity, microbial component and microbial structure. The potential correlation between the bacteria with pocket probing depth (PPD) of peri-implant calculated by Spearman correlation analysis.@*RESULTS@#The α diversity of submucosal microbial of health group was significantly lower than that in peri-implantitis group (Chao1 index: 236.85±66.13 vs. 150.54±57.43, P < 0.001; Shannon index: 3.42±0.48 vs. 3.02±0.65, P=0.032). Principal coordinated analysis showed that the submucosal microbial structure had significant difference between healthy and peri-implantitis groups [R2=0.243, P=0.001, analysis of similarities (ANOSIM)]. Compared with healthy implants, relative abundance of periodontal pathogens were higher in peri-implantitis, including members of the red complex (Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola) and some members of orange complex (Precotella intermedia, Eubacterium nodatum, Parvimonas micra), as well as some new periodontal pathogens, such as Fillifactor alocis, Fretibacterium fastidiosum, Desulfobulbus sp._HMT_041, and Porphyromonas endodontalis. Spearman correlation analysis revealed that the relative abundance of Treponema denticola (r=0.686, P < 0.001), Tannerella forsythia (r=0.675, P < 0.001), Fretibacterium sp. (r=0.671, P < 0.001), Desulfobulbus sp._HMT_041 (r=0.664, P < 0.001), Filifactor alocis (r=0.642, P < 0.001), Fretibacterium fastidiosum (r=0.604, P < 0.001), Porphyromonas gingivalis (r=0.597, P < 0.001), Porphyromonas endodontalis (r=0.573, P < 0.001) were positive correlated with PPD. While the relative abundance of Rothia aeria (r=-0.615, P < 0.001) showed negatively correlation with PPD.@*CONCLUSION@#Marked differences were observed in the microbial profiles of healthy implants and peri-implantitis. The members of red and orange complex as well as some new periodontal pathogens seem to play an important role in peri-implant disease. Compared with healthy implants, the submucosal microbial of peri-implantitis were characterized by high species richness and diversity.

Community structure of endophytic fungi in roots and leaves of Fagopyrum mill and Avena sativa in a Chinese northern cold region

In order to explore the endophytic fungi of Fagopyrum Mill and Avena sativa, Illumina Miseq high-throughput sequencing was used to analyze the community structure and diversity of endophytic fungi in leaves and roots of buckwheat and oat at the mature stage. The results of community structure showed that there were 205 operational taxonomic units (OTUs) in buckwheat roots and 181 OTUs in buckwheat leaves based on 97% sequence similarity level. There were 152 OTUs and 127 OTUs in the root and the leaf of oat, respectively. At the phylum level, Ascomycota and Basidiomycota were the dominant endophytic fungi in buckwheat roots and leaves, while Ascomycota was the dominant endophytic fungus in oat roots and leaves. Alpha diversity analysis showed that the Ace index, Chao index and Shannon index of buckwheat roots were higher than that of buckwheat leaves, and the three indices of oat roots were also higher than that of oat leaves, indicating that the richness and diversity of endophytic fungi community in roots were higher than that in leaves. Biomarkers were found by significant difference analysis in buckwheat and oat. The endophytic functional groups of buckwheat and oat were mainly distributed in Pathotroph and Saprotroph. The results of this study laid a foundation for fully exploiting the dominant endophytic fungal resources of buckwheat and oat and further developing microbial fertilizers.

Identificación y secuenciación de SARS-CoV-2 en una leona africana (Panthera leo), bajo el cuidado técnico profesional, en Colombia

Objetivo Identificar y caracterizar el virus SARS-CoV-2 en una leona africana (Panthera leo), hembra, de edad avanzada, que presentó por varios meses signos relacionados con enfermedad respiratoria atípica. Métodos Se tomaron muestras de hisopados nasales 23 días después de haber reportado secreción nasal inicial. Se realizó la detección del virus SARS-Cov2 mediante RT-qPCR y posteriormente se caracterizó el genoma completo mediante secuencia Illumina. Resultados Desde el punto de vista clínico, los resultados encontrados en las muestras de sangre no mostraron cambios evidentes que se pudieran relacionar con el virus o con todos los signos descritos desde el inicio del caso. Para la secuenciación genómica los análisis mostraron una alineación múltiple comparativa entre los tres genomas (muestra Leona, FIP u NC_045512 [Wu han]) por medio de Mauve, centrado en los genes Spike, E y M (archivo complementario, parte B). Se logró identificar 5 segmentos muy similares entre Leona y NC_045512 (Wuhan). Conclusiones Es necesario adelantar más investigaciones para estandarizar el diagnóstico de esta patología en los animales. Así mismo, se requieren estudios genómicos en estas especies. Además, se evidenció con la revisión del estado de la cuestión que existen muchos vacíos del conocimiento en la implicación zoonótica de la pandemia y en el conocimiento de este virus en animales domésticos y silvestres, lo que supone un reto importante para las investigaciones de aquí en adelante.

Objective To identify and characterize the SARS-CoV-2 virus in an elderly African lioness (Panthera leo) that presented signs related to atypical respiratory disease for several months. Methods Nasal swab samples were taken 23 days after infection. have reported initial nasal discharge. Results The SARS-Cov2 virus was detected by RT-qPCR and the complete genome was subsequently characterized by Illumina sequencing. The results found in the blood samples did not show obvious changes that could be related to the virus or to the signs described from the beginning of the case. For genomic sequencing the analyzes showed a comparative multiple alignment between the three genomes (sample Leona, FIP or NC_045512 (Wu han)) by means of Mauve, focusing on the Spike, E and M genes (Supplementary file, part B); 5 very similar segments between Leona and NC_045512 (Wuhan) was identified. Conclusions It is necessary to carry out more research to standardize the diagnosis of this pathology in animals and guarantee access to it. Also, genomic studies in these species. Additionally, it was evidenced with the literature review that there are many knowledge gaps in the zoonotic implication of the Pandemic and in the knowledge of this virus in domestic and wild animals, which represents an important challenge for research from now on.

Preliminary study on the biological characteristics of intestinal flora in IgA nephropathy based on high-throughput sequencing / 中华肾脏病杂志

Objective:To study the structure and diversity of intestinal flora in IgA nephropathy (IgAN) patients, and to explore the correlation of intestinal microorganisms with clinical indicators and renal pathology.Methods:Fifteen IgAN patients in the First Affiliated Hospital of Baotou Medical College from May 2020 to September 2020 were retrospectively enrolled as IgAN group, and 8 healthy families and 7 health checkups were enrolled as healthy control group. Illumina high-throughput sequencing technology was performed for DNA sequencing in the 16S rDNA-V4 region of all bacteria in the feces sample. QIIME 2 was used to process and analyze original sequence, compared with Greengenes (V138) database. The DADA2 software was called to denoise the data, which was equivalent to a 100% similarity cluster (OTU was a 97% similarity cluster). PCoA was used to analyze the structure and diversity of intestinal flora. Spearman correlation or Pearson correlation analysis was used to analyze the correlation of differential flora with renal pathology and clinical indicators.Results:(1) The intestinal microbial β diversity in IgAN patients was significantly different from that in healthy controls ( P=0.010). (2) Compared with the healthy control group, the numbers of intestinal flora species in IgAN group were significantly increased in 1 phylum, 3 families and 22 genus. At the levels from phylum to family, the species numbers of Firmicutes and Ruminococcaceae in IgAN patients reduced than those in healthy controls and the species numbers of Chloroflexi, Gaiellaceae, Staphylococcaceae and Family-XⅢ in IgAN patients increased than those in healthy controls (all P<0.05). At the genus level, compared with the healthy controls, the species number of Subdoligranulum in IgAN patients was significantly reduced ( P=0.020), and the species number of Ruminococcus- gnavus- group was significantly increased ( P=0.004). (3) At the phylum level of the species number, Firmicutes in IgAN patients was positively correlated to albumin (ALB) ( r=0.637, P=0.037) and IgG ( r=0.452, P=0.046), Gemmatimonadetes was negatively correlated to serum creatinine ( r=-0.453, P=0.045), Verrucomicrobia was negatively correlated to IgM ( r=-0.450, P=0.046), and Patescibacteria was positively correlated to IgA ( r=0.469, P=0.037). At the genus level of the species number, Ruminococcus- gnavus- group ( r=-0.614, P=0.004) and Megamonas ( r=-0.451, P=0.042) were negatively correlated to ALB; Subdoligranulum was positively correlated to ALB ( r=0.563, P=0.009); Dialister was negatively correlated to C3 ( r=-0.427, P=0.041) and was positively correlated to IgA ( r=0.434, P=0.035); Veillonella was positively correlated to estimated glomerular filtration rate ( r=0.452, P=0.043). The species numbers of Eisenbergiella ( r=-0.850, P=0.007), Holdemania ( r=-0.845, P=0.008), Flavonifractor ( r=-0.845, P=0.008), and Ruminiclostridium- 9 ( r=-0.845, P=0.008) were negatively correlated to glomerulosclerosis or adhesion (S) of Oxford classification; the species number of Fusicatenibacter was negatively correlated to mesangial hypercellularity ( r=-0.845, P=0.008); the number of Coprococcus- 2 was positively correlated to S ( r=0.738, P=0.037) and tubular atrophy or interstitial fibrosis ( r=0.756, P=0.030). (4) Random forest model was built with Ruminococcus- gnavus- group and Subdoligranulum, after fitting the area under the receiver operating characteristic curve was 0.927. Conclusions:The intestinal flora of IgAN patients is different from that in healthy subjects. Changes of intestinal flora in IgAN patients are related to clinical indicators and renal pathology. In particular, Ruminococcus- gnavus- group and Subdoligranulum may play an important role in IgAN.

High-throughput sequencing identifies salivary microbiota in Chinese caries-free preschool children with primary dentition / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

OBJECTIVES@#The study aimed at identifying salivary microbiota in caries-free Chinese preschool children using high-throughput sequencing.@*METHODS@#Saliva samples were obtained from 35 caries-free preschool children (18 boys and 17 girls) with primary dentition, and 16S ribosomal DNA (rDNA) V3-V4 hypervariable regions of the microorganisms were analyzed using Illumina MiSeq.@*RESULTS@#At 97% similarity level, all of these reads were clustered into 334 operational taxonomic units (OTUs). Among these, five phyla (Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes, and Candidate division TM7) and 13 genera (@*CONCLUSIONS@#Our results revealed the diversity and composition of salivary microbiota in caries-free preschool children, with little difference between male and female subjects. Identity of the core microbiome, coupled with prediction of gene function, deepens our understanding of oral microbiota in caries-free populations and provides basic information for associating salivary microecology and oral health.

High-throughput sequencing reveals the molecular mechanisms determining the stay-green characteristic in soybeans

Senescence is an internally systematized degeneration process leading to death in plants. Leaf yellowing, oneof the most prominent features of plant aging may lead to reduced crop yields. The molecular mechanism ofresponses to senescence in soybean leaves is not completely clear. In our research, two soybean varieties wereselected with different stay-green traits: stay-green variety (BN106) and non-stay-green variety (KF14). RNAsamples extracted from the leaves of two varieties were sequenced and compared using high-throughputsequencing. Six key enzyme genes in chlorophyll degradation pathways were studied to analyze the changes intheir expression at seedling, flowering and maturation stage. Meanwhile, the construction of the genetictransformation process had been constructed to identify the function of putative gene by RNA-interference. Atotal of 4329 DEGs were involved in 52 functional groups and 254 KEGG pathways. Twelve genes encodingsenescence-associated and inducible chloroplast stay-green protein showed significant differential expression.MDCase and PAO have a significant expression in BN106 that may be the key factors affecting the maintenance of green characteristics. In addition, the function of GmSGRs has been identified by genetic transformation. The loss of GmSGRs may cause soybean seeds to change from yellow to green. In summary, ourresults revealed fundamental information about the molecular mechanism of aging in soybeans with differentstay-green characteristics. The work of genetic transformation lays a foundation for putative gene functionstudies that could contribute to postpone aging in soybeans

Detección de virus de ARN en cultivos de tomate del Oriente Antioqueño mediante secuenciación de alto rendimiento y RT-qPCR / Detection of RNA viruses in tomato fields in eastern Antioquia using high-throughput sequencing (HTS) and RT-qPCR

RESUMEN En Antioquia, el cultivo de tomate (Solanum lycopersicum) se ve afectado por diversas enfermedades virales, que ocasionan la disminución en la calidad de los frutos y de los rendimientos; sin embargo, pocos estudios han identificado, a nivel de especie, los agentes causales de dichas enfermedades. En los últimos años, la secuenciación de alto rendimiento (HTS), se ha convertido en una herramienta eficiente de diagnóstico de fitopatógenos, permitiendo la detección y la caracterización genómica de un alto número de virus, en diferentes plantas. En este trabajo, se evaluó la presencia de virus de ARN infectando tomate var. Chonto del oriente Antioqueño, mediante HTS y RT-PCR, en tiempo real (RT-qPCR), en muestras de tejido foliar y en semillas. El análisis de HTS indicó la infección de los virus Potato virus S (PVS), Potato virus Y (PVY), Potato yellow vein virus (PYVV), Potato virus X (PVX), Southern tomato virus (STV) y Bell pepper endornavirus (BPEV), en los cultivos de tomate de esta región, obteniéndose los genomas completos de PYVV, STV y BPEV. Las pruebas de RT-qPCR indicaron la presencia de PYVV en el 100% de las muestras foliares analizadas, mientras que PVX, PVY, STV y PVS, se encontraron en niveles de 94,4, 77,8, 72,2 y 5,6%, respectivamente. La evaluación de estos virus en lotes de semilla sexual comercial y no comercial y en sus plantas derivadas evidenció la presencia de cinco virus en dicho material, con niveles de prevalencia del 13 al 93% e infecciones mixtas, que incluyeron combinaciones, desde dos a cinco virus.

ABSTRACT In Antioquia, the tomato crop (Solanum lycopersicum) is seriously affected by a wide range of viral diseases that affect yield and the quality of fruits. Despite of this, there are few studies aimed at identifying these viruses at the species level. With the advent of High-throughput sequencing (HTS) methods, it is now possible to achieve an efficient characterization of viruses infecting plant hosts. In this work, the presence of RNA viruses infecting tomato var. Chonto in eastern Antioquia was tested using HTS and Real-time RT-PCR (RT-qPCR) in leaf tissues and seeds. HTS revealed infection with Potato virus S (PVS), Potato virus Y (PVY), Potato yellow vein virus (PYVV), Potato virus X (PVX), Southern tomato virus (STV) and Bell pepper endornavirus (BPEV). Complete genome sequences were obtained for PYVV, STV and BPEV. RT-qPCR showed prevalence of 100%, 94.4%, 77.8%, 72.2% and 5.6% for PYVV, PVX, PVY, STV and PVS in leaf samples, respectively. These viruses were also found infecting commercial and informal seeds and in their seedlings with a prevalence between 13 and 93%. Mixed infections were found to combine a mixture of two to five viruses.

Analysis of microbial community characteristics of Panax notoginseng based on high-throughput sequencing technology / 中草药

Objective: The purpose of this study was to obtain the bacterial communities structures of fresh and dry Panax notoginseng. Methods: The bacterial were sequenced by Illumina MiSeq high-throughput sequencing technology, and the biological information was analyzed. Results: A total of 637 OTUs were obtained, of which 15 were in common, 484 were from fresh P. notoginseng and 123 were from dry P. notoginseng respectively. The larger phylum of fresh sample was Proteobacteria (59.7%) and Firmicutes (40.1%) while the dry sample were Firmicutes (99.8%). The main genus of fresh sample was Enterobacter (37.4%) and Alkaliphilus (11.5%), while dry sample were Bacillus (54.6%) and Paenibaciiius (44.9%). The functional prediction of KEGG showed that the population of bacteria metabolizing terpenoids and polyketones of dry sample was higher than that of fresh sample. Conclusion: The diversity of microbiota in fresh P. notoginseng was higher than that of dry sample; The species with the metabolic function of interpenoids and polyketones of dry P. notoginseng was higher than that of fresh sample, which provides an important reference for screening the microorganism for biotransformation of saponins in P. notoginseng.

Anti-breast cancer mechanism of transcriptome analysis of ebracteolatain A / 中草药

Objective: To research the mechanism of ebracteolatain A against breast cancer cells, screening the genes with significant changes using second-generation sequencing, and explore the anti-breast cancer mechanism of action of ebracteolatain A at the transcriptomics level. Methods: The acetyl phloroglucinol compound ebracteolatain A was extracted from Euphorbia ebracteolata, interferencing with MCF7 cells (luminal A type of breast cancer cells) to observe differential gene expression between the interfered cells and normal cells. High-throughput transcriptome sequencing and data analysis were performed on three groups of control groups and three experimental groups using Illumina Hi-Seq sequencing technology. Results: A total of 123 656 848, 123 974 262 available reads were obtained in the control group and experimental group, respectively, the reads on the reference genome were 119 762 214, 119 881 622, respectively, accounting for 96.85% and 96.69% of the total; Two groups of transcriptome controls were available: the total number of differential genes was 1 695, of which 770 were up-regulated, 925 were down-regulated, and 3 874 genes were clearly annotated. Bio-enrichment analysis was carried out by using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). GO analysis found that these 3 874 genes mainly involved in biological processes (1 270), cell composition (1 322) and molecular function (1 282), 45 subcategories of three major categories, including cell growth and development, signaling protein activity, membrane and regulation of gene expression. KEGG analysis revealed that the differentially expressed genes involved 263 signaling pathways; The main metabolic pathways were: PI3K-Akt signaling pathway, MAPK signaling pathway, carbohydrate metabolism, myocardial system and cellular reproductive system and etc. Conclusion: The results showed that 1 695 differential genes were screened and identified by Illumina Hi-Seq sequencing technology, and the relationship between the genes of ebracteolatain A and MCF7 cells was further understood, which provided some theoretical cornerstones for breast cancer treatment.

Complete mitogenome sequencing of Andaman buffalo: an endangered germplasm of Andaman and Nicobar Islands, India

Andaman buffalo is an indigenous buffalo of Andaman and Nicobar Islands, India. Over the last decade, it has witnessed a rapid decline in population, necessitating its immediate characterization and conservation. The present study reports the complete mitogenome profile of Andaman buffalo which is 16,359 bp in length and comprised of 37 genes, including 13 protein-coding genes (PCGs), 22 transfer RNAs and two ribosomal RNAs. In addition, one A + T rich region (D-loop) was also present. A biasness towards A and T base was observed in all the genes. All the PCGs except ND6 were present on heavy strand. Start codons for all the 13 PCGs were ATN codon and abbreviated/truncated stop codons were observed in ND1, ND2, COX3, ND3 and ND4. The phylogenetic analysis revealed that the Andaman buffalo is closely related to buffalo from India and China. The results from this study will help in sketching the conservation plan of the threatened breed.

Transcriptome sequencing and de novo analysis of the northern snakehead, Ophiocephalus argus

Northern snakehead, Ophiocephalus argus Cantor, is an endemic freshwater fish in China. However, wild stocks of O. argus are dwindling sharply. Further, water conservancy projects, environmental pollution and human activities have caused the decrease of wild stocks, which has attracted much attention. Here, we have investigated the genomic information of O. argus using IlluminaHiseq 4000 sequencing. The transcriptomes of O. argus were sequenced by Illumina technology. A total of 67,564 sequences from 79,500,964 paired-end reads were generated, 33,710 unigenes were annotated based on protein databases (NCBI nonredundant (NR) databases). In total, 7182 unigenes had the clusters of orthologous group (COG) classifications, 33,710 unigenes were assigned to 59 gene ontology (GO) terms. Further, a total of 21,464 simple sequence repeats (SSRs) from 67,564 unigenes and 113,518 single nucleotide polymorphism (SNP) sites among 335 Mclean reads were yielded for O. argus based on a transcriptome-wide search. The new transcriptome data which is presented in this study for O. argus will provide valuable information for gene discovery and downstream applications, such as phylogenetic analysis, gene-expression profiling and identification of genetic markers (SSRs andSNP).

Complete and de novo assembly of the Leishmania braziliensis (M2904) genome

Leishmania braziliensis is the etiological agent of American mucosal leishmaniasis, one of the most severe clinical forms of leishmaniasis. Here, we report the assembly of the L. braziliensis (M2904) genome into 35 continuous chromosomes. Also, the annotation of 8395 genes is provided. The public availability of this information will contribute to a better knowledge of this pathogen and help in the search for vaccines and novel drug targets aimed to control the disease caused by this Leishmania species.

Key microbial populations involved in anaerobic degradation of phenol and p-cresol using different inocula

Background: Anaerobic digestion is an alternative bioprocess used to treat effluents containing toxic compounds such as phenol and p-cresol. Selection of an adequate sludge as inoculum containing an adapted microbial consortium is a relevant factor to improve the removal of these pollutants. The objective of this study is to identify the key microorganisms involved in the anaerobic digestion of phenol and p-cresol and elucidate the relevance of the bamA gene abundance (a marker gene for aromatic degraders) in the process, in order to establish new strategies for inocula selection and improve the system's performance. Results: Successive batch anaerobic digestion of phenol and p-cresol was performed using granular or suspended sludge. Granular sludge in comparison to suspended sludge showed higher degradation rates both for phenol (11.3 ± 0.7 vs 8.1 ± 1.1 mg l-1 d-1) and p-cresol (7.8 ± 0.4 vs 3.7 ± 1.0 mg l-1 d-1). After three and four re-feedings of phenol and p-cresol, respectively, the microbial structure from both sludges was clearly different from the original sludges. Anaerobic digestion of phenol and p-cresol generated an abundance increase in Syntrophorhabdus genus and bamA gene, together with hydrogenotrophic and aceticlastic archaea. Analysis of results indicates that differences in methanogenic pathways and levels of Syntrophorhabdus and bamA gene in the inocula, could be the causes of dissimilar degradation rates between each sludge. Conclusions: Syntrophorhabdus and bamA gene play relevant roles in anaerobic degradation of phenolics. Estimation of these components could serve as a fast screening tool to find the most acclimatized sludge to efficiently degrade mono-aromatic compounds.

The genetic diversity of wild and cultivated Manila clam (Ruditapes philippinarum) revealed by 29 novel microsatellite markers

Background: Microsatellite loci often used as a genetic tool for estimating genetic diversity population variation in a wide variety of different species. The application of microsatellite markers in genetics and breeding includes investigating the genetic differentiation of wild and cultured populations, assessing and determining the genetic relationship of different populations. The aim of this work is to develop several microsatellite markers via highthroughput sequencing and characterize these markers in commercially important bivalve Ruditapes philippinarum. Results: Among the two populations of R. philippinarum studied, 110 alleles were detected. The number of alleles at the cultured population ranged from 3 to 17 (mean NA = 6.897) and wild population ranged from 2 to 15 (mean NA = 6.793). The observed and expected heterozygosities of cultured population ranged from 0.182 to 0.964, and from 0.286 to 0.900, with an average of 0.647 and 0.692, respectively. The observed and expected heterozygosities of wild population ranged from 0.138 to 1.000, and from 0.439 to 0.906, with an average of 0.674 and 0.693, respectively. The polymorphism information content ranged from 0.341 to 0.910 with an average of 0.687. Sixteen and thirteen microsatellite loci deviated significantly from Hardy­Weinberg equilibrium after correction for multiple tests in cultured and wild population, respectively. Conclusions: Twenty-nine novel microsatellite loci were developed using Illumina paired-end shotgun sequencing and characterized in two population of R. philippinarum.

Development of Polymorphic Simple Sequence Repeat Markers using High-Throughput Sequencing in Button Mushroom (Agaricus bisporus)

The white button mushroom (Agaricus bisporus) is one of the most widely cultivated species of edible mushroom. Despite its economic importance, relatively little is known about the genetic diversity of this species. Illumina paired-end sequencing produced 43,871,558 clean reads and 69,174 contigs were generated from five offspring. These contigs were subsequently assembled into 57,594 unigenes. The unigenes were annotated with reference genome in which 6,559 unigenes were associated with clusters, indicating orthologous genes. Gene ontology classification assigned many unigenes. Based on genome data of the five offspring, 44 polymorphic simple sequence repeat (SSR) markers were developed. The major allele frequency ranged from 0.42 to 0.92. The number of genotypes and the number of alleles ranged from 1 to 4, and from 2 to 4, respectively. The observed heterozygosity and the expected heterozygosity ranged from 0.00 to 1.00, and from 0.15 to 0.64, respectively. The polymorphic information content value ranged from 0.14 to 0.57. The genetic distances and UPGMA clustering discriminated offspring strains. The SSR markers developed in this study can be applied in polymorphism analyses of button mushroom and for cultivar discrimination.

Deep sequencing analysis of toad Rhinella schneideri skin glands and partial biochemical characterization of its cutaneous secretion

Animal poisons and venoms are sources of biomolecules naturally selected. Rhinella schneideri toads are widespread in the whole Brazilian territory and they have poison glands and mucous gland. Recently, protein from toads' secretion has gaining attention. Frog skin is widely known to present great number of host defense peptides and we hypothesize toads present them as well. In this study, we used a RNA-seq analysis from R. schneideri skin and biochemical tests with the gland secretion to unravel its protein molecules. Methods: Total RNA from the toad skin was extracted using TRizol reagent, sequenced in duplicate using Illumina Hiseq2500 in paired end analysis. The raw reads were trimmed and de novo assembled using Trinity. The resulting sequences were submitted to functional annotation against non-redundant NCBI database and Database of Anuran Defense Peptide. Furthermore, we performed caseinolytic activity test to assess the presence of serine and metalloproteases in skin secretion and it was fractionated by fast liquid protein chromatography using a reverse-phase column. The fractions were partially sequenced by Edman's degradation. Results: We were able to identify several classes of antimicrobial peptides, such as buforins, peroniins and brevinins, as well as PLA2, lectins and galectins, combining protein sequencing and RNA-seq analysis for the first time. In addition, we could isolate a PLA2 from the skin secretion and infer the presence of serine proteases in cutaneous secretion. Conclusions: We identified novel toxins and proteins from R. schneideri mucous glands. Besides, this is a pioneer study that presented the in depth characterization of protein molecules richness from this toad secretion. The results obtained herein showed evidence of novel AMP and enzymes that need to be further explored.(AU)

Benefits of procyanidins on gut microbiota in Bama minipigs and implications in replacing antibiotics

Several studies have reported the effect of absorption of procyanidins and their contribution to the small intestine. However, differences between dietary interventions of procyanidins and interventions via antibiotic feeding in pigs are rarely reported. Following 16S rRNA gene Illumina MiSeq sequencing, we observed that both procyanidin administration for 2 months (procyanidin-1 group) and continuous antibiotic feeding for 1 month followed by procyanidin for 1 month (procyanidin-2 group) increased the number of operational taxonomic units, as well as the Chao 1 and ACE indices, compared to those in pigs undergoing antibiotic administration for 2 months (antibiotic group). The genera Fibrobacter and Spirochaete were more abundant in the antibiotic group than in the procyanidin-1 and procyanidin-2 groups. Principal component analysis revealed clear separations among the three groups. Additionally, using the online Molecular Ecological Network Analyses pipeline, three co-occurrence networks were constructed; Lactobacillus was in a co-occurrence relationship with Trichococcus and Desulfovibrio and a co-exclusion relationship with Bacillus and Spharerochaeta. Furthermore, metabolic function analysis by phylogenetic investigation of communities by reconstruction of unobserved states demonstrated modulation of pathways involved in the metabolism of carbohydrates, amino acids, energy, and nucleotides. These data suggest that procyanidin influences the gut microbiota and the intestinal metabolic function to produce beneficial effects on metabolic homeostasis.

Analysis of microbial community of heat resistant microorganisms in Chinese herbal pieces / 中国中药杂志

To investigate the composition and diversity of heat resistant microorganisms in contaminated Chinese herbal pieces. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) protein fingerprinting and 16S rRNA high-throughput sequencing of Illumina Miseq were used to analyze the heat resistant microbial community of 9 varieties of Chinese herbal pieces. Stem pieces (Spatholobi Caulis, Tetrapanacis Medulla, Stachyuri Medulla) showed highest detection rate and most species of contaminants; However fruit pieces (Schisandrae Sphenantherae Fructus) had the lowest detection rate and least species of contaminants; among root pieces, the detection rate and number of contaminants species were lower in Tuber Dioscoreae Persimilis and Rehmanniae Radix Praeparata. The heat resistant microbial community was mainly of Bacillaceae and Paenibacillaceae, and Bacillus showed the highest detection rate among them, followed by Brevibacillus, Paenibacillus, and Solibacillus. The rest genus in high-throughput sequencing analysis included Enterobacter, Brevundimonas, Leuconostoc, Methylobacterium, Dechloromonas, Pantoea, Klebsiella, and Erwinia. There were potential risk factors in heat resistant microbial community of Chinese herbal pieces, so we shall improve the microbial limit standard, strictly control the pathogenic bacteria in the product, and strengthen the supervision and management in production and circulation of Chinese herbal pieces.

Diversity of endophytic bacteria in Astragali Radix analyzed by Illumina MiSeq high-throughput sequencing technology / 中草药

Objective This study aimed to analyze the diversity of endophytic bacterial communities, and to provide reference to the oriented processing technology, which helps to screen bacteria fermentation and conversion in the active ingredients of Astragali Radix. Methods The 16S rDNA V3-V4 region of Astragali Radix was sequenced by Illumina MiSeq high-throughput sequencing technology, and the abundance of species and other biological information were analyzed. Results The results showed that the numbers of effective sequences and OTUs for sample were 40 051 and 967, respectively. The number of sequencing was close to saturation, and the sequencing data volume was reasonable. The endophytic bacteria of Astragali Radix mainly belonged to Anderseniella, Bacillus, Burkholderia, Acinetobacter, Stenotrophomonas, and Oceanobacillus. Conclusion The diversity of endophytic bacteria was low in Astragali Radix. The dominant population of endophytic bacteria in Astragali Radix belongs to Anderseniella and Bacillus.

Transcriptome sequencing analysis of gene expression of flavonoid biosynthesis in Ginkgo biloba / 中草药

Objective The transcriptome sequencing was used to analyze the expression of key genes of flavonoid biosynthesis in different stages of growth and development of Ginkgo biloba. Methods The leaves of young trees and adult trees of G. biloba in different periods were used as the test material. Transcriptome sequencing analysis was carried out by using Illumina HiSeq 2000, and analyses of gene functional annotation of Unigene and expression characteristics of key genes for biosynthesis of G. biloba flavonoids were also performed. Results A total of 43 073 Unigene were obtained by transcriptome sequencing, of which 35 179 were annotated and 5 117 genes were screened by differential gene expression. Fifty candidate genes were screened by analyzing KEGG pathway related to flavonoid synthesis. The expression patterns of 50 candidate genes were analyzed. It was found that the key genes of flavonoid synthesis were all highly expressed in young leaves of G. biloba, but there was no significant difference in the leaves between adult and young trees at same time. The 13 genes closely related to the synthesis of flavonoids were analyzed. Among them, the expression of C4H, CHS, ANS, ANR, and FOMT genes was high, and the expression of F3’H, F3’5’H, and FLS genes was relatively low. Conclusion Through transcriptome sequencing, we screened and analyzed the key genes of flavonoid biosynthesis of G. biloba and their expression characteristics, which provided the theoretical basis of molecular pharmacology for improving the yield of ginkgo flavonoids.