Genetic diversity of the S-type small subunit ribosomal RNA gene of Plasmodium knowlesi isolates from Sabah,Malaysian Borneo and Peninsular Malaysia

Objective:To determine the genetic diversity of Plasmodium(P.)knowlesi isolates from Sabah,Malaysian Borneo and Peninsular Malaysia,targeting the S-type SSU rRNA gene and including aspects of natural selection and haplotype.Methods:Thirty-nine blood samples infected with P.knowlesi were collected in Sabah,Malaysian Borneo and Peninsular Malaysia.The S-type SSU rRNA gene was amplified using polymerase chain reaction,cloned into a vector,and sequenced.The natural selection and haplotype of the S-type SSU rRNA gene sequences were determined using DnaSP v6 and illustrated using NETWORK v10.This study's 39 S-type SSU rRNA sequences and eight sequences from the Genbank database were subjected to phylogenetic analysis using MEGA 11.Results:Overall,the phylogenetic analysis showed no evidence of a geographical cluster of P.knowlesi isolates from different areas in Malaysia based on the S-type SSU rRNA gene sequences.The S-type SSU rRNA gene sequences were relatively conserved and with a purifying effect.Haplotype sharing of the S-type SSU rRNA gene was observed between the P.knowlesi isolates in Sabah,Malaysian Borneo,but not between Sabah,Malaysian Borneo and Peninsular Malaysia.Conclusions:This study suggests that the S-type SSU rRNA gene of P.knowlesi isolates in Sabah,Malaysian Borneo,and Peninsular Malaysia has fewer polymorphic sites,representing the conservation of the gene.These features make the S-type SSU rRNA gene suitable for comparative studies,such as determining the evolutionary relationships and common ancestry among P.knowlesi species.

Correlation analysis of breast fibroadenoma and the intestinal flora based on 16S rRNA sequencing

Objective To analyze the characteristics of the intestinal microflora in patients with breast fibroadenoma using 16S ribosomal RNA(rRNA)high-throughput sequencing.Methods Fecal samples from 20 patients with breast fibroadenoma and 36 healthy subjects were randomly collected and analyzed using high-throughput sequencing technology for 16S rRNA V4 region sequencing,and the alpha diversity(Chao index,Shannon index)was calculated using Mothur(v.1.39.5)software.Beta diversity was analyzed using QIIME(v1.80).SPSS software(version 23.0)and the t-test of two independent samples were used to analyze differences in the abundance of bacteria between the two groups.Results Compared with that in the healthy control group,theαdiversity of the intestinal microflora in breast fibroadenoma patients increased,but the difference was not statistically significant(P>0.05).At the phylum level,significant differences were observed between the two groups.The abundance of Firmicutes was higher in the breast fibroadenoma group(P<0.05),whereas the abundance of Synergistetes was higher in the healthy control group(P<0.005).A total of five bacterial genera showed significant differences between the two groups:the breast fibroadenoma group showed higher levels of Bautia(P<0.005),Coprococcus(P<0.005),Roseburia(P<0.05),and Ruminococcus(P<0.005),whereas Sutterella was more abundant in the healthy control group than in the breast fibroadenoma group(P<0.05).Conclusion The diversity and abundance of the intestinal flora in patients with breast fibroadenoma are significantly different from those in healthy subjects,suggesting that an imbalance in the intestinal flora is correlated with the occurrence of breast fibroadenoma.

Mass spectrometry profiling analysis enables the identification of new modifications in ribosomal RNA

Ribosomal RNAs(rRNAs) provide the structural framework of ribosomes and play critical roles in protein translation.In ribosome biogenesis,rRNAs acquire various modifications that can influence the structure and catalytic activity of ribosomes.However,rRNA modifications in plants have yet to be fully defined.Herein,we proposed a method to purify rRNAs by a successive isolation with different strategies,including poly A-based m RNA depletion and agarose gel electrophoresis-based purification,with which highly pure rRNAs could be obtained.In addition,we developed a liquid chromatography-electrospray ionization-tandem mass spectrometry(LC-ESI-MS/MS) method to systematically profile and characterize modifications from the isolated highly pure plant 18S rRNA and 25S rRNA.LC-ESI-MS/MS analysis showed that 10 and 12 kinds of modifications were present in plant 18S rRNA and 25S rRNA,respectively.Notably,among these identified modifications,2 kinds of modifications of N^(2),N^(2)-dimethylguanosine(m^(2,2)G)and N^(6),N^(6)-dimethyladenosine(m^(6,6)A) in 18S rRNA,and 4 kinds of modifications of m^(2,2)G,m^(6,6)A,N7-methylguanosine(m^(7)G) and 3-methyluridin(m^(3)U) in 25S rRNA,were first reported to be present in plants.Moreover,exposure of Arabidopsis thaliana to cadmium(Cd) led to significant changes of modifications in both 18S rRNA and 25S rRNA of plants,indicating that rRNA modifications play important roles in response to environmental stress.The discovery of new modifications in plant rRNAs improves the spectra of plant rRNA modifications and may promote the investigation of the functional roles of plant ribosomes in regulating gene expression.

Application of PCR-DGGE in Research of Bacterial Diversity in Drinking Water

Objective To analyze the structure of bacteria in drinking water by molecular biological techniques, Methods DNA of bacteria in drinking water was directly extracted without culture. 16S ribosomal DNA fragments, including V-6, -7, and -8 regions, were amplified with universal primers （EUBf933CJC and EUBr1387） and analyzed by DGGE. Results DGGE indicated that amplification products could be separated, The results showed that DGGE could be used in the separation of different microbial 16SrRNA genes extracted from drinkng water. Though there were special bacteria in different water samples, the predominant bacteria were essentially the same. Three sequences of the reclaimed specific bands were obtained, and phylogenetic tree of these bands was made. Conclusion Bacterial diversity in drinking water is identified by molecular biological techniques.

Detection of Prorocentrum donghaiense using sandwich hybridization integrated with nuclease protection assay

Prorocentrum donghaiense is an important harmful algae bloom （HAB） causing creature in China＇s seas, and the conventional visual detection can not cope with long-term monitoring and highthroughput sampling projects. An assay for P. donghaiense with sandwich hybridization integrated with nuclease protection assay （NPA-SH） was established. Tests with mixed samples and spiked field ones confirmed its good specificity and sensitivity. The cell number of P. donghaiense correlated well with the optical density, and the regression equation is y=4× 10^- 6x＋ 0.694 9, in which x is the cell number, and y is the optical density, with r2=0.953 5. These results show that the NPA-SH method has good feasibility in the detection of P. donghaiense. Results of NPA-SH and microscopy are excellent for each sample. The NPA-SH method was a simple way in quantitative detection of P. donghaiense, and the whole process could be finished in about six hours, which provided a new approach in high-throughput sampling and long-term monitoring of P. donghaiense.

Microbial community changes in the digestive tract of the clam Meretrix petechialis in response to Vibrio parahaemolyticus challenge

Disease in clams frequently occurred over the last decade and has become a serious threat to the clam aquaculture industry and natural stocks.Mass clam mortality events were reported to be associated with the presence of opportunistic pathogen vibrio.However,the complexity of infection that occurs in the natural environment remains poorly understood.In this study,we smulated a natural disease outbreak by vibrio immersion infection to study the diversity and dynamics of microbiota in the digestive tract of clam Meretrix petechialis during the infection process.Dramatic changes in operational taxonomic unit richness and phylum composition of the bacterial communities were observed during pathogen invasion.In addition,we investigated the potential relationship between microbiota dynamics and host status during disease progression.Results reveal that,at the end stage of vibrio infection,interindividual variation in the digestive tract microbiota increased,as did the diff erence in individual health status.The moribund clams displayed signs of microbial community shifts to low diversity,and the microbial community was characterized by mass proliferation of a few operational taxonomic units.

Molecular Identification of a Species in Genus Nannochloropsis

Nannochloropsis is a genus of marine eukaryotic unicellular algae,which belongs to class Eustigmatophyceae.The spe-cies of Nannochloropsis which are fine rotifer feed and rich in eicosapentaenoic acid(EPA)are economically important.Species in this genus are usually 2-5μm in size and are morphologically similar,which makes their identification difficult.We obtained a monoclone of Nannochloropsis with plating method in this study.DNA was extracted and the quality was determined by restriction enzyme digestion and spectrophotometer analysis.The DNA extracted was used to amplify the sequences of 18S ribosomal RNA gene,ITS region of ribosomal RNA transcription unit and rbcL gene.The phylogenetic analysis was carried out by constructing the neighbor-joining trees with Tamura-Nei distances.The phylogenetic analysis showed that the monoclone is N.oceanica.

Essential role of NbNOG1 in ribosomal RNA processing^FA

Summary Nucleolar GTP-binding protein 1 （NOG1） is a highly conserved GTPase first reported in Trypanosoma as required for ribosome biogenesis. We characterized NbNOG1, a Nicotiana benthamiana NOG1 ortholog sharing more than 4570 amino acid identity with Trypanosoma, yeast, and human NOG1. N. benthamiana plants silenced for NbNO0 were stunted and produced sterile flowers.

A single degenerated primer significantly improves COX1 barcoding performance in soil nematode community profiling

A new COX1 primer for soil nematode metabarcoding was designed,and this primer outperforms other commonly used COX1 primer pairs in species recovery and quantity of PCR products.•The lack of reference database is the main reason that led to the low species recovery in COX1 metabarcoding.•We expanded current NCBI database by adding 51 newly generated COX1 reference sequences.Microscopic nematodes play important roles in soil ecosystems and often serve as bioindicators of soil health.The identification of soil nematodes is often difficult due to their limited diagnostic characters and high phenotypic plasticity.DNA barcoding and metabarcoding techniques are promising but lack universal primers,especially for mitochondrial COX1 gene.In this study a degenerated COX1 forward primer COIFGED was developed.The primer pair(COIFGED/JB5GED)outperforms other four commonly used COX1 primer pairs in species recovery and quantity of polymerase chain reaction(PCR)products.In metabarcoding analysis,the reads obtained from the new primer pair had the highest sequencing saturation threshold and amplicon sequence variant(ASV)diversity in comparison to other COX1 as well as 18S rRNA primers.The annotation of ASVs suggested the new primer pair initially recovered 9 and 6 out of 25 genera from mock communities,respectively,outperformed other COX1 primers,but underperformed the widely used 18S NF1/18Sr2b primers(16 out of 25 genera).By supplementing the COX1 database with our reference sequences,we recovered an additional 6 mock community species bringing the tally closer to that obtained with 18S primers.In summary,our newly designed COX1 primers significantly improved species recovery and thus can be supplementary or alternative to the conventional 18S metabarcoding.

IRESfinder:Identifying RNA internal ribosome entry site in eukaryotic cell using framed k-mer features

In eukaryotic cells, initiation of protein translation is to recruit the ribosome to a specific mRNA, which is generally dependent on the 5＇ cap structure. However, protein translation can also be initiated in a cap-independent manner by using a cis-regulatory element termed the internal ribosome entry site （IRES）. The first experimentally validated IRES was reported in the poliovirus （Pelletier and Sonenberg, 1988）. Then eukaryotic cellular mRNAs were also validated to contain IRES elements.

Changes in the gut microbiota of osteoporosis patients based on 16S rRNA gene sequencing:a systematic review and meta-analysis

Background:Osteoporosis(OP)has become a major public health issue,threatening the bone health of middle-aged and elderly people from all around the world.Changes in the gut microbiota(GM)are correlated with the maintenance of bone mass and bone quality.However,research results in this field remain highly controversial,and no systematic review or meta-analysis of the relationship between GM and OP has been conducted.This paper addresses this shortcoming,focusing on the difference in the GM abundance between OP patients and healthy controls based on previous 16S ribosomal RNA(rRNA)gene sequencing results,in order to provide new clinical reference information for future customized prevention and treatment options of OP.Methods:According to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses(PRISMA),we comprehensively searched the databases of Pub Med,Web of Science,Embase,Cochrane Library,and China National Knowledge Infrastructure(CNKI).In addition,we applied the R programming language version 4.0.3 and Stata 15.1 software for data analysis.We also implemented the Newcastle-Ottawa Scale(NOS),funnel plot analysis,sensitivity analysis,Egger’s test,and Begg’s test to assess the risk of bias.Results:This research ultimately considered 12 studies,which included the fecal GM data of 2033 people(604 with OP and 1429 healthy controls).In the included research papers,it was observed that the relative abundance of Lactobacillus and Ruminococcus increased in the OP group,while the relative abundance for Bacteroides of Bacteroidetes increased(except for Ireland).Meanwhile,Firmicutes,Blautia,Alistipes,Megamonas,and Anaerostipes showed reduced relative abundance in Chinese studies.In the linear discriminant analysis Effect Size(LEfSe)analysis,certain bacteria showed statistically significant results consistently across different studies.Conclusions:This observational meta-analysis revealed that changes in the GM were correlated with OP,and variations in some advantageous GM might involve regional differences.

Taxonomy and phylogeny of the section Chaetoceros(Chaetocerotaceae,Bacillariophyta),with description of two new species

Chaetoceros Ehrenberg is one of the most diverse genera of planktonic diatoms.The species in section Chaetoceros are characterized by cells and setae having numerous chloroplasts and being widely distributed.However,the delimitations of some species are problematic because of limited morphological information in the classical descriptions.Monoclonal strains of the section Chaetoceros were established,morphological features were studied using light and electron microscopy,and the hypervariable D 1-D 3 region of the nuclear ribosomal large subunit gene was sequenced to address phylogenetic relationships.Fifteen species belonging to the section Chaetoceros were recorded,including two new species,C.hainanensis sp.nov.and C.tridiscus sp.nov.Chaetoceros hainanensis was characterized by straight chains,narrowly lanceolate to hexagonal apertures,sibling setae diverging in nearly right angles,stipule-shaped spines on terminal setae and arrowhead-shaped spines on intercalary setae.C.tridiscus had short straight chains,narrowly lanceolate apertures,arrowhead-shaped spines and circular poroids arranged in a grid pattern on terminal and intercalary setae.The phylogenetic analyses revealed six groups formed by 19 species within the section Chaetoceros,which was found to be monophyletic.The subdivision of the section is still not well understood.The morphological characters within each group varied considerably and molecular information on more species are needed to enrich the phylogenetic profiling.

New Edges of RNA Adenosine Methylation Modifications

Recently an article published in Molecular Cell reveals the mechanism of a nuclear N6-methyladenosine（m^6A）reader,the YTH domain-containing protein 1（YTHDC1）,in regulating pre-m RNA splicing[1].Meanwhile,two additional articles published in Nature and Nature Chemical Biology report the

Microbial community structure of different tongue fur types in patients with chronic gastritis

OBJECTIVE:To explore the correlation between tongue and oral microbiota,we studied the microbial community structure of different tongue coating types in patients with chronic gastritis.METHODS:16S rDNA gene sequencing and bioinformatics analysis were used to study the dynamic changes and correlation of microbial flora in patients with chronic gastritis,healthy people,and patients with different tongue fur.In addition,it was also discussed between the severity of gastritis and the microflora of tongue fur.RESULTS:The microbial diversity of tongue fur in patients with chronic gastritis was significantly different from healthy controls.There were significant changes in bacterial communities’diversity and relative abundance between extra tongue fur in patients but not in healthy people.Oral bacteria with relative abundance>1%and P<0.05 among different tongue fur flora were dominant bacteria,including 12 phyla such as Bacteroidetes,Proteobacteria and Firmicutes,and 256 genera such as Neisseria,Prevotella_7 and Haemophilus.CONCLUSIONS:The changes in oral flora in patients with chronic gastritis were related to tongue fur.Therefore,the significant microbiota might enlighten further study on the correlation between tongue inspection and oral microbiota in patients with chronic gastritis.

外来入侵植物刺萼龙葵(Solanum rostratum Dunal)根际土壤真菌多样性及其次生代谢产物的化感作用研究

【目的】分析外来入侵植物刺萼龙葵根际土壤真菌群落多样性,了解其根际可培养真菌次生代谢产物的植物生长调节活性,并从中筛选具有开发为植物生长调节剂及生物除草剂潜力的菌株。【方法】采集刺萼龙葵根际土壤,提取总DNA,利用高通量测序技术分析土壤真菌群落多样性;同时,利用纯培养手段获得可培养真菌菌株,以乙酸乙酯萃取其发酵产物,并以单子叶植物早熟禾和双子叶植物反枝苋为受试植物,检测其次生代谢产物的促生/抑生活性。【结果】子囊菌门真菌广泛存在于刺萼龙葵根际土壤中,且占绝对优势。Alpha多样性分析发现,刺萼龙葵根际土壤中真菌群落有着非常高的多样性和丰富性。此外,菌株SR02、SR05和SR13对受试植物具有较强的生长抑制作用。【结论】刺萼龙葵根际土壤真菌优势类群明显,尖孢镰刀菌和链格孢在真菌群落中占优势地位;从根际土壤中发现多个具有显著生长促进或抑制作用的菌株,其在刺萼龙葵根际土壤中所起到的生理生态学意义,以及其次生代谢产物是否具有开发为植物生长调节剂及生物农药的潜力,值得进行深入的研究。

Dietary supplementation with Clostridium butyricum improves growth performance of broilers by regulating intestinal microbiota and mucosal epithelial cells

Clostridium butyricum has been widely considered an antibiotic substitute in recent years.It can promote growth performance,improve the immune response and enhance the intestinal barrier function of the host.In the present study,1-d-old Arbor Acres(AA)broilers were fed C.butyricum(1×109 cfu/kg)for 28 d.The transcriptomic characteristics of epithelial cells of the cecal mucosa were determined by RNA-sequence,and the cecal microbiota composition was explored by 16 S ribosomal RNA gene sequencing.The changes in the intestinal mucosa of broilers were then analyzed by tissue staining.Gene Ontology(GO)annotations identified substance transport and processes and pathways that might participate in intestinal development and cell viability.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis revealed that the differentially expressed genes are involved in numerous pathways related to amino acid and vitamin metabolism and antioxidant and defensive functions,among others.The relative expression of some genes associated with intestinal barrier function(claudins 2,15,19,and 23,tight junction proteins 1,2,and 3 and mucin 1)was significantly increased in the treatment group(P<0.05 or P<0.01).Moreover,the proportion of Firmicutes was higher in the C.butyricum-treated group,whereas the proportion of Proteobacteria was higher in the control group.At the genus level,the relative abundances of Butyricicoccus and Lactobacillus,among other bacteria,were increased after C.butyricum supplementation.The tissue staining analysis showed that the cecal mucosa of broilers was significantly ameliorated after the addition of C.butyricum(P<0.05 or P<0.01).These results showed that dietary supplementation with C.butyricum can enhance the antioxidant capacity,mucosal barrier function,and stabilize the cecal microbiota,resulting in improving the growth performance.

Identification of Anamorph Yeast of Tremella aurantialba and Optimization of Medium Composition for Production of Exopolysaccharides

A yeast-like fungus strain B1 isolated from wild fungus Tremella aurantialba was identified and initially characterized. Two phylogenetic trees were generated based on the sequences of large subunit ribosomal RNA gene D1/D2 regions and internal transcribed spacer (ITS) regions of related fungi, respectively. The analysis of D1/D2 regions and ITS sequences showed that fungus B1 was clustered together with T. aurantialba, T. aurantia and T. microspore in the phylogenetic trees. Both the morphological characteristic and phylogenetic analysis established that fungus B1 was one of the anamorph strains of T. aurantialba and belongs to Tremella genus. A fermentation medium for exopolysaccharides (EPS) production by T. aurantialba B1 . Plackett-Burmen design was used to evaluate the effects of different components in the culture medium. Glucose and yeast extract have significant influence on the EPS production. The concentrations of two factors were optimized subsequently using central composite design and response surface analysis. The results showed that 49.2 g/L glucose and 10.4 g/L yeast extract could lead to the maximum production of EPS (4.99 g/L). The optimized medium led to a 1.5-fold enhancement of the production of EPS by T. aurantialba B1 , as compared with that without optimization.

A new contribution to the taxonomy and phylogeny of the ciliate genus Spirostomum(Alveolata,Ciliophora,Heterotrichea),with comprehensive descriptions of two species from wetlands in China

Species of the ciliate genus Spirostomum Ehrenberg,1834 are distributed worldwide and have a research history spanning more than two centuries.However,species delimitation and phylogenetic relationships within this genus are still uncertain due to the paucity of stable morphologic characters for species separation and the unavailability of accompanying morphological data for most molecular sequences in public databases.In the present study,S.yagiui Shigenaka,1959(three populations)and S.caudatum(Müller,1786)Delphy,1939(one population)were investigated using morphological and molecular methods for the first time in China.Detailed morphological data for the two species were documented,and improved diagnoses were supplied based on a combination of previous studies and the current work.It should be highlighted that there were three different atypical morphotypes identified in a Ningbo population of S.yagiui which may represent various stages in conjugative reproduction.Molecular phylogenies based on 18S,ITS1-5.8S-ITS2,and 28S rRNA gene sequences show that the genus Spirostomum is monophyletic,however,the internal relationships inferred from different genes were poorly resolved but suggest that the species with a moniliform macronucleus comprise an early-diverging clade within this genus.Finally,the global distribution of Spirostomum is summarized based on previous and present studies.