Impact of Different Rates of Nitrogen Supplementation on Soil PhysicochemicalProperties and Microbial Diversity in Goji Berry

Goji berry(Lycium barbarum L.)is substantially dependent on nitrogen fertilizer application,which can signifi-cantly enhance fruit yield and Goji berry industrial development in Ningxia,China.This study aimed to analyze the functions of differential nitrogen application rates including low(N1),medium(N2),and high(N3)levels in soil microbial community structure(bacterial and fungal)at 2 diverse soil depths(0-20,20-40 cm)through high-throughput sequencing technology by targeting 16S RNA gene and ITS1&ITS2 regions.All the observed physicochemical parameters exhibited significant improvement(p<0.05)with increased levels of nitrogen and the highest values for most parameters were observed at N2.However,pH decreased(p<0.05)gradually.The alpha and beta diversity analyses for bacterial and fungal communities’metagenome displayed more similarities than differences among all groups.The top bacterial and fungal phyla and genera suggested no obvious(p>0.05)differences among three group treatments(N1,N2,and N3).Furthermore,the functional enrichment analysis demonstrated significant(p<0.05)enrichment of quorum sensing,cysteine and methionine metabolism,and transcriptional machinery for bacterial communities,while various saprotrophic functional roles for fungal communities.Conclusively,moderately reducing the use of N-supplemented fertilizers is conducive to increasing soil nitrogen utilization rate,which can contribute to sustainable agriculture practices through improved soil quality,and microbial community structure and functions.

P16 METHYLATION OF THE COLORECTAL CANCER AND ASSOCIATION WITH DUKES STAGES

Objective: To explore whether methylation of the CpG island in the promoter of the p16 tumor suppressor gene was associated with clinicopathological characteristics of the colorectal cancer patients. Methods: Methylation-specific PCR (MSP) was used to detect p16 methylation of the colorectal cancer patients. Results: In 58 sporadic colorectal cancer, 43.1% of the tumors had detectable p16 methylation. Dukes’ stage was associated with p16 methylation status. Dukes C, D patients (75%) were more likely to contain methylated p16 compared with Dukes A, B patients (13.3%). Conclusion: p16 methylation plays a role in the carcinogenesis of a subset of colorectal cancer. P16 methylation might be considered as a prognostic indicator.

Polymorphism in the upstream regulatory region of human papilloma virus type 16 from the cervical cancer biopsies in Xinjiang Uygur women

To investigate the mutations in the upstream regulatory region (URR) of human papillomavirus type 16 (HPV-16) from the cervical cancer biopsies in Xinjiang Uygur women and its relationship to the high incidence of cervical cancer in the southern Xinjiang, the tissue DNA was extracted from the cervical cancer biopsies, and the URR segment of HPV-16 DNA was amplified, sequenced and analyzed. Thereafter, the polymorphism of URR in HPV-16 was then analyzed. It was demonstrated that the positive rate detected for the presence of URR in HPV-16 was 89.47% (17/19). Compared with the previously published sequence in URR of prototype HPV-16, some mutations were detected in the sequence of URR. The mutations in 17 URR fragments of HPV-16 could be divided into 11 patterns (XJU-1 to XJU-11) at nucleic acid level, in which each of XJU-1 and XJU-4 accounted for 23.53% (4/17), and other patterns of mutation accounted for 5.88% (1/17) . In comparison with the URR of prototype HPV-16, the DNA identity of these patterns was 98.50%-99.68% . In these 17 URR fragments, two point mutations occurred at position 7192 (G to T) and position 7520 (G to A) and they appeared to be constant in Xinjiang area. These two mutations were ubiquitous in the Asia-American type and conferred strong infection activity and carcinogenicity of this virus. In addition, the mutations at position 7729 (A to C), position 7843 (A to G) and position 7792 (C to T) could enhance its transcription activity considerably. It is concluded that some mutations occur in URR gene of HPV-16 in the cervical cancer biopsies taken from Uygur women in Xinjiang area, suggesting that certain relationship exists among the mutations in URR of HPV-16, the phylogeny of HPV-16 and the high incidence of cervical cancer in southern part of Xinjiang area.

Dynamic changes of microbial community diversity in a photohydrogen producing reactor monitored by PCR-DGGE

A PCR-DGGE (denaturing gradient gel electrophoresis of polymerase chain reaction) protocol was used for monitoring the dynamic changes in the microbial population during photohydrogen production. Total DNA was extracted directly from the mixed bacterial community in the reactor and subjected to PCR with V3-16S rDNA and pufM gene primers, and the amplifications were then analyzed by DGGE. The DGGE patterns demonstrated the dynamics of community structure and the shift of microbial diversity, which correspond...

Rapid Identification of Pathogenic Bacteria by means of TwoConservative Gene Loci′ Specific PCR-CE-RFLP

To establish a rapid identification method for common pathogenic bacteria on the basis of molecular biology and to construct a preliminary Polymerase Chain Reaction-Capillary Electrophoresis - Restriction Fragment Length Polymorphism (PCR-CE-RFLP) database of bacteria isolated from clinical specimens frequently, 183 strains collected from clinical samples belonging to 12 genera and 19 species whose biochemical characterizations corresponded to the typical ones were examined. The genomic DNAs were amplified by two pairs of fluorescence labeled primers aiming at 16S rRNA gene and 16S-23S rRNA spacer region gene respectively at the same time. PCR products were then digested by restriction endonuclease HaeⅢ incompletely before taking capillary electrophoresis. The results with the PCR-CE-RFLP patterns of 16S rRNA genes were just alike within some genera, but when it comes to 16S-23S rRNA spacer region genes, each bacterium showed a unique pattern, which can be distinguished from each other easily. It seems that PCR-CE-RFLP patterns of 16S rRNA gene could only be used to classify the bacteria into family level, whereas the data of 16S-23S rRNA spacer region gene could be utilized to identify the whole microorganisms as precisely as the species level. In spite of the data of the spacer region gene alone can be sufficiently to verify the whole bacteria, we insist that the 16S rRNA gene could be of some assistant in case that there should be lots of families of bacteria, in which some similar ones, with the same RFLP data of 16S-23S rRNA spacer region gene, may coexist. This study proves that the utility of PCR-CE-RFLP is a convenient, rapid method to identify pathogenic bacteria, and is also a quick diagnosis measure for application to clinical use.

Identification of Streptococcus species and Haemophilus influenzae by direct sequencing of PCR products from 16S-23SrDNA intergenic spacer regions

Objective To set up a rapid and simple method for identificating bacteria by 16S 23SrDNA intergenic spacer regions (ISRs) Methods Polymorphic products of PCR from ISRs were selected on agarose gel and sequenced directly using purified fragments by excising the gel without cloning Nucleotide sequences were compared with GenBank databases and analyzed by DNAMAN program Results There was only a single product in streptococcus genus after PCR amplification of 16S 23SrDNA ISRs Five streptococcal species were obtained from 7 strains of streptococcus Two major amplicons were consistently generated for 8 strains of Haemophilus influenzae (H influenzae) The sequence data showed that they all belonged to H influenzae type b on GenBank databases Conclusion PCR and direct sequencing of 16S 23SrDNA ISRs were very successful methods for bacterial species identification

High-throughput sequencing identifies salivary microbiota in Chinese caries-free preschool children with primary dentition

Objectives:The study aimed at identifying salivary microbiota in caries-free Chinese preschool children using highthroughput sequencing.Methods:Saliva samples were obtained from 35 caries-free preschool children(18 boys and 17 girls)with primary dentition,and 16 S ribosomal DNA(r DNA)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)and13 genera(Streptococcus,Rothia,Granulicatella,Prevotella,Enterobacter,Veillonella,Neisseria,Staphylococcus,Janthinobacterium,Pseudomonas,Brevundimonas,Devosia,and Gemella)were the most dominant,constituting 99.4%and 89.9%of the salivary microbiota,respectively.The core salivary microbiome comprised nine genera(Actinomyces,Capnocytophaga,Gemella,Granulicatella,Lachnoanaerobaculum,Neisseria,Porphyromonas,Rothia,and Streptococcus).Analysis of microbial diversity and community structure revealed a similar pattern between male and female subjects.The difference in microbial community composition between them was mainly attributed to Neisseria(P=0.023).Furthermore,functional prediction revealed that the most abundant genes were related to amino acid transport and metabolism.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 cariesfree populations and provides basic information for associating salivary microecology and oral health.