Difference Analysis Based on 16S rRNA Sequencing of Different Soil Bacterial Communities.

ABSTRACT: Objective To study the structure and differences of bacterial communities in different soils, and to explore the effectiveness of 16S rRNA sequencing in identification of different soil. Methods Soil samples from 7 places in Shanghai were collected, then bacterial genomic DNA were extracted from them. The fragments of hypervariable region from 16S rRNA sequences were sequenced with high-throughput sequencing techniques. The results were quantified or visualized with bioinformatics software. The differences in diversity and abundance among the three kinds of bacterial communities in soil samples from grassland, forests and beaches were compared and analyzed. Results The statistical differences that existed among the alpha diversity indexes of bacterial communities in soil samples of grassland, forests and beaches had statistical significance. The relative abundance and diversity of bacterial communities in these three kinds of soil were significantly different. Grassland soil had higher Acidobacteria abundance, forest soil had higher Proteobacteria abundance, beach soil had higher Actinobacteria abundance. However, the differences in soil bacterial communities in artificial grasslands, natural grasslands and industrial district grasslands did not have statistical significance. Conclusion 16S rRNA sequencing can effectively distinguish different soils. This method may be able to provide clues for first crime scene inference in criminal cases.

[The effect of downregulation of MCT1 on the proliferation of glioma cells].

Objective: To investigate the molecular mechanism of down-regulation of monocarboxylic acid transporter 1 (MCT1) on the proliferation inhibition of glioma cell. Methods: siMCT1, siMCT4 and negative control siRNA were transfected into glioma cell lines including U-251 and U-87. The proliferation activities of U-251 and U-87 cells were detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay and clonogenic assay. Glucose consumption and lactic acid efflux of U-251 and U-87 cells were determined by spectrophotometry.Western blot was used to detect the expressions of MCT1, MCT4, human glucose transporter 1 (GLUT1), GLUT4, tuberous sclerosis associated protein (TSC2), p-TSC2, 4E binding protein 1 (4EBP1), p-4EBP1, ribosomal S6 protein kinase (S6) and p-S6 protein in U-251 and U-87 cells. Results: Compared with negative control group, siMCT1 and siMCT4 significantly inhibited the expressions of MCT1 and MCT4 protein in U-251 and U-87 cells (both P<0.05). However, only knockdown of MCT1, the proliferation activities of U-251 and U-87 cells significantly decreased (P<0.05). The clone formation rates of U-251 and U-87 cells decreased to (55.20±3.27)% and (68.33±4.58) %, respectively (P<0.05). The glucose consumption of U-251 and U-87 cells in the negative control group at 72 hours were (82.65±6.66) pmol/L and (63.33±5.27) pmol/L, respectively, significantly higher than (31.70±3.17) pmol/L and (26.41±3.19) pmol/L of the siMCT1 transfected group (P<0.05). The extracellular lactate flow of U-251 and U-87 cells in negative control group at 72 h were (155.49±8.15) mmol/L and (135.37±8.21) mmol/L, respectively, significantly higher than (42.69±4.66) mmol/L and (38.91±4.83) mmol/L of the siMCT1 transfected group (P<0.05). Western blot analysis showed that knockdown of MCT1 significantly decreased the protein levels of GLUT1 p-TSC2, p-4EBP1 and p-S6 in U-251 and U-87 cells. Conclusions: Downregulation of MCT1 expression can inhibit the proliferation of glioma cells. Deletion of MCT1 inhibits the glycolysis and metabolism of glioma cells through regulating the mTOR signaling pathway.

MiR-32-5p regulates the proliferation and metastasis of cervical cancer cells by targeting HOXB8.

OBJECTIVE: To investigate the effects of miR-32-5p on the biological behaviors of cervical cancer (CCa), the relevant mechanism was studied in CCa cell lines (HeLa) in vitro. PATIENTS AND METHODS: The expression level of miR-32-5p was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). TargetScan, miRDB, microRNA databases and Luciferase method were conducted to predict and validate the target gene of miR-32-5p; the effects of miR-32-5p on cell proliferation, clone formation, invasion and migration capacity were analyzed in vitro study. RESULTS: We found miR-32-5p to be significantly inhibited in CCa tissues and cells. Bioinformatics approach together with Luciferase method screened Homeobox B8 (HOXB8) as a downstream regulatory target of miR-32-5p. Besides, HOXB8 was incredibly high expression in CCa tissues and cells. After transfection in HeLa cells by miR-32-5p mimics, HOXB8 expression was indicated to be negatively correlated with miR-32-5p both in qRT-PCR and Western blot (WB) assays. The subsequent experiments showed that decreased expression of HOXB8 resulting from up-regulation of miR-32-5p could weaken the cell proliferation, clone formation, invasion and migration ability of HeLa cells. CONCLUSIONS: MiR-32-5p could inhibit the cellular malignant behavior through regulating the expression of HOXB8 in HeLa cells. We provide a new clue for the study of molecular mechanisms of CCa. MiR-32-5p/HOXB8 axis might serve as potential target for the clinical diagnosis and treatment of CCa.

Progress in the 16S rRNA Gene Sequencing in Forensic Science.

Forensic microorganism is one of the hotspots of forensic science research. Due to its conservatism and specificity, the 16S rRNA gene is found to be an ideal marker for forensic identification. With the rapid development of high throughput sequencing technology, the research on microorganisms has been gradually applied to many fields such as environment and health care. In the field of forensic science, the results of forensic microbiology research, represented by 16S rRNA gene sequencing, are also gradually applied to forensic practice, such as biological samples identification, individual identification, postmortem interval estimation, and regional inference, which not only provide clues for the investigation of cases but also complement and assist traditional methods. This paper describes the research methods and related sequencing technologies of 16S rRNA gene sequencing, summarizes its research progress, and discusses the application value and potential of 16S rRNA in forensic science.

[Establishment of DNA Genetic Marker Identification System for Plant Evidence].

OBJECTIVES: To establish a species identification system based on DNA genetic markers for plant evidence. METHODS: Two hundred common plants in Shanghai were collected and identified by morphological characteristics. The primers of gene segments rbcL, matK, and ITS were designed and amplified. The PCR amplicon was detected by agarose gel electrophoresis. After the sequencing, the universality and the identification capacity of the three markers were evaluated. RESULTS: The success rate of amplification was in order of rbcL （99.5%） > matK （92.5%） > ITS （86.0%）. The identification capacity of the combination of rbcL and matK was better than that of rbcL or matK, by which most plant species could be identified to the genus or higher. ITS was not suitable to be a unique marker because of its unstable result, but it still could be a powerful supplement. The identification capacity of the combination of rbcL, matK and ITS was higher than that of rbcL and matK, by which most plant species could be identified to the genus or lower. CONCLUSIONS: The identification system with the combination of rbcL, matK and ITS as markers has excellent universality for plant evidence, which can distinguish most plant species to the genus or lower.

[Efficiency Analysis of EX16+10Y Kit on Detection of the Uygur Population in Xinjiang Province].

OBJECTIVES: To analyse the efficiency of EX16+10Y kit on the forensic detection of the Uygur in Xinjiang province. METHODS: The blood samples were extracted from 4 620 male individuals of Uygur in Xinjiang province, and amplified by EX16+10Y kit. The typing of amplification products was performed by 3130xl genetic analyzer. RESULTS: The genotyping graphs of 15 autosomal STR loci and 10 Y-chromosomal STR loci from 4 620 male individuals of Uygur in Xinjiang province were acquired completely. The genotype distribution of 15 autosomal STR loci was consistent with Hardy-Weinberg equilibrium. The heterozygosity, polymorphism information content and discrimination power of STR loci were 0.637-0.838, 0.580-0.860 and 0.811-0.978, respectively. There were 766 haplotypes in 10 Y -chromosomal STR loci. CONCLUSIONS: The test results of EX16+10Y kit is accurate and trustworthy, which can simultaneously be used for the individual identification and the screening of paternal pedigree in practical work.

[Analysis of related factors of recurrent otitis media with effusion in children].

Objective:To investigate the influencing factors of recurrent episodes of otitis media with effusion in children.Method:A retrospective summary of the clinical data of 210 cases of children with otitis media with effusion, 75 cases of recurrence after treatment, 135 cases were recovered, the recurrence of the related factors and after symptomatic treatment effect is analyzed.Result:Logistic regression analysis results found that adenoid hypertrophy (Ⅲ°, Ⅳ°), tonsil hypertrophy (Ⅳ°) and sinusitis (including choanal polyp), a positive allergens, upper respiratory tract infection, the stomach esophagus regurgitation, cleft palate, younger age has significant effect on recurrence of otitis media with effusion, have significant difference (P< 0.05). And the influence of duration, gender, passive smoking history and previous medical history of otitis media with effusion has no obvious statistical significance (P> 0.05). Through the comparison among different age groups, adenoidectomy Ⅲ °, Ⅳ ° hypertrophy tract infections in > 3-6 years old group has significant effect (P< 0.05), recurrent respiratory tract infections in less than 3 years old group and the group of children aged > 3-6 years OME recurrence has significant effect (P< 0.05). By tympanocentesis or tympanostomy tube insertion and according to different conditions to take symptomatic treatment, 75 cases (123 ears) were cured 96 ears (78.05%), 19 ears were improved (15.45%), the total effective rate was 93.50%, ineffective in 8 ears (6.50%).Conclusion:Adenoid hypertrophy (Ⅲ°, Ⅳ°), tonsil hypertrophy (Ⅳ°), sinusitis, nasal polyps, allergic diseases and upper respiratory tract infection gastroesophageal reflux, cleft palate and younger age may be adverse factors related to recurrent otitis media with effusion in children, the clinical doctors should pay attention to these symptoms, according to different causes, adopt individualized treatment plan, make children get the best treatment as soon as possible.

The influence of emission control on particle number size distribution and new particle formation during China's V-Day parade in 2015.

Temporary strict emission control strategies were conducted to ensure good air quality for China's V-Day parade (August 20-September 3, 2015) in Beijing and nearby cities. The influence of the emission control on particle number size distribution (PNSD) was evaluated based on the long-term measurements of PNSD at a rural site (Shangdianzi) located northeast of Beijing. This study also presented the comparison results of PNSD during the parade in 2015 and the Olympics in 2008 (August 8-23), as well as the same period without strict emission control in 2010-2013 (August 20-September 3). Compared with the same period in 2010-2013 and 2008 Olympics, the accumulation mode particle number concentration showed a significant reduction in 2015, and the PM1 mass concentration decreased by approximately 60-90%. The alleviation of the PM1 was also associated with the weather conditions. The back trajectories analysis results showed that the southerly air mass passing through the polluted areas accounted for 14% of the total back trajectories in 2015, which contributed to approximately 60% in the other years. During the control period in 2015, there were six new particle formation (NPF) events observed, with a higher frequency, but a lower formation rate and growth rate than the same period in 2010-2013. The comparison of the condensation sink (CS), sulfuric acid, solar radiation and relative humidity among the different years indicated that at Shangdianzi station, the first factor in determining the NPF occurrence was the CS, and the second factor could be the concentration level of precursor vapors participating in the NPF event (e.g., sulfuric acid).

Source Identification of Human Biological Materials and Its Prospect in Forensic Science.

Source identification of human biological materials in crime scene plays an important role in reconstructing the crime process. Searching specific genetic markers to identify the source of different human biological materials is the emphasis and difficulty of the research work of legal medical experts in recent years. This paper reviews the genetic markers which are used for identifying the source of human biological materials and studied widely, such as DNA methylation, mRNA, microRNA, microflora and protein, etc. By comparing the principles and methods of source identification of human biological materials using different kinds of genetic markers, different source of human biological material owns suitable marker types and can be identified by detecting single genetic marker or combined multiple genetic markers. Though there is no uniform standard and method for identifying the source of human biological materials in forensic laboratories at present, the research and development of a series of mature and reliable methods for distinguishing different human biological materials play the role as forensic evidence which will be the future development direction.

[Identification of Vaginal Fluid Using Microbial Signatures].

OBJECTIVES: To investigate the specific microbial signatures in vaginal fluid. METHODS: Vaginal fluid （16 samples）, saliva （16 samples）, feces （16 samples）, semen （8 samples）, peripheral blood （8 samples）, urine （5 samples）, and nasal secretion （4 samples） were collected respectively. The 16S rRNA genes of Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus iners, and Atopobium vaginae were amplified. PCR production was detected via a 3130xl Genetic Analyzer. RESULTS: The detected number of Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus iners, and Atopobium vaginae were 15, 5, 8, 14, and 3 in all vaginal fluid samples, respectively. Lactobacillus crispatus and Lactobacillus jensenii existed specifically in vaginal fluid. CONCLUSIONS: There is a potential application value to detect Lactobacillus crispatus and Lactobacillus jensenii for the identification of vaginal fluid.

[Research Progress on Gene Alterations of Amelogenin Locus in Gender Identification].

There are two kinds of amelogenin gene mutation, including mutation in primer-binding region of amelogenin gene and micro deletion of Y chromosome encompassing amelogenin gene, and the latter is more common. The mechanisms of mutation in primer-binding region of amelogenin gene is nucleotide point mutation and the mechanism of micro deletion of Y chromosome encompassing amelogenin gene maybe non-allelic homologous recombination or non-homologous end-joining. Among the population worldwide, there is a notably higher frequency of amelogenin gene mutations in Indian population, Sri Lanka population and Nepalese population which reside within the Indian subcontinent. Though amelogenin gene mutations have little impact on fertility and phenotype, they might cause incorrect result in gender identification. Using composite-amplification kit which including autosomal STR locus, amelogenin gene locus and multiple Y-STR locus, could avoid wrong gender identification caused by amelogenin gene mutation.

Effects of different remifentanil target concentrations on MAC BAR of sevoflurane in gynaecological patients with CO2 pneumoperitoneum stimulus.

BACKGROUND: To investigate the effects of different target plasma remifentanil concentrations on the minimum alveolar concentration of sevoflurane (MAC) for blocking adrenergic response (BAR) during laparoscopic gynaecological surgery with carbon dioxide insufflation. METHODS: Seventy-five gynaecological patients with ASA I-II undergoing laparoscopic surgery were randomly assigned to three groups. Anaesthesia was induced by sevoflurane, and 0.1 mg kg(-1) of vecuronium i.v. was injected to facilitate tracheal intubation. After intubation the target plasma concentrations of remifentanil in Groups 1, 2, and 3 were adjusted to 0, 1, and 2 ng ml(-1), respectively. The changes in haemodynamics were observed before and after the creation of carbon dioxide pneumoperitoneum. The MAC BAR of sevoflurane in each group was determined by using an up-and-down sequential-allocation technique, and blood samples were collected at corresponding time points to determine the concentrations of remifentanil, norepinephrine, and epinephrine. RESULTS: In Groups 1, 2 and 3, the MAC BAR of sevoflurane was 4.6% (CI 95%: 4.3-4.9%), 2.4% (CI 95%: 2.2-2.6%), and 1.7% (CI 95%: 1.4-2.1%), respectively. No significant differences were found in the increase of norepinephrine, epinephrine, and mean arterial pressure after compared with before insufflation of pneumoperitoneum among the three groups. CONCLUSIONS: Remifentanil can effectively decrease the sevoflurane concentration to block sympathetic adrenergic response to CO2 pneumoperitoneum stimulus. At similar MAC BAR the haemodynamic and adrenergic response is not affected by the infused remifentanil concentration. CLINICAL TRIAL REGISTRATION: The number of this clinical trial registry is ChiCTR-TRC-13004005, and the Universal Trial Number is U1111-1151-5630.

Genetic expression of an amyloid peptide fragment and analysis of formylated products.

The model amyloid peptide AAKLVFF was expressed as a His-tagged fusion protein with the immunoglobulin-binding domain B1 of streptococcal protein G (GB1), a small (56 residues), stable, single-domain protein. It is shown that expression of this model amyloid peptide is possible and is not hindered by aggregation. Formylation side reactions during the CNBr cleavage are investigated via synthesis of selectively formylated peptides.

The HumD21S11 system of short tandem repeat DNA polymorphisms in Japanese and Chinese.

HumD21S11 is a short tandem repeat DNA polymorphic system with a complex basic structure of (TCTA)4-6 (TCTG)5-6 (TCTA)3 TA (TCTA)3 TCA (TCTA)2 TCCA TA (TCTA)n. Using the allelic ladder prepared by us, the distribution of alleles among Japanese and Chinese was investigated, and four new alleles 28.2, 34, 35.2, and 36.2, were discovered. DNA sequencing was performed on the newly found alleles as well as on family samples and led to the discovery of different gene structures within alleles 28 and 32. Forensic materials, including hairs and seminal stains, were tested in parallel with blood samples from the same individual and were successfully typed for D21S11.

[Study on the variable activity of nucleolar organizer regions in lymphocytes from patients with carcinoma].

The activity of nucleolar organizer regions (NOR) in the peripheral lymphocytes from patients with lung, gastric, intestinal or breast carcinoma was studied with a combined method of Ag-staining and G-band. The frequencies of Ag-NOR were higher in chromosome 15 and sum total from patients with lung cancer, lower in chromosome 14 from patients with breast cancer, and lower in chromosome 14 but higher in chromosome 22 from patients with gastric cancer, as compared with those of controls. No significant variation of the frequencies of Ag-NOR was found in the patients with intestinal cancer. The results indicate that a dominant Ag-stained NOR model is shown in the patients with different carcinoma, i.e., the active expression of rRNA genes may present a speciality concerning the location of carcinoma.

Complete culture medium is better than low folate medium for detecting increased chromosome aberrations in smokers in 48-h lymphocyte cultures.

We compared chromosome breakage in parallel, 48-h lymphocyte cultures established from smokers and nonsmokers using minimal essential medium (MEM) and MEM without folate (MEM-FA). There was a statistically significant, higher frequency of aberrations for smokers than for nonsmokers in cells cultured in MEM, but not in those cultured in MEM-FA. Thus, these data support the recommendation of the World Health Organization (1985) that population monitoring studies for exposure assessment should not use a low folate medium.

Increased chromosome fragility as a consequence of blood folate levels, smoking status, and coffee consumption.

Chromosome fragility in 96 h, low-folate cultures was found to be associated with smoking status, coffee consumption, and blood folate level. The higher proportion of cells with chromosome aberrations in cigarette smokers was attributable to lower red cell folate levels in smokers compared with nonsmokers. There was a positive linear relationship between the average cups of coffee consumed per day and the proportion of cells with aberrations. This association was independent of the effects of smoking and red cell folate level. These data suggest that smoking history, coffee consumption, and red cell folate level are important considerations for the design and interpretation of fragile site studies in cancer cytogenetics.