Pyrosequencing Analysis of Oral Microbiota Shifting in Various Caries States in Childhood

Dental caries is one of the most prevalent childhood diseases worldwide, but little is known about the dynamic characteristics of oral microbiota in the development of dental caries. To investigate the shifting bacterial profiles in different caries states, 60 children (3–7-year-old) were enrolled in this study, including 30 caries-free subjects and 30 caries-active subjects. Supragingival plaques were collected from caries-active subjects on intact enamel, white spot lesions and carious dentin lesions. Plaques from caries-free subjects were used as a control. All samples were analyzed by 454 pyrosequencing based on 16S rRNA gene V1-V3 hypervariable regions. A total of 572,773 pyrosequencing reads passed the quality control and 25,444 unique phylotypes were identified, which represented 18 phyla and 145 genera. Reduced bacterial diversity in the cavitated dentin was observed as compared with the other groups. Thirteen genera (including Capnocytophaga, Fusobacterium, Porphyromonas, Abiotrophia, Comamonas, Tannerella, Eikenella, Paludibacter, Treponema, Actinobaculum, Stenotrophomonas, Aestuariimicrobium, and Peptococcus) were found to be associated with dental health, and the bacterial profiles differed considerably depending on caries status. Eight genera (including Cryptobacterium, Lactobacillus, Megasphaera, Olsenella, Scardovia, Shuttleworthia, Cryptobacterium, and Streptococcus) were increased significantly in cavitated dentin lesions, and Actinomyces and Corynebacterium were present at significant high levels in white spot lesions (P?<?0.05), while Flavobacterium, Neisseria, Bergeyella, and Derxia were enriched in the intact surfaces of caries individuals (P?<?0.05). Our results showed that oral bacteria were specific at different stages of caries progression, which contributes to informing the prevention and treatment of childhood dental caries.     

Utilization of Extracorporeal Membrane Oxygenation Alleviates Intestinal Ischemia–Reperfusion Injury in Prolonged Hemorrhagic Shock Animal Model

Intestinal ischemia–reperfusion injury is one of the main factors leading to multiple organ failure after resuscitation of prolonged hemorrhagic shock; however, the current conventional fluid resuscitation still cannot effectively reduce intestinal injury caused by prolonged hemorrhagic shock. To investigate the effect of ECMO resuscitation on alleviating intestinal ischemia–reperfusion injury in a prolonged hemorrhagic shock rabbit model. Thirty New Zealand white rabbits were randomly divided into three groups: control group, conventional fluid resuscitation group, and ECMO resuscitation group. The prolonged hemorrhagic shock model was established by keeping the arterial blood pressure from 31 to 40 mmHg for 3 h through the femoral artery bleeding, and performing the resuscitation for 2 h by conventional fluid resuscitation and ECMO resuscitation, respectively. Chiu’s score of intestinal injury, serum lactate and TNF-α levels, intestinal mucosamyeloperoxidase (MPO) activity, intercellular adhesion molecule (ICAM-1), and Claudin-1expression were detected. The mean arterial blood pressure in Group 2 was significantly higher after resuscitation than in Group 1, but serum lactate and inflammatory cytokines TNF-α level were significantly lower. And Chiu’s score of intestinal injury and myeloperoxidase (MPO) activity level and ICAM-1 expression were significantly lower in the ECMO resuscitation group, in which the Claudin-1 levels were significantly increased. ECMO resuscitation for the prolonged hemorrhagic shock improves tissue perfusion and reduces the systemic inflammation, and thus alleviates intestinal damage caused by prolonged hemorrhagic shock.     

Highly interactive nature of flower‐specific enhancers and promoters,and its potential impact on tissue‐specific expression and engineering of multiple genes or agronomic traits

Molecular stacking enables multiple traits to be effectively engineered in crops using a single vector. However, the co‐existence of distinct plant promoters in the same transgenic unit might, like their mammalian counterparts, interfere with one another. In this study, we devised a novel approach to investigate enhancer–promoter and promoter–promoter interactions in transgenic plants and demonstrated that three of four flower‐specific enhancer/promoters were capable of distantly activating a pollen‐ and stigma‐specific Pps promoter (fused to the cytotoxic DT‐A gene) in other tissues, as revealed by novel tissue ablation phenotypes in transgenic plants. The NtAGI1 enhancer exclusively activated stamen‐ and carpel‐specific DT‐A expression, thus resulting in tissue ablation in an orientation‐independent manner; this activation was completely abolished by the insertion of an enhancer‐blocking insulator (EXOB) between the NtAGI1 enhancer and Pps promoter. Similarly, AGL8 and AP1Lb1, but not AP1La, promoters also activated distinct tissue‐specific DT‐A expression and ablation, with the former causing global growth retardation and the latter ablating apical inflorescences. While the tissue specificity of the enhancer/promoters generally defined their activation specificities, the strength of their activity in particular tissues or developmental stages appeared to determine whether activation actually occurred. Our findings provide the first evidence that plant‐derived enhancer/promoters can distantly interact/interfere with one another, which could pose potential problems for the tissue‐specific engineering of multiple traits using a single‐vector stacking approach. Therefore, our work highlights the importance of adopting enhancer‐blocking insulators in transformation vectors to minimize promoter–promoter interactions. The practical and fundamental significance of these findings will be discussed.     

Association between DAPK1 Promoter Methylation and Cervical Cancer: A Meta-Analysis

Background

Death-associated protein kinase1 (DAPK1) is an important tumor suppressor gene. DNA methylation can inactivate genes, which has often been observed in the carcinogenesis of cervical cancer. During the past several decades, many studies have explored the association between DAPK1 promoter methylation and cervical cancer. However, many studies were limited by the small samples size and the findings were inconsistent among them. Thus, we conducted a meta-analysis to assess the association between DAPK1 promoter methylation and cervical cancer.

Methods

We systematically searched eligible studies in the PubMed, Web of Science, EMBASE and CNKI databases. Using meta-regression, subgroup analysis and sensitivity analysis, we explored the potential sources of heterogeneity. The odds ratio (OR) and 95% confidence interval (95% CI) were calculated by Meta-Analysis in R.

Results

A total of 15 studies from 2001 to 2012, comprising 818 tumor tissues samples and 671 normal tissues samples, were analyzed in this meta-analysis. The frequencies of DAPK1 promoter methylation ranged from 30.0% to 78.6% (median, 59.3%) in cervical cancer tissue and 0.0% to 46.7% (median, 7.8%) in normal cervical tissue. The pooled OR was 19.66 (95%CI = 8.72–44.31) with the random effects model, and heterogeneity was found through the sensitivity analysis. The I² = 60% (P = 0.002) decreased to I² = 29.2% (P = 0.144) when one heterogeneous study was excluded, and the pooled OR increased to 21.80 (95%CI = 13.44–35.36) with the fixed effects model.

Conclusion

The results suggested a strong association between DAPK1 promoter methylation and cervical cancer. This study also indicated that DAPK1 promoter methylation may be a biomarker during cervical carcinogenesis that might serve as an early indication of cervical cancer.     

Species-Specific Detection and Identification of Fusarium Species Complex,the Causal Agent of Sugarcane Pokkah Boeng in China

Background

Pokkah boeng disease caused by the Fusarium species complex results in significant yield losses in sugarcane. Thus, the rapid and accurate detection and identification of the pathogen is urgently required to manage and prevent the spreading of sugarcane pokkah boeng.

Methods

A total of 101 isolates were recovered from the pokkah boeng samples collected from five major sugarcane production areas in China throughout 2012 and 2013. The causal pathogen was identified by morphological observation, pathogenicity test, and phylogenetic analysis based on the fungus-conserved rDNA-ITS. Species-specific TaqMan real-time PCR and conventional PCR methods were developed for rapid and accurate detection of the causal agent of sugarcane pokkah boeng. The specificity and sensitivity of PCR assay were also evaluated on a total of 84 isolates of Fusarium from China and several isolates from other fungal pathogens of Sporisorium scitamineum and Phoma sp. and sugarcane endophyte of Acremonium sp.

Result

Two Fusarium species (F. verticillioides and F. proliferatum) that caused sugarcane pokahh boeng were identified by morphological observation, pathogenicity test, and phylogenetic analysis. Species-specific TaqMan PCR and conventional PCR were designed and optimized to target their rDNA-ITS regions. The sensitivity of the TaqMan PCR was approximately 10 pg of fungal DNA input, which was 1,000-fold over conventional PCR, and successfully detected pokkah boeng in the field-grown sugarcane.

Conclusions/Significance

This study was the first to identify two species, F. verticillioides and F. proliferatum, that were causal pathogens of sugarcane pokkah boeng in China. It also described the development of a species-specific PCR assay to detect and confirm these pathogens in sugarcane plants from mainland China. This method will be very useful for a broad range of research endeavors as well as the regulatory response and management of sugarcane pokkah boeng.     

Construction of an Integrated Map of Haliotis diversicolor Using Microsatellite Markers

Small abalone, Haliotis diversicolor, is naturally distributed along the coastal waters of East Asia from Japan to the Philippines. It is an economically important maricultured species in southern China and Taiwan. Genetic linkage maps for small abalone were constructed using a total of 308 simple sequence repeat markers including 297 novel markers. Segregation data on 96 progeny were genotyped using a pseudo-testcross strategy. Sixteen linkage groups were identified in both female and male maps, consistent with the haploid chromosome number. The female linkage map covered 758.3 cM, with an average interval of 5.2 cM. The male linkage map spanned a total genetic distance of 676.2 cM, with an average interval of 4.5 cM. An integrated map was constructed by incorporating the homologous parental linkage groups, resulting in 16 linkage groups with a total of 762.1 cM. Genome coverage of the integrated linkage map was approximately 80.7%. The genetic linkage maps of small abalone may facilitate marker-assisted selection and quantitative trait loci mapping.     

洋葱卵细胞的分离

将洋葱的胚珠置于酶液中酶解50~110 min后剥去其珠被,可清楚地看到珠心中的胚囊轮廓。用解剖针将珠心从中部横切,然后挤压其珠孔部位,卵器细胞从胚珠的切口处逸出。再用显微操作仪的玻璃针将卵细胞和两个助细胞分开,达到分离洋葱卵细胞的目的。酶对分离卵细胞具有重要作用,在最佳的酶液浓度[0.02%果胶酶Y23、0.08%果胶酶(Serva)、0.05%纤维素酶和0.05%半纤维素酶]下酶解胚珠110 min后,解剖1 h可从24个胚珠中分离出10个卵细胞(41.67%)。随着胚囊的发育,两个助细胞的体积出现明显的二形性。洋葱生活卵细胞的分离为开展洋葱离体受精建立了基础,也为研究洋葱卵器细胞的发育创造了条件。     

半褶织纹螺(Nassarius semiplicatus)及其生活环境中分离培养的细菌毒性测试与种类分析

在中国沿海地区,因食用织纹螺导致的中毒事件时有发生.最近的研究表明,河豚毒素及其衍生物是织纹螺中主要的致毒成分.但是,对于织纹螺中河豚毒素的来源还不清楚.[目的]本研究尝试分离、培养和鉴定织纹螺及其生活环境中的细菌,并对其毒性进行分析,为探明织纹螺中河豚毒素的可能来源提供科学依据.[方法]先后于2006年6月13日和19日在江苏省盐城采集织纹螺样品,应用小鼠生物法对织纹螺样品的毒性进行了测试;从织纹螺体内及其生活环境中分离细菌,并选择部分菌株进行了室内培养;以直接竞争酶联免疫分析方法(ELISA)对培养菌株中的河豚毒素进行了检测;通过对细菌16S核糖体DNA(rDNA)部分序列的测定,对有毒菌株进行了初步的种灯分析.[结果]实验结果表明,采集的织纹螺为半褶织纹螺,两次采集样品的毒性分别为247 MU(mouse unit,小鼠单位)和270MU/100g组织(湿重).对14个菌株进行了毒性检测,其中有毒细菌9株.产毒菌株的毒性普遍较低,毒性范围为15～96 ng/g.有毒菌株核糖体序列与弧菌(Vibrio)、希瓦氏菌(Shewanella)、动性球菌(Planococcus)、海单胞菌(Marinomonas)、发光杆菌(Photobacterium)等菌属有较高的相似性,可能具有较近的亲缘关系.[结论]研究发现半褶织纹螺体内及其生活环境中存在能够产生河豚毒素的细菌,说明织纹螺中的河豚毒素可能与其体内及其生活环境中的细菌有关,有必要进行深入研究.     

Physiological and epigenetic analyses of <Emphasis Type="Italic">Brassica napus</Emphasis> seed germination in response to salt stress

Salinity stress significantly affects plant growth and development because of osmotic stress, ion toxicity, and nutrient imbalance. Therefore, salinity stress becomes a serious threat to rapeseed production in agriculture. Plants evolved a series of complex mechanisms, including morphological changes, physiological adjustment, and gene expression regulation, at a molecular level to adapt to salt stress. Epigenetic regulations, including DNA methylation and histone modification, play a major role in tuning gene expression in plant response to environmental stimuli. Although many progresses have been reported in plant response to salt stress, the epigenetic changes in Brassica napus under salt stress are far from being understood. A series of physiological parameters, including water content, proline content, malondialdehyde content, electrolyte leakage, and antioxidant enzyme activities, under different concentrations (0, 25, 50, and 100 mM) of NaCl treatment in “Yangyou 9” was determined at the germination stage. Immunofluorescent staining and high-performance liquid chromatography-assisted quantification were conducted to analyze the level and distribution patterns of DNA and histone methylation under salt stress. Results of morphological and physiological analyses under salt stress indicated that 25 mM NaCl treatment promoted the growth of “Yangyou 9” seedlings, whereas 50 and 100 mM NaCl treatments inhibited the growth of “Yangyou 9” seedlings. Epigenetic investigations showed that 25 mM NaCl mediated the enrichment of H3K4me3, as well as decreases in H3K9me2 and 5-methylcytosine (5-mC), whereas 50 and 100 mM NaCl induced increases in H3K9me2 and 5-mC and a decrease in H3K4me3. Overall, this study offers new insights into the epigenetic changes in salt stress response in rapeseed, and this information would be propitious to engineer crops with enhanced salt tolerance.