环境样品中DNA的分离纯化和文库构建

采用研磨 /冻融和SDS/蛋白酶K热处理等理化方法 ,直接从性质不同的环境样品中提取和纯化混合基因组DNA。所获得纯品DNA的产量为每克样品 2～ 1 6μg。对纯品DNA进行限制性内切酶处理后 ,构建了以pUC1 8为载体的DNA文库。建库效率为从每克环境样品获得约 1 0 3～ 1 0 4 个含 3～ 8kb外源随机插入片段的克隆。通过DNA序列测定和基因注释 ,对从文库中随机选取的克隆进行了分析 ,发现外源插入片段均含序列未见报道的新基因。本文所做的尝试对于保存、研究和开发未培养微生物基因资源具有意义     

Endosymbiont metacommunities,mtDNA diversity and the evolution of the Bemisia tabaci (Hemiptera: Aleyrodidae) species complex

Bemisia tabaci, an invasive pest that causes crop damage worldwide, is a highly differentiated species complex, divided into biotypes that have mainly been defined based on mitochondrial DNA sequences. Although endosymbionts can potentially induce population differentiation, specialization and indirect selection on mtDNA, studies have largely ignored these influential passengers in B. tabaci, despite as many as seven bacterial endosymbionts have been identified. Here, we investigate the composition of the whole bacterial community in worldwide populations of B. tabaci, together with host genetic differentiation, focusing on the invasive B and Q biotypes. Among 653 individuals studied, more than 95% of them harbour at least one secondary endosymbiont, and multiple infections are very common. In addition, sequence analyses reveal a very high diversity of facultative endosymbionts in B. tabaci, with some bacterial genus being represented by more than one strain. In the B and Q biotypes, nine different strains of bacteria have been identified. The mtDNA‐based phylogeny of B. tabaci also reveals a very high nucleotide diversity that partitions the two ITS clades (B and Q) into six CO1 genetic groups. Each genetic group is in linkage disequilibrium with a specific combination of endosymbionts. All together, our results demonstrate the rapid dynamics of the bacterial endosymbiont–host associations at a small evolutionary scale, questioning the role of endosymbiotic communities in the evolution of the Bemisia tabaci species complex and strengthening the need to develop a metacommunity theory of inherited endosymbionts.     

Canopy interactions and physical stress gradients in subtidal communities

Species interactions are integral drivers of community structure and can change from competitive to facilitative with increasing environmental stress. In subtidal marine ecosystems, however, interactions along physical stress gradients have seldom been tested. We observed seaweed canopy interactions across depth and latitudinal gradients to test whether light and temperature stress structured interaction patterns. We also quantified interspecific and intraspecific interactions among nine subtidal canopy seaweed species across three continents to examine the general nature of interactions in subtidal systems under low consumer pressure. We reveal that positive and neutral interactions are widespread throughout global seaweed communities and the nature of interactions can change from competitive to facilitative with increasing light stress in shallow marine systems. These findings provide support for the stress gradient hypothesis within subtidal seaweed communities and highlight the importance of canopy interactions for the maintenance of subtidal marine habitats experiencing environmental stress.     

Structures of Streptococcus pneumoniae PiaA and Its Complex with Ferrichrome Reveal Insights into the Substrate Binding and Release of High Affinity Iron Transporters

Iron scarcity is one of the nutrition limitations that the Gram-positive infectious pathogens Streptococcus pneumoniae encounter in the human host. To guarantee sufficient iron supply, the ATP binding cassette (ABC) transporter Pia is employed to uptake iron chelated by hydroxamate siderophore, via the membrane-anchored substrate-binding protein PiaA. The high affinity towards ferrichrome enables PiaA to capture iron at a very low concentration in the host. We presented here the crystal structures of PiaA in both apo and ferrichrome-complexed forms at 2.7 and 2.1 Å resolution, respectively. Similar to other class III substrate binding proteins, PiaA is composed of an N-terminal and a C-terminal domain bridged by an α-helix. At the inter-domain cleft, a molecule of ferrichrome is stabilized by a number of highly conserved residues. Upon ferrichrome binding, two highly flexible segments at the entrance of the cleft undergo significant conformational changes, indicating their contribution to the binding and/or release of ferrichrome. Superposition to the structure of Escherichia coli ABC transporter BtuF enabled us to define two conserved residues: Glu119 and Glu262, which were proposed to form salt bridges with two arginines of the permease subunits. Further structure-based sequence alignment revealed that the ferrichrome binding pattern is highly conserved in a series of PiaA homologs encoded by both Gram-positive and negative bacteria, which were predicted to be sensitive to albomycin, a sideromycin antibiotic derived from ferrichrome.     

Diabetes with hypertension as risk factors for adult dengue hemorrhagic fever in a predominantly dengue serotype 2 epidemic: a case control study

Background

Dengue hemorrhagic fever (DHF) is a severe form of dengue, characterized by bleeding and plasma leakage. A number of DHF risk factors had been suggested. However, these risk factors may not be generalized to all populations and epidemics for screening and clinical management of patients at risk of developing DHF. This study explored demographic and comorbidity risk factors for DHF in adult dengue epidemics in Singapore in year 2006 (predominantly serotype 1) and in year 2007–2008 (predominantly serotype 2).

Methods

A retrospective case-control study was conducted with 149 DHF and 326 dengue fever (DF) patients from year 2006, and 669 DHF and 1,141 DF patients from year 2007–2008. Demographic and reported comorbidity data were collected from patients previously. We performed multivariate logistic regression to assess the association between DHF and demographic and co-morbidities for year 2006 and year 2007–2008, respectively.

Results

Only Chinese (adjusted odds ratio [AOR] = 1.90; 95% confidence interval [CI]: 1.01–3.56) was independently associated with DHF in year 2006. In contrast, age groups of 30–39 years (AOR = 1.41; 95% CI:1.09–1.81), 40–49 years (AOR = 1.34; 95% CI:1.09–1.81), female (AOR = 1.57; 95% CI:1.28–1.94), Chinese (AOR = 1.67; 95% CI:1.24–2.24), diabetes (AOR = 1.78; 95% CI:1.06–2.97), and diabetes with hypertension (AOR = 2.16; 95%CI:1.18–3.96) were independently associated with DHF in year 2007–2008. Hypertension was proposed to have effect modification on the risk of DHF outcome in dengue patients with diabetes. Chinese who had diabetes with hypertension had 2.1 (95% CI:1.07–4.12) times higher risk of DHF compared with Chinese who had no diabetes and no hypertension.

Conclusions

Adult dengue patients in Singapore who were 30–49 years, Chinese, female, had diabetes or diabetes with hypertension were at greater risk of developing DHF during epidemic of predominantly serotype 2. These risk factors can be used to guide triaging of patients who require closer clinical monitoring and early hospitalization in Singapore, when confirmed in more studies.     

Type III secretion and effectors shape the survival and growth pattern of Pseudomonas syringae on leaf surfaces

The bacterium Pseudomonas syringae pv syringae B728a (PsyB728a) uses a type III secretion system (T3SS) to inject effector proteins into plant cells, a process that modulates the susceptibility of different plants to infection. Analysis of GREEN FLUORESCENT PROTEIN-expressing PsyB728a after spray inoculation without additives under moderate relative humidity conditions permitted (1) a detailed analysis of this strain's survival and growth pattern on host (Nicotiana benthamiana) and nonhost (tomato [Solanum lycopersicum]) leaf surfaces, (2) an assessment of the role of plant defenses in affecting PsyB728a leaf surface (epiphytic) growth, and (3) the contribution of the T3SS and specific effectors to PsyB728a epiphytic survival and growth. On host leaf surfaces, PsyB728a cells initially persist without growing, and show an increased population only after 48 h, unless plants are pretreated with the defense-inducing chemical benzothiazole. During the persistence period, some PsyB728a cells induce a T3SS reporter, whereas a T3SS-deficient mutant shows reduced survival. By 72 h, rare invasion by PsyB728a to the mesophyll region of host leaves occurs, but endophytic and epiphytic bacterial growths are not correlated. The effectors HopZ3 and HopAA1 delay the onset of epiphytic growth of PsyB728a on N. benthamiana, whereas they promote epiphytic survival/growth on tomato. These effectors localize to distinct sites in plant cells and likely have different mechanisms of action. HopZ3 may enzymatically modify host targets, as it requires residues important for the catalytic activity of other proteins in its family of proteases. Thus, the T3SS, HopAA1, HopZ3, and plant defenses strongly influence epiphytic survival and/or growth of PsyB728a.     

Lipoteichoic acid induces surfactant protein-A biosynthesis in human alveolar type II epithelial cells through activating the MEK1/2-ERK1/2-NF-κB pathway

Background

Asthma is a chronic inflammatory disease of the airways but recent studies have shown that alveoli are also subject to pathophysiological changes. This study was undertaken to compare hydrogen peroxide (H₂O₂) concentrations in different parts of the lung using a new technique of fractioned breath condensate sampling.

Methods

In 52 children (9-17 years, 32 asthmatic patients, 20 controls) measurements of exhaled nitric oxide (FE_NO), lung function, H₂O₂ in exhaled breath condensate (EBC) and the asthma control test (ACT) were performed. Exhaled breath condensate was collected in two different fractions, representing mainly either the airways or the alveoli. H₂O₂ was analysed in the airway and alveolar fractions and compared to clinical parameters.

Results

The exhaled H₂O₂ concentration was significantly higher in the airway fraction than in the alveolar fraction comparing each single pair (p = 0.003, 0.032 and 0.040 for the whole study group, the asthmatic group and the control group, respectively). Asthma control, measured by the asthma control test (ACT), correlated significantly with the H₂O₂ concentrations in the alveolar fraction (r = 0.606, p = 0.004) but not with those in the airway fraction in the group of children above 12 years. FE_NO values and lung function parameters did not correlate to the H₂O₂ concentrations of each fraction.

Conclusion

The new technique of fractionated H₂O₂ measurement may differentiate H₂O₂ concentrations in different parts of the lung in asthmatic and control children. H₂O₂ concentrations of the alveolar fraction may be related to the asthma control test in children.