利用串联亲和纯化的方法筛选耻垢分枝杆菌中MutM的相互作用蛋白

Mut M(formamidopyrimidine-DNA glycosylase,Fpg)是原核生物碱基切除修复系统(BER)中同时具有DNA糖苷酶和脱嘌呤/脱嘧啶AP裂解酶活性的一种双功能酶,不但可以识别DNA损伤,而且能切除损伤的碱基,从而参与到许多种损伤的修复过程.除了高致突变率的8-羟基鸟嘌呤(8-oxoguanine,8-oxo G)外,Mut M在其他损伤修复中具体作用机制还不清楚.本研究主要以耻垢分枝杆菌(M.smegmatis)为研究对象,利用串联亲和纯化技术和质谱相结合的方法对可能与Mut M相互作用的蛋白因子进行发现和鉴定,并于体外用Far-western和GST pull-down方法对鉴定出的蛋白DEAD-box rna helicase、Rps C、Uvr A与Mut M的相互作用进行了验证.实验结果表明,利用串联亲和纯化方法来发现Mut M相互作用的蛋白是切实可行的.本研究为进一步深入研究Mut M在其参与的损伤修复中的具体机制提供了切入点.     

The interactive effects of soil transplant into colder regions and cropping on soil microbiology and biogeochemistry

Soil transplant into warmer regions has been shown to alter soil microbiology. In contrast, little is known about the effects of soil transplant into colder regions, albeit that climate cooling has solicited attention in recent years. To address this question, we transplanted bare fallow soil over large transects from southern China (subtropical climate zone) to central (warm temperate climate zone) and northern China (cold temperate climate zone). After an adaptation period of 4 years, soil nitrogen components, microbial biomass and community structures were altered. However, the effects of soil transplant on microbial communities were dampened by maize cropping, unveiling a negative interaction between cropping and transplant. Further statistical analyses with Canonical correspondence analysis and Mantel tests unveiled annual average temperature, relative humidity, aboveground biomass, soil pH and NH₄⁺‐N content as environmental attributes closely correlated with microbial functional structures. In addition, average abundances of amoA‐AOA (ammonia‐oxidizing archaea) and amoA‐AOB (ammonia‐oxidizing bacteria) genes were significantly (P < 0.05) correlated with soil nitrification capacity, hence both AOA and AOB contributed to the soil functional process of nitrification. These results suggested that the soil nitrogen cycle was intimately linked with microbial community structure, and both were subjected to disturbance by soil transplant to colder regions and plant cropping.     

Burden of Invasive Group B Streptococcus Disease and Early Neurological Sequelae in South African Infants

Introduction

Group B Streptococcus (GBS) is a leading cause of neonatal sepsis and meningitis. We aimed to evaluate the burden of invasive early-onset (0–6 days of life, EOD) and late-onset (7–89 days, LOD) GBS disease and subsequent neurological sequelae in infants from a setting with a high prevalence (29.5%) of HIV among pregnant women.

Methods

A case-control study was undertaken at three secondary-tertiary care public hospitals in Johannesburg. Invasive cases in infants <3 months age were identified by surveillance of laboratories from November 2012 to February 2014. Neurodevelopmental screening was done in surviving cases and controls at 3 and 6 months of age.

Results

We identified 122 cases of invasive GBS disease over a 12 month period. Although the incidence (per 1,000 live births) of EOD was similar between HIV-exposed and HIV-unexposed infants (1.13 vs. 1.46; p = 0.487), there was a 4.67-fold (95%CI: 2.24–9.74) greater risk for LOD in HIV-exposed infants (2.27 vs. 0.49; p<0.001). Overall, serotypes Ia, Ib and III constituted 75.8% and 92.5% of EOD and LOD, respectively. Risk factors for EOD included offensive draining liquor (adjusted Odds Ratio: 27.37; 95%CI: 1.94–386.50) and maternal GBS bacteriuria (aOR: 8.41; 95%CI: 1.44–49.15), which was also a risk-factor for LOD (aOR: 3.49; 95%CI: 1.17–10.40). The overall case fatality rate among cases was 18.0%. The adjusted odds for neurological sequelae at 6 months age was 13.18-fold (95%CI: 1.44–120.95) greater in cases (13.2%) than controls (0.4%).

Discussion

The high burden of invasive GBS disease in South Africa, which is also associated with high case fatality rates and significant neurological sequelae among survivors, is partly due to the heightened risk for LOD in infants born to HIV-infected women. An effective trivalent GBS conjugate vaccine targeted at pregnant women could prevent invasive GBS disease in this setting.     

Subcutaneous and Segmental Fat Loss with and without Supportive Supplements in Conjunction with a Low-Calorie High Protein Diet in Healthy Women

Background

Weight loss benefits of multi-ingredient supplements in conjunction with a low-calorie, high-protein diet in young women are unknown. Therefore, the purpose of this study was to investigate the effects of a three-week low-calorie diet with and without supplementation on body composition.

Methods

Thirty-seven recreationally-trained women (n = 37; age = 27.1 ± 4.2; height = 165.1 ± 6.4; weight = 68.5 ± 10.1; BMI = 25.1 ± 3.4) completed one of the following three-week interventions: no change in diet (CON); a high-protein, low-calorie diet supplemented with a thermogenic, conjugated linoleic acid (CLA), a protein gel, and a multi-vitamin (SUP); or the high-protein diet with isocaloric placebo supplements (PLA). Before and after the three-week intervention, body weight, %Fat via dual X-ray absorptiometry (DXA), segmental fat mass via DXA, %Fat via skinfolds, and skinfold thicknesses at seven sites were measured.

Results

SUP and PLA significantly decreased body weight (SUP: PRE, 70.47 ± 8.01 kg to POST, 67.51 ± 8.10 kg; PLA: PRE, 67.88 ± 12.28 kg vs. POST, 66.38 ± 11.94 kg; p ≤ 0.05) with a greater (p ≤ 0.05) decrease in SUP than PLA or CON. SUP and PLA significantly decreased %Fat according to DXA (SUP: PRE, 34.98 ± 7.05% to POST, 32.99 ± 6.89%; PLA: PRE, 34.22 ± 6.36% vs. POST, 32.69 ± 5.84%; p ≤ 0.05), whereas only SUP significantly decreased %Fat according to skinfolds (SUP: PRE, 27.40 ± 4.09% to POST, 24.08 ± 4.31%; p ≤ 0.05). SUP significantly (p ≤ 0.05) decreased thicknesses at five skinfolds (chest, waist, hip, subscapular, and tricep) compared to PLA, but not at two skinfolds (axilla and thigh).

Conclusions

The addition of a thermogenic, CLA, protein, and a multi-vitamin to a three-week low-calorie diet improved weight loss, total fat loss and subcutaneous fat loss, compared to diet alone.     

The BD FACSPresto Point of Care CD4 Test Accurately Enumerates CD4+ T Cell Counts

Objective

Currently 50% of ART eligible patients are not yet receiving life-saving antiretroviral therapy (ART). Financial constraints do not allow most developing countries to adopt a universal test and offer ART strategy. Decentralizing CD4+ T cell testing may, therefore, provide greater access to testing, ART, and better patient management. We evaluated the technical performance of a new point-of-care CD4+ T cell technology, the BD FACSPresto, in a field methods comparison study.

Methods

264 HIV-positive patients were consecutively enrolled and included in the study. The BD FACSPresto POC CD4+ T cell technology was placed in two rural health care facilities and operated by health care facility staff. We compared paired finger-prick and venous samples using the BD FACSPresto and several existing reference technologies, respectively.

Results

The BD FACSPresto had a mean bias of 67.29 cells/ul and an r² of 0.9203 compared to the BD FACSCalibur. At ART eligibility thresholds of 350 and 500 cells/ul, the sensitivity to define treatment eligibility were 81.5% and 77.2% and the specificities were 98.9% and 100%, respectively. Similar results were observed when the BD FACSPresto was compared to the BD FACSCount and Alere Pima. The coefficient of variation (CV) was less than 7% for both the BD FACSCalibur and BD FACSPresto. CD4+ T cell testing by nurses using the BD FACSPresto at rural health care facilities showed high technical similarity to test results generated by laboratory technicians using the BD FACSPresto in a high functioning laboratory.

Conclusions

The BD FACSPresto performed favorably in the laboratory setting compared to the conventional reference standard technologies; however, the lower sensitivities indicated that up to 20% of patients tested in the field in need of treatment would be missed. The BD FACSPresto is a technology that can allow for greater decentralization and wider access to CD4+ T cell testing and ART.     

Evaluation of Molecular Methods To Improve the Detection of Burkholderia pseudomallei in Soil and Water Samples from Laos

Burkholderia pseudomallei is the cause of melioidosis, a severe and potentially fatal disease of humans and animals. It is endemic in northern Australia and Southeast Asia and is found in soil and surface water. The environmental distribution of B. pseudomallei worldwide and within countries where it is endemic, such as the Lao People''s Democratic Republic (Laos), remains unclear. However, this knowledge is important to our understanding of the ecology and epidemiology of B. pseudomallei and to facilitate public health interventions. Sensitive and specific methods to detect B. pseudomallei in environmental samples are therefore needed. The aim of this study was to compare molecular and culture-based methods for the detection of B. pseudomallei in soil and surface water in order to identify the optimal approach for future environmental studies in Laos. Molecular detection by quantitative real-time PCR (qPCR) was attempted after DNA extraction directly from soil or water samples or after an overnight enrichment step. The positivity rates obtained by qPCR were compared to those obtained by different culture techniques. The rate of detection from soil samples by qPCR following culture enrichment was significantly higher (84/100) than that by individual culture methods and all culture methods combined (44/100; P < 0.001). Similarly, qPCR following enrichment was the most sensitive method for filtered river water compared with the sensitivity of the individual methods and all individual methods combined. In conclusion, molecular detection following an enrichment step has proven to be a sensitive and reliable approach for B. pseudomallei detection in Lao environmental samples and is recommended as the preferred method for future surveys.     

Dynamic Succession of Groundwater Functional Microbial Communities in Response to Emulsified Vegetable Oil Amendment during Sustained In Situ U(VI) Reduction

A pilot-scale field experiment demonstrated that a one-time amendment of emulsified vegetable oil (EVO) reduced groundwater U(VI) concentrations for 1 year in a fast-flowing aquifer. However, little is known about how EVO amendment stimulates the functional gene composition, structure, and dynamics of groundwater microbial communities toward prolonged U(VI) reduction. In this study, we hypothesized that EVO amendment would shift the functional gene composition and structure of groundwater microbial communities and stimulate key functional genes/groups involved in EVO biodegradation and reduction of electron acceptors in the aquifer. To test these hypotheses, groundwater microbial communities after EVO amendment were analyzed using a comprehensive functional gene microarray. Our results showed that EVO amendment stimulated sequential shifts in the functional composition and structure of groundwater microbial communities. Particularly, the relative abundance of key functional genes/groups involved in EVO biodegradation and the reduction of NO₃⁻, Mn(IV), Fe(III), U(VI), and SO₄²⁻ significantly increased, especially during the active U(VI) reduction period. The relative abundance for some of these key functional genes/groups remained elevated over 9 months. Montel tests suggested that the dynamics in the abundance, composition, and structure of these key functional genes/groups were significantly correlated with groundwater concentrations of acetate, NO₃⁻, Mn(II), Fe(II), U(VI), and SO₄²⁻. Our results suggest that EVO amendment stimulated dynamic succession of key functional microbial communities. This study improves our understanding of the composition, structure, and function changes needed for groundwater microbial communities to sustain a long-term U(VI) reduction.     

Syk Is Recruited to Stress Granules and Promotes Their Clearance through Autophagy

Syk is a cytoplasmic kinase that serves multiple functions within the immune system to couple receptors for antigens and antigen-antibody complexes to adaptive and innate immune responses. Recent studies have identified additional roles for the kinase in cancer cells, where its expression can either promote or suppress tumor cell growth, depending on the context. Proteomic analyses of Syk-binding proteins identified several interacting partners also found to be recruited to stress granules. We show here that the treatment of cells with inducers of stress granule formation leads to the recruitment of Syk to these protein-RNA complexes. This recruitment requires the phosphorylation of Syk on tyrosine and results in the phosphorylation of proteins at or near the stress granule. Grb7 is identified as a Syk-binding protein involved in the recruitment of Syk to the stress granule. This recruitment promotes the formation of autophagosomes and the clearance of stress granules from the cell once the stress is relieved, enhancing the ability of cells to survive the stress stimulus.