Proteomic and targeted qPCR analyses of subsurface microbial communities for presence of methane monooxygenase

The Test Area North (TAN) site at the Idaho National Laboratory near Idaho Falls, ID, USA, sits over a trichloroethylene (TCE) contaminant plume in the Snake River Plain fractured basalt aquifer. Past observations have provided evidence that TCE at TAN is being transformed by biological natural attenuation that may be primarily due to co-metabolism in aerobic portions of the plume by methanotrophs. TCE co-metabolism by methanotrophs is the result of the broad substrate specificity of microbial methane monooxygenase which permits non-specific oxidation of TCE in addition to the primary substrate, methane. Arrays of experimental approaches have been utilized to understand the biogeochemical processes driving intrinsic TCE co-metabolism at TAN. In this study, aerobic methanotrophs were enumerated by qPCR using primers targeting conserved regions of the genes pmoA and mmoX encoding subunits of the particulate MMO (pMMO) and soluble MMO (sMMO) enzymes, respectively, as well as the gene mxa encoding the downstream enzyme methanol dehydrogenase. Identification of proteins in planktonic and biofilm samples from TAN was determined using reverse phase ultra-performance liquid chromatography (UPLC) coupled with a quadrupole-time-of-flight (QToF) mass spectrometer to separate and sequence peptides from trypsin digests of the protein extracts. Detection of MMO in unenriched water samples from TAN provides direct evidence of intrinsic methane oxidation and TCE co-metabolic potential of the indigenous microbial population. Mass spectrometry is also well suited for distinguishing which form of MMO is expressed in situ either soluble or particulate. Using this method, pMMO proteins were found to be abundant in samples collected from wells within and adjacent to the TCE plume at TAN.     

The RNA binding domain of influenza A virus NS1 protein affects secretion of tumor necrosis factor alpha, interleukin-6, and interferon in primary murine tracheal epithelial cells

Primary differentiated respiratory epithelial cell cultures closely model the in vivo environment and allow for studies of innate immune responses generated specifically by epithelial cells, the primary cell type infected by human influenza A virus strains. We used primary murine tracheal epithelial cell (mTEC) cultures to investigate antiviral and cytokine responses to influenza A virus infection, focusing on the contribution of the RNA binding domain of the NS1 protein. rWSN NS1 R38A replication is attenuated in mTEC cultures; however, viral antigen is detected predominantly in ciliated cells, similar to wild-type virus. NS1 and NS1 R38A proteins display a primarily cytoplasmic localization in infected mTEC cultures. Increased production of tumor necrosis factor alpha, interleukin-6, and beta interferon is observed during rWSN NS1 R38A infection, and cytokines are secreted in a directional manner. Cytokine pretreatment of mTEC cultures and Vero cells suggest that rWSN NS1 R38A is more sensitive to the presence of antiviral/inflammatory cytokines than wild-type virus. Our results demonstrate that the RNA binding domain is a critical regulator of both cytokine production and cytokine sensitivity during influenza A virus infection of primary tracheal epithelial cells.     

Isolation and characterization of Cr(VI) reducing Cellulomonas spp. from subsurface soils: implications for long-term chromate reduction

Microbial enrichments from Cr(VI) contaminated and uncontaminated US Department of Energy Hanford Site sediments produced Cr(VI) reducing consortia when grown in the presence of Cr(VI) with acetate, D-xylose or glycerol as a carbon and energy source. Eight of the nine isolates from the consortia were Gram positive and four of these were identified by 16S rRNA sequence homology and membrane fatty acid composition as belonging to the genus Cellulomonas. Two strains, ES6 and WS01, were further examined for their ability to reduce Cr(VI) under growth and non-growth conditions. During fermentative growth on D-xylose, ES6 and WS01 decreased aqueous Cr(VI) concentrations from 0.04 mM Cr(VI) to below the detection limit (0.002 mM Cr(VI)) in less than three days and retained their ability to reduce Cr(VI) even after four months of incubation. Washed ES6 and WS01 cells also reduced Cr(VI) under non-growth conditions for over four months, both with and without the presence of an exogenous electron donor. K-edge XANES spectroscopy confirmed the reduction of Cr(VI) to Cr(III). The ability to reduce Cr(VI) after growth had stopped and in the absence of an external electron donor, suggests that stimulation of these types of organisms may lead to effective long-term, in situ passive reactive barriers for Cr(VI) removal. Our results indicate that Cr(VI) reduction by indigenous Cellulomonas spp. may be a potential method of in situ bioremediation of Cr(VI) contaminated sediment and groundwater.     

Cell-free complements in vivo expression of the E. coli membrane proteome

Reconstituted cell-free (CF) protein expression systems hold the promise of overcoming the traditional barriers associated with in vivo systems. This is particularly true for membrane proteins, which are often cytotoxic and due to the nature of the membrane, difficult to work with. To evaluate the potential of cell-free expression, we cloned 120 membrane proteins from E. coli and compared their expression profiles in both an E. coli in vivo system and an E. coli-derived cell-free system. Our results indicate CF is a more robust system and we were able to express 63% of the targets in CF, compared to 44% in vivo. To benchmark the quality of CF produced protein, five target membrane proteins were purified and their homogeneity assayed by gel filtration chromatography. Finally, to demonstrate the ease of amino acid labeling with CF, a novel membrane protein was substituted with selenomethionine, purified, and shown to have 100% incorporation of the unnatural amino acid. We conclude that CF is a novel, robust expression system capable of expressing more proteins than an in vivo system and suitable for production of membrane proteins at the milligram level.     

Statistical Cluster Analysis of the British Thoracic Society Severe Refractory Asthma Registry: Clinical Outcomes and Phenotype Stability

Background

Severe refractory asthma is a heterogeneous disease. We sought to determine statistical clusters from the British Thoracic Society Severe refractory Asthma Registry and to examine cluster-specific outcomes and stability.

Methods

Factor analysis and statistical cluster modelling was undertaken to determine the number of clusters and their membership (N = 349). Cluster-specific outcomes were assessed after a median follow-up of 3 years. A classifier was programmed to determine cluster stability and was validated in an independent cohort of new patients recruited to the registry (n = 245).

Findings

Five clusters were identified. Cluster 1 (34%) were atopic with early onset disease, cluster 2 (21%) were obese with late onset disease, cluster 3 (15%) had the least severe disease, cluster 4 (15%) were the eosinophilic with late onset disease and cluster 5 (15%) had significant fixed airflow obstruction. At follow-up, the proportion of subjects treated with oral corticosteroids increased in all groups with an increase in body mass index. Exacerbation frequency decreased significantly in clusters 1, 2 and 4 and was associated with a significant fall in the peripheral blood eosinophil count in clusters 2 and 4. Stability of cluster membership at follow-up was 52% for the whole group with stability being best in cluster 2 (71%) and worst in cluster 4 (25%). In an independent validation cohort, the classifier identified the same 5 clusters with similar patient distribution and characteristics.

Interpretation

Statistical cluster analysis can identify distinct phenotypes with specific outcomes. Cluster membership can be determined using a classifier, but when treatment is optimised, cluster stability is poor.     

Acute inhibition of superoxide formation and Rac1 activation by nitric oxide and iloprost in human vascular smooth muscle cells in response to the thromboxane A2 analogue, U46619

BACKGROUND: The over-production of superoxide (O(2)(-)) derived from NADPH oxidase (NOX) plays a central role in cardiovascular diseases. By contrast, nitric oxide (NO) and prostacyclin (PGI(2)) are vasculoprotective. The effect of the NO donor, NONOate and iloprost on O(2)(-) formation, p47(phox) and Rac(1) activation in human vascular smooth muscle cells (hVSMCs) was investigated. METHODS: hVSMCs were incubated with 10nM thromboxane A(2) analogue, U46619 for 16h, and then with apocynin (a NOX inhibitor), NONOate or iloprost for 1h and O(2)(-) measured spectrophometrically. The role of cyclic AMP and cyclic GMP was examined by co-incubation of drugs with protein kinase (PK) A and G inhibitors listed above. Rac(1) was studied using pull-down assays. RESULTS: NONOate and iloprost inhibited O(2)(-) formation, acutely, effects blocked by inhibition of PKG and PKA, respectively. Rac(1) and p47(phox) activation and translocation to the plasma membrane was completely inhibited by NONOate and iloprost, effects again reversed by co-incubation with PKG or PKA inhibitors. CONCLUSIONS: NO and PGI(2) block the acute activity of NOX in hVSMCs via the cGMP-PKG axis (for NO) and by the cAMP-PKA axis (for iloprost) through inhibition of Rac(1) and p47(phox) translocation. These findings have implications in the pathophysiology and treatment of CVD.     

云南切梢小蠹对初侵害云南松挥发物的电生理和行为反应

云南切梢小蠹Tomicus yunnanensis(Kirkendall and Faccoli)是一种蛀害云南松Pinus yunnanensis的本土害虫.为深入了解其寄主选择机制,用顶空动态法和浸提法分别提取了初侵染云南松针叶和松脂的挥发性化合物,并用气相色谱-质谱联用技术(GC-MS)、气相色谱-昆虫触角电位测量系统(GC-EAD)、生测法鉴定和筛选了对云南切梢小蠹具有活性功能的成分.结果表明:云南松针叶和松脂中共有18种化合物,均为萜烯类物质,但两者化学成分的构成有显著差异.针叶中单萜类占99.98％,主要是 α-蒎烯(80.82％)、β-蒎烯(8.78％)、D-柠檬烯(4.77％)、莰烯(2.86％)和β-月桂烯(1.42％),而松脂以单萜类和双萜类为主,前者以α-蒎烯(21.38％)、3-蒈烯(21.42％)和异松油烯(2.78％)为主要成分,后者仅有长叶松酸(51.13％)一种.云南切梢小蠹对α-蒎烯、β-蒎烯、3-蒈烯、γ-萜品烯和4-烯丙基苯甲醚有触角电位反应,其中α-蒎烯、3-蒈烯和γ-萜品烯具引诱作用,4-烯丙基苯甲醚和β-蒎烯则为驱避功能.研究可为开发植物源引诱剂或与性信息素结合进行种群监测和诱杀提供科学依据.     

Mapping global cropland and field size

A new 1 km global IIASA‐IFPRI cropland percentage map for the baseline year 2005 has been developed which integrates a number of individual cropland maps at global to regional to national scales. The individual map products include existing global land cover maps such as GlobCover 2005 and MODIS v.5, regional maps such as AFRICOVER and national maps from mapping agencies and other organizations. The different products are ranked at the national level using crowdsourced data from Geo‐Wiki to create a map that reflects the likelihood of cropland. Calibration with national and subnational crop statistics was then undertaken to distribute the cropland within each country and subnational unit. The new IIASA‐IFPRI cropland product has been validated using very high‐resolution satellite imagery via Geo‐Wiki and has an overall accuracy of 82.4%. It has also been compared with the EarthStat cropland product and shows a lower root mean square error on an independent data set collected from Geo‐Wiki. The first ever global field size map was produced at the same resolution as the IIASA‐IFPRI cropland map based on interpolation of field size data collected via a Geo‐Wiki crowdsourcing campaign. A validation exercise of the global field size map revealed satisfactory agreement with control data, particularly given the relatively modest size of the field size data set used to create the map. Both are critical inputs to global agricultural monitoring in the frame of GEOGLAM and will serve the global land modelling and integrated assessment community, in particular for improving land use models that require baseline cropland information. These products are freely available for downloading from the http://cropland.geo-wiki.org website.     

The specific and sensitive detection of bacterial pathogens within 4 h using bacteriophage amplification

This paper describes a novel approach, termed the 'phage amplification assay', for the rapid detection and identification of specific bacteria. The technique is based on the phage lytic cycle with plaque formation as the assay end-point. It is highly sensitive, quantitative and gives results typically within 4 h. The assay comprises four main stages : (1) phage infection of target bacterium ; (2) destruction of exogenous phage ; (3) amplification of phage within infected host and (4) plaque formation from infected host with the aid of helper bacteria. A key component of this assay is a potent virucidal agent derived from natural plant extracts, pomegranate rind extract (PRE). In combination with ferrous sulphate PRE can bring about an 11 log-cycle reduction in phage titre within 3 min. This is achieved without any injury to the infected target bacteria. Subsequently, any resulting plaques are derived only from infected target organisms. Data are presented for a range of bacterial hosts including Pseudomonas aeruginosa, Salmonella typhimurium and Staphylococcus aureus. The detection limit for Ps. aeruginosa was 40 bacteria ml⁻¹ in a time of 4 h and 600 bacteria m⁻¹ for Salm. typhimurium. Application of the principles of this technology to other bacterial genera is discussed.