The impact of temperature, pH and environmental heterogeneity on prokaryotic diversity in Yellowstone National Park thermal springs

Yellowstone National Park (YNP) is one of the largest and most diverse hydrothermal areas on Earth. Extensive culture-independent studies in YNP thermal springs have shown dramatic taxonomic and metabolic diversity in microbial communities. We conducted a survey of bacterial communities along temperature gradients in three alkaline springs with similar geochemistries at the local scale. With these data, we investigated the influence of environmental variables on bacterial community diversity and assemblages along a broad temperature range using high throughput 454 pyrosequencing. Previous studies have suggested that pH is the driver of microbial diversity in thermal springs among geographical regions or at the global scale, whereas the temperature is an important factor controlling microbial diversity in springs within a similar pH range or within an individual geographical region. Our results revealed that temperature was the most important environmental variable to shape the structure of bacterial communities at the local scale. Similar community structure was observed in the sites with similar temperatures, irrespective of the origin of the thermal spring. The results of this study expand our current knowledge of the role of temperature in controlling community structure in YNP alkaline thermal springs. In addition to temperature, pH is also a crucial factor in determining microbial assemblages in thermal springs. Although thermal springs in YNP are characterized by a broad range of pH (1-10), the pH distribution is bimodal. Most of springs are either vapor-dominated acidic springs or water-dominated neutral to slightly alkaline springs. Yet the intermediate pH (e.g., pH 4-5) habitats have been overlooked. The dearth of information on microbial communities in the intermediate pH sites has still hindered an understanding of the whole picture of microbial diversity in YNP thermal springs. We conducted the first metagenomic investigation of microbial phylogenetic and functional diversity in a pH 4 and low temperature site. We uncovered a high proportion of Chloroflexi, Bacteroidetes, Proteobacteria and Firmicutes in this site. Functional comparison indicated that the community was enriched in the COG functions related to energy production and conversion, transcription and carbohydrate transport, possibly to result from high microbial dynamics. This is the first study to examine a pH 4 and low temperature habitat, which is a key step towards an understanding of the whole picture of microbial diversity in YNP thermal springs. Local environmental heterogeneity may also be responsible for the assemblages and distribution of some microbial groups. We examined microbial diversity and community composition in ten filamentous thermal springs with similar physiochemical properties in the Shoshone area by using the barcoded amplicon pyrosequencing approach. Despite all these samples from the same type of springs, statistical analyses suggest that the relatively small variation of environmental variables such as temperature, pH and conductivity among the springs can shape microbial composition. Additionally, we conducted a metagenomic analysis on a previously uninvestigated high temperature and pH filamentous habitat. The results indicated that the site was exclusively dominated by the family Aquificaceae, and functions related to aerobic respiration and amino acid synthesis were enriched. The research presented here is an in-depth investigation of thermal springs, enhanced by high-throughput community sequencing and a combination of environmental data. The work will fill the knowledge gap for microbial communities in uninvestigated habitats and offer a basis for understanding microbial ecology in global thermal springs

Expression and Purification of Recombinant Hemoglobin in Escherichia coli

Recombinant DNA technologies have played a pivotal role in the elucidation of structure-function relationships in hemoglobin (Hb) and other globin proteins. Here we describe the development of a plasmid expression system to synthesize recombinant Hbs in Escherichia coli, and we describe a protocol for expressing Hbs with low intrinsic solubilities. Since the α- and β-chain Hbs of different species span a broad range of solubilities, experimental protocols that have been optimized for expressing recombinant human HbA may often prove unsuitable for the recombinant expression of wildtype and mutant Hbs of other species.As a test case for our expression system, we produced recombinant Hbs of the deer mouse (Peromyscus maniculatus), a species that has been the subject of research on mechanisms of Hb adaptation to hypoxia. By experimentally assessing the combined effects of induction temperature, induction time and E. coli expression strain on the solubility of recombinant deer mouse Hbs, we identified combinations of expression conditions that greatly enhanced the yield of recombinant protein and which also increased the efficiency of post-translational modifications.Our protocol should prove useful for the experimental study of recombinant Hbs in many non-human animals. One of the chief advantages of our protocol is that we can express soluble recombinant Hb without co-expressing molecular chaperones, and without the need for additional reconstitution or heme-incorporation steps. Moreover, our plasmid construct contains a combination of unique restriction sites that allows us to produce recombinant Hbs with different α- and β-chain subunit combinations by means of cassette mutagenesis

Expression and Purification of Recombinant Hemoglobin in \u3ci\u3eEscherichia coli\u3c/i\u3e

Background: Recombinant DNA technologies have played a pivotal role in the elucidation of structure-function relationships in hemoglobin (Hb) and other globin proteins. Here we describe the development of a plasmid expression system to synthesize recombinant Hbs in Escherichia coli, and we describe a protocol for expressing Hbs with low intrinsic solubilities. Since the alpha- and beta-chain Hbs of different species span a broad range of solubilities, experimental protocols that have been optimized for expressing recombinant human HbA may often prove unsuitable for the recombinant expression of wildtype and mutant Hbs of other species. Methodology/Principal Findings: As a test case for our expression system, we produced recombinant Hbs of the deer mouse (Peromyscus maniculatus), a species that has been the subject of research on mechanisms of Hb adaptation to hypoxia. By experimentally assessing the combined effects of induction temperature, induction time and E. coli expression strain on the solubility of recombinant deer mouse Hbs, we identified combinations of expression conditions that greatly enhanced the yield of recombinant protein and which also increased the efficiency of post-translational modifications. Conclusion/Significance: Our protocol should prove useful for the experimental study of recombinant Hbs in many nonhuman animals. One of the chief advantages of our protocol is that we can express soluble recombinant Hb without coexpressing molecular chaperones, and without the need for additional reconstitution or heme-incorporation steps. Moreover, our plasmid construct contains a combination of unique restriction sites that allows us to produce recombinant Hbs with different alpha- and beta-chain subunit combinations by means of cassette mutagenesis

Numerical Simulation of Acoustic Resonance Enhancement for Mean Flow Wind Energy Harvester as Well as Suppression for Pipeline

Acoustic resonance in closed side branches should be enhanced to improve the efficiency of wind energy harvesting equipment or thermo-acoustic engine. However, in gas pipeline transportation systems, this kind of acoustic resonance should be suppressed to avoid fatigue damage to the pipeline. Realizable k-ε delayed detached eddy simulations (DDES) were conducted to study the effect of different branch pipe shapes on acoustic resonance. At some flow velocities, the pressure amplitude of the simulation results is twice as large as that of the experimental results, but the simulation can accurately capture the flow velocity range where acoustic resonance occurs. The results prove the feasibility of the method of the equivalent diameter of the circular cross-section pipe and the square cross-section pipe to predict acoustic resonance. The pressure pulsation amplitude of acoustic resonance in a square cross-section pipe is significantly increased than that in a circular square cross-section pipe, indicating that the square cross-section branch configuration can be more conducive to improving the efficiency of wind energy harvesting. The influence of the angle between the branch and the main pipe on the acoustic resonance was studied for the first time, which has an obvious influence on the acoustic resonance. It is found that the design of a square wind energy harvester is better than that of a circular one; meanwhile, changing the branch angle can increase or suppress the acoustic resonance, which can improve the utilization efficiency of the acoustic resonance and provide a new method for suppressing the acoustic resonance

A Multisource Monitoring Data Coupling Analysis Method for Stress States of Oil Pipelines under Permafrost Thawing Settlement Load

Thaw settlement is one of the common geohazard threats for safe operation of buried pipelines crossing permafrost regions, as pipes need to bear additional bending stress induced by settlement load. In the presented study, a novel coupled data analysis method was proposed for stress state estimation of buried steel pipeline under thawing settlement load. Multisource data including pipe bending strain derived by inertial measurement unit, pipe longitudinal strain derived by strain gauges, and thawing displacement loads derived by soil temperature monitoring were used to estimate the pipe’s mechanical states. Based on the derived data, finite element method-based pipe soil interaction model was established to predict pipe’s actual stress distribution. A monitored pipe segment of one crude oil pipeline in northeast China operated since 2010 was adopted as a prototype for the investigation, monitoring data derived in the last ten years was employed to predict the settlement loading, and relative accurate stress results was obtained via the established pipe soil interaction model. The mean absolute error (MAE) of the predicted pipe stresses compared with the monitoring results in 2014, 2017, and 2018 are 5.77%, 12.13%, and 13.55%, respectively. Based on the analyzed stress results, it can be found that the investigated pipe was subjected to an increasing settlement load from 2010–2016, made the bending stress increased up to 149.5 MPa. While after 2016, due to the depth of frost soil in this area is no more than 3.5 m, the thawing settlement load almost remained constant after 2016. As the investigated pipe is made by X65 line pipe steel, the von-Mises stress in pipe is much smaller than the allowable one indicating pipe’s structural safety status so far. The proposed method can also be referenced in the status monitoring of buried pipeline crossing other geological hazard regions

Determination of the human antibody response to the neutralization epitopes encompassing amino acids 313-327 and 432-443 of hepatitis C virus E1E2 glycoproteins.

It has been reported that monoclonal antibodies (MAbs) to the E1E2 glycoproteins may have the potential to prevent hepatitis C virus (HCV) infection. The protective epitopes targeted by these MAbs have been mapped to the regions encompassing amino acids 313-327 and 432-443. In this study, we synthesized these two peptides and tested the reactivity of serum samples from 336 patients, 210 of which were from Chronic Hepatitis C (CHC) patients infected with diverse HCV genotypes. The remaining 126 samples were isolated from patients who had spontaneously cleared HCV infection. In the chronic HCV-infected group (CHC group), the prevalence of human serum antibodies reactive to epitopes 313-327 and 432-443 was 24.29% (51 of 210) and 4.76% (10 of 210), respectively. In the spontaneous clearance group (SC group), the prevalence was 0.79% (1 of 126) and 12.70% (16 of 126), respectively. The positive serum samples that contained antibodies reactive to epitope 313-327 neutralized HCV pseudoparticles (HCVpp) bearing the envelope glycoproteins of genotypes 1a or 1b and/or 4, but genotypes 2a, 3a, 5 and 6 were not neutralized. The neutralizing activity of these serum samples could not be inhibited by peptide 313-327. Six samples (SC17, SC38, SC86, SC92, CHC75 and CHC198) containing antibodies reactive to epitope 432-443 had cross-genotype neutralizing activities. The neutralizing activity of SC38, SC86, SC92 and CHC75 was partially inhibited by peptide 432-443. However, the neutralizing activity of sample SC17 for genotype 4HCVpp and sample CHC198 for genotype 1b HCVpp were not inhibited by the peptide. This study identifies the neutralizing ability of endogenous anti-HCV antibodies and warrants the exploration of antibodies reactive to epitope 432-443 as sources for future antibody therapies

Neutralizing capacity of serum samples reactive to epitope 313–327 to genotype 2a HCVcc.

<p>HCVcc neutralization assays were performed in the presence of diluted 313–327 epitope-reactive samples (1∶100 dilution), and the HCV NS3-postive foci were calculated after 72 hours. (A) Ten 313–327 epitope-reactive samples can neutralize JFH-1 virus stocks. (B) Ten 313–327 epitope-reactive samples can neutralize J6/JFH-1 virus stocks.</p

Neutralizing activity of serum samples reactive to epitope 313–327 cannot be blocked by corresponding exogenous peptide.

<p>Serum samples were cultured in the presence of sera at a dilution resulting in approximately 50% inhibition of HCVcc infection (dilutions varied between 1∶200 and 1∶1600). (A) The neutralizing activity of ten samples reactive to epitope 313–327to JFH-1 virus stockscannot be blocked by peptide 313–327. (B) The neutralizing activity of ten samples reactive to epitope 313–327to J6/JFH-1 virus stockscannot be blocked by peptide 313–327. Shaded bars and filled bars represent control peptide and peptide 313–327, respectively.</p