Bioaerosol biomonitoring: Sampling optimization for molecular microbial ecology

Bioaerosols (or biogenic aerosols) have largely been overlooked by molecular ecologists. However, this is rapidly changing as bioaerosols play key roles in public health, environmental chemistry and the dispersal ecology of microbes. Due to the low environmental concentrations of bioaerosols, collecting sufficient biomass for molecular methods is challenging. Currently, no standardized methods for bioaerosol collection for molecular ecology research exist. Each study requires a process of optimization, which greatly slows the advance of bioaerosol science. Here, we evaluated air filtration and liquid impingement for bioaerosol sampling across a range of environmental conditions. We also investigated the effect of sampling matrices, sample concentration strategies and sampling duration on DNA yield. Air filtration using polycarbonate filters gave the highest recovery, but due to the faster sampling rates possible with impingement, we recommend this method for fine ‐scale temporal/spatial ecological studies. To prevent bias for the recovery of Gram‐positive bacteria, we found that the matrix for impingement should be phosphate‐buffered saline. The optimal method for bioaerosol concentration from the liquid matrix was centrifugation. However, we also present a method using syringe filters for rapid in‐field recovery of bioaerosols from impingement samples, without compromising microbial diversity for high ‐throughput sequencing approaches. Finally, we provide a resource that enables molecular ecologists to select the most appropriate sampling strategy for their specific research question.     

The cold-inducible RNA-binding protein migrates from the nucleus to cytoplasmic stress granules by a methylation-dependent mechanism and acts as a translational repressor

The cold-inducible RNA-binding protein (CIRP) is a nuclear 18-kDa protein consisting of an amino-terminal RNA Recognition Motif (RRM) and a carboxyl-terminal domain containing several RGG motifs. First characterized for its overexpression upon cold shock, CIRP is also induced by stresses such as UV irradiation and hypoxia. Here, we investigated the expression as well as the subcellular localization of CIRP in response to other stress conditions. We demonstrate that oxidative stress leads to the migration of CIRP to stress granules (SGs) without alteration of expression. Stress granules are dynamic cytoplasmic foci at which stalled translation initiation complexes accumulate in cells subjected to environmental stress. Relocalization of CIRP into SGs also occurs upon other cytoplasmic stresses (osmotic pressure or heat shock) as well as in response to stresses of the endoplasmic reticulum. CIRP migration into SGs is independent from TIA-1 which has been previously reported to be a general mediator of SG formation, thereby suggesting the existence of multiple pathways leading to SG formation. Moreover, deletion mutants revealed that both RGG and RRM domains can independently promote CIRP migration into SGs. However, the methylation of arginine residues in the RGG domain is necessary for CIRP to exit the nucleus to be further recruited into SGs. By RNA-tethering experiments, we also show that CIRP down-regulates mRNA translation and that this activity is carried by the carboxyl-terminal RG-enriched domain. Altogether, our findings further reveal the diversity of mechanisms by which CIRP is regulated by environmental stresses and provide new insights into CIRP cytoplasmic function.     

Assembly of the <Emphasis Type="Italic">Tc1</Emphasis> and <Emphasis Type="Italic">mariner</Emphasis> transposition initiation complexes depends on the origins of their transposase DNA binding domains

In this review, we focus on the assembly of DNA/protein complexes that trigger transposition in eukaryotic members of the IS630–Tc1–mariner (ITm) super-family, the Tc1- and mariner-like elements (TLEs and MLEs). Elements belonging to this super-family encode transposases with DNA binding domains of different origins, and recent data indicate that the chimerization of functional domains has been an important evolutionary aspect in the generation of new transposons within the ITm super-family. These data also reveal that the inverted terminal repeats (ITRs) at the ends of transposons contain three kinds of motif within their sequences. The first two are well known and correspond to the cleavage site on the outer ITR extremities, and the transposase DNA binding site. The organization of ITRs and of the transposase DNA binding domains implies that differing pathways are used by MLEs and TLEs to regulate transposition initiation. These differences imply that the ways ITRs are recognized also differ leading to the formation of differently organized synaptic complexes. The third kind of motif is the transposition enhancers, which have been found in almost all the functional MLEs and TLEs analyzed to date. Finally, in vitro and in vivo assays of various elements all suggest that the transposition initiation complex is not formed randomly, but involves a mechanism of oriented transposon scanning. Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at . An erratum to this article can be found at     

Reversible hexacoordination of alpha-hemoglobin-stabilizing protein (AHSP)/alpha-hemoglobin Versus pressure. Evidence for protection of the alpha-chains by their chaperone

Using high hydrostatic pressure or hydrogen peroxide as perturbing agents, we demonstrate a protective effect of the chaperone AHSP for the alpha-chains of Hb. High pressure induces an irreversible aggregation of the ferrous deoxy alpha-chains, whereas the AHSP/alpha-Hb complex shows reversible hexacoordination of the alpha-Hb without protein aggregation. Upon pressure release, the relaxation kinetics of the transition from the hexacoordinated to pentacoordinated form of alpha-Hb in the presence of AHSP exhibit a biphasic shape. High pressure did not induce dissociation of alpha-Hb from its chaperone, as evidenced by the ligand binding kinetics that show a unique rate for the AHSP/alpha-Hb complex. For both free alpha-Hb and the AHSP/alpha-Hb complex, the bimolecular rate constant of CO binding (k(CO)(on)) versus pressure exhibits a bell shape, attributed to the transition of the rate-determining step from the chemical barrier to the migration of CO within the protein matrix. These results reveal a plasticity of the alpha-Hb active site in the presence of the chaperone and indicate that the AHSP was still active at 300 MPa. The ferric state of the AHSP/alpha-Hb complex shows hexacoordination even at atmospheric pressures, indicating a His-Fe-His binding scheme as previously observed in neuroglobin and cytoglobin. The reaction with hydrogen peroxide of ferric alpha-Hb within the complex also demonstrates a protection against aggregation.     

Internalization of weight bias: Implications for binge eating and emotional well-being

Objective: This study examined the relationship between internalization of negative weight‐based stereotypes and indices of eating behaviors and emotional well‐being in a sample of overweight and obese women. Research Method and Procedures: The sample was comprised of 1013 women who belonged to a national, non‐profit weight loss organization. Participants completed an on‐line battery of self‐report questionnaires measuring frequency of weight stigmatization and coping responses to deal with bias and symptoms of depression and self‐esteem, attitudes about weight and obesity, and binge eating behaviors. In addition, participants were asked to list the most common weight‐based stereotypes and whether they believed them to be true or false. Results: Participants who believed that weight‐based stereotypes were true reported more frequent binge eating and refusal to diet in response to stigma experiences compared with those who reported stereotypes to be false. The degree to which participants believed stereotypes to be true or false was not related to types or amount of stigma experiences reported, self‐esteem, depression, or attitudes toward obese persons. In addition, engaging in weight loss strategies as a response to bias was not predicted by stereotype beliefs or by actual stigma experiences, regardless of the amount or types of stigma reported. Discussion: These findings suggest that obese individuals who internalize negative weight‐based stereotypes may be particularly vulnerable to the negative impact of stigma on eating behaviors and also challenge the notion that stigma may motivate obese individuals to engage in efforts to lose weight. This study highlights a new area of research that warrants attention to better understand weight stigma and its potential consequences for health.     

Speech and non-speech audio-visual illusions: a developmental study

It is well known that simultaneous presentation of incongruent audio and visual stimuli can lead to illusory percepts. Recent data suggest that distinct processes underlie non-specific intersensory speech as opposed to non-speech perception. However, the development of both speech and non-speech intersensory perception across childhood and adolescence remains poorly defined. Thirty-eight observers aged 5 to 19 were tested on the McGurk effect (an audio-visual illusion involving speech), the Illusory Flash effect and the Fusion effect (two audio-visual illusions not involving speech) to investigate the development of audio-visual interactions and contrast speech vs. non-speech developmental patterns. Whereas the strength of audio-visual speech illusions varied as a direct function of maturational level, performance on non-speech illusory tasks appeared to be homogeneous across all ages. These data support the existence of independent maturational processes underlying speech and non-speech audio-visual illusory effects.     

Mascot file parsing and quantification (MFPaQ), a new software to parse, validate, and quantify proteomics data generated by ICAT and SILAC mass spectrometric analyses: application to the proteomics study of membrane proteins from primary human endothelial cells

Proteomics strategies based on nanoflow (nano-) LC-MS/MS allow the identification of hundreds to thousands of proteins in complex mixtures. When combined with protein isotopic labeling, quantitative comparison of the proteome from different samples can be achieved using these approaches. However, bioinformatics analysis of the data remains a bottleneck in large scale quantitative proteomics studies. Here we present a new software named Mascot File Parsing and Quantification (MFPaQ) that easily processes the results of the Mascot search engine and performs protein quantification in the case of isotopic labeling experiments using either the ICAT or SILAC (stable isotope labeling with amino acids in cell culture) method. This new tool provides a convenient interface to retrieve Mascot protein lists; sort them according to Mascot scoring or to user-defined criteria based on the number, the score, and the rank of identified peptides; and to validate the results. Moreover the software extracts quantitative data from raw files obtained by nano-LC-MS/MS, calculates peptide ratios, and generates a non-redundant list of proteins identified in a multisearch experiment with their calculated averaged and normalized ratio. Here we apply this software to the proteomics analysis of membrane proteins from primary human endothelial cells (ECs), a cell type involved in many physiological and pathological processes including chronic inflammatory diseases such as rheumatoid arthritis. We analyzed the EC membrane proteome and set up methods for quantitative analysis of this proteome by ICAT labeling. EC microsomal proteins were fractionated and analyzed by nano-LC-MS/MS, and database searches were performed with Mascot. Data validation and clustering of proteins were performed with MFPaQ, which allowed identification of more than 600 unique proteins. The software was also successfully used in a quantitative differential proteomics analysis of the EC membrane proteome after stimulation with a combination of proinflammatory mediators (tumor necrosis factor-alpha, interferon-gamma, and lymphotoxin alpha/beta) that resulted in the identification of a full spectrum of EC membrane proteins regulated by inflammation.     

Characterization of a novel method for the production of single-span membrane proteins in Escherichia coli

The large-scale production and isolation of recombinant protein is a central element of the biotechnology industry and many of the products have proved extremely beneficial for therapeutic medicine. Escherichia coli is the microorganism of choice for the expression of heterologous proteins for therapeutic application, and a range of high-value proteins have been targeted to the periplasm using the well characterized Sec protein export pathway. More recently, the ability of the second mainstream protein export system, the twin-arginine translocase, to transport fully-folded proteins into the periplasm of not only E. coli, but also other Gram-negative bacteria, has captured the interest of the biotechnology industry. In this study, we have used a novel approach to block the export of a heterologous Tat substrate in the later stages of the export process, and thereby generate a single-span membrane protein with the soluble domain positioned on the periplasmic side of the inner membrane. Biochemical and immuno-electron microscopy approaches were used to investigate the export of human growth hormone by the twin-arginine translocase, and the generation of a single-span membrane-embedded variant. This is the first time that a bonafide biotechnologically relevant protein has been exported by this machinery and visualized directly in this manner. The data presented here demonstrate a novel method for the production of single-span membrane proteins in E. coli.