Single-molecule force spectroscopy of mycobacterial adhesin-adhesin interactions

The heparin-binding hemagglutinin (HBHA) is one of the few virulence factors identified for Mycobacterium tuberculosis. It is a surface-associated adhesin that expresses a number of different activities, including mycobacterial adhesion to nonphagocytic cells and microbial aggregation. Previous evidence indicated that HBHA is likely to form homodimers or homopolymers via a predicted coiled-coil region located within the N-terminal portion of the molecule. Here, we used single-molecule atomic-force microscopy to measure individual homophilic HBHA-HBHA interaction forces. Force curves recorded between tips and supports derivatized with HBHA proteins exposing their N-terminal domains showed a bimodal distribution of binding forces reflecting the formation of dimers or multimers. Moreover, the binding peaks showed elongation forces that were consistent with the unfolding of α-helical coiled-coil structures. By contrast, force curves obtained for proteins exposing their lysine-rich C-terminal domains showed a broader distribution of binding events, suggesting that they originate primarily from intermolecular electrostatic bridges between cationic and anionic residues rather than from specific coiled-coil interactions. Notably, similar homophilic HBHA-HBHA interactions were demonstrated on live mycobacteria producing HBHA, while they were not observed on an HBHA-deficient mutant. Together with the fact that HBHA mediates bacterial aggregation, these observations suggest that the single homophilic HBHA interactions measured here reflect the formation of multimers that may promote mycobacterial aggregation.     

Folding and turnover of human iron regulatory protein 1 depend on its subcellular localization

Aconitases are iron-sulfur hydrolyases catalysing the interconversion of citrate and isocitrate in a wide variety of organisms. Eukaryotic aconitases have been assigned additional roles, as in the case of the metazoan dual activity cytosolic aconitase-iron regulatory protein 1 (IRP1). This human protein was produced in yeast mitochondria to probe IRP1 folding in this organelle where iron-sulfur synthesis originates. The behaviour of human IRP1 was compared with that of genuine mitochondrial (yeast or human) aconitases. All enzymes were functional in yeast mitochondria, but IRP1 was found to form dense particles as detected by electron microscopy. MS analysis of purified inclusion bodies evidenced the presence of human IRP1 and alpha-ketoglutarate dehydrogenase complex component 1 (KGD1), one of the subunits of alpha-ketoglutarate dehydrogenase. KGD1 triggered formation of the mitochondrial aggregates, because the latter were absent in a KGD1(-) mutant, but it did not efficiently do so in the cytosol. Despite the iron-binding capacity of IRP1 and the readily synthesis of iron-sulfur clusters in mitochondria, the dense particles were not iron-rich, as indicated by elemental analysis of purified mitochondria. The data show that proper folding of dual activity IRP1-cytosolic aconitase is deficient in mitochondria, in contrast to genuine mitochondrial aconitases. Furthermore, efficient clearance of the aggregated IRP1-KGD1 complex does not occur in the organelle, which emphasizes the role of molecular interactions in determining the fate of IRP1. Thus, proper folding of human IRP1 strongly depends on its cellular environment, in contrast to other members of the aconitase family.     

A way forward with eco evo devo: an extended theory of resource polymorphism with postglacial fishes as model systems

A major goal of evolutionary science is to understand how biological diversity is generated and altered. Despite considerable advances, we still have limited insight into how phenotypic variation arises and is sorted by natural selection. Here we argue that an integrated view, which merges ecology, evolution and developmental biology (eco evo devo) on an equal footing, is needed to understand the multifaceted role of the environment in simultaneously determining the development of the phenotype and the nature of the selective environment, and how organisms in turn affect the environment through eco evo and eco devo feedbacks. To illustrate the usefulness of an integrated eco evo devo perspective, we connect it with the theory of resource polymorphism (i.e. the phenotypic and genetic diversification that occurs in response to variation in available resources). In so doing, we highlight fishes from recently glaciated freshwater systems as exceptionally well‐suited model systems for testing predictions of an eco evo devo framework in studies of diversification. Studies on these fishes show that intraspecific diversity can evolve rapidly, and that this process is jointly facilitated by (i) the availability of diverse environments promoting divergent natural selection; (ii) dynamic developmental processes sensitive to environmental and genetic signals; and (iii) eco evo and eco devo feedbacks influencing the selective and developmental environments of the phenotype. We highlight empirical examples and present a conceptual model for the generation of resource polymorphism – emphasizing eco evo devo, and identify current gaps in knowledge.     

Circumventing senescence is associated with stem cell properties and metformin sensitivity

Most cancers arise in old individuals, which also accumulate senescent cells. Cellular senescence can be experimentally induced by expression of oncogenes or telomere shortening during serial passage in culture. In vivo, precursor lesions of several cancer types accumulate senescent cells, which are thought to represent a barrier to malignant progression and a response to the aberrant activation of growth signaling pathways by oncogenes (oncogene toxicity). Here, we sought to define gene expression changes associated with cells that bypass senescence induced by oncogenic RAS. In the context of pancreatic ductal adenocarcinoma (PDAC), oncogenic KRAS induces benign pancreatic intraepithelial neoplasias (PanINs), which exhibit features of oncogene‐induced senescence. We found that the bypass of senescence in PanINs leads to malignant PDAC cells characterized by gene signatures of epithelial‐mesenchymal transition, stem cells, and mitochondria. Stem cell properties were similarly acquired in PanIN cells treated with LPS, and in primary fibroblasts and mammary epithelial cells that bypassed Ras‐induced senescence after reduction of ERK signaling. Intriguingly, maintenance of cells that circumvented senescence and acquired stem cell properties was blocked by metformin, an inhibitor of complex I of the electron transport chain or depletion of STAT3, a protein required for mitochondrial functions and stemness. Thus, our studies link bypass of senescence in premalignant lesions to loss of differentiation, acquisition of stemness features, and increased reliance on mitochondrial functions.     

Responses of soil microbial community and enzymes during plant-assisted biodegradation of di-(2-ethylhexyl) phthalate and pyrene

Abstract

A pot experiment was conducted to explore the plant-assisted degradation efficiency of di-(2-ethylhexyl) phthalate (DEHP) and pyrene. Three plant species: Ceylon spinach, sunflower, and leaf mustard were cultivated in co-contaminated soils under three contamination levels: control (T0), 20?mg kg^?1 (T20), and 50?mg kg^?1 (T50). The results showed that a higher DEHP and pyrene degradation efficiency was observed evidently in planted cases, increasing from 42 to 53–59% (T0), 61 to 65–76% (T20) and 52 to 68–78% (T50) for DEHP, and from 22 to 30–49% (T0), 58 to 62–72% (T20), and 54 to 57–70% (T50) for pyrene. Under T20 contamination level, soil phospholipid fatty-acid analysis depicted the increased microbial biomass in rhizosphere, especially the arbuscular mycorrhizal fungus that is effective for the degradation of organic pollutants. The study also revealed that the activities of dehydrogenase, acid phosphomonoesterase, urease, and phenol oxidase negatively correlated with pollutant concentration. In general, the removal rate of DEHP and pyrene was highest in the soil planted with leaf mustard for each contamination level considered. For soils at T20 level, sunflower and leaf mustard appeared as interesting phytoremediation plants due to the improved removal rates of organic pollutants and the soil microbial activity.     

A Function Essential to Viral Entry Underlies the Hepatitis B Virus “a” Determinant

The hepatitis B virus (HBV) particles bear a receptor-binding site located in the pre-S1 domain of the large HBV envelope protein. Using the hepatitis delta virus (HDV) as a surrogate of HBV, a second infectivity determinant was recently identified in the envelope proteins antigenic loop (AGL), and its activity was shown to depend upon cysteine residues that are essential for the structure of the HBV immunodominant “a” determinant. Here, an alanine-scanning mutagenesis approach was used to precisely map the AGL infectivity determinant to a set of conserved residues, which are predicted to cluster together with cysteines in the AGL disulfide bridges network. Several substitutions suppressed both infectivity and the “a” determinant, whereas others were infectivity deficient with only a partial impact on antigenicity. Interestingly, G145R, a substitution often arising under immune pressure selection and detrimental to the “a” determinant, had no effect on infectivity. Altogether, these findings indicate that the AGL infectivity determinant is closely related to, yet separable from, the “a” determinant. Finally, a selection of HDV entry-deficient mutations were introduced at the surface of HBV virions and shown to also abrogate infection in the HBV model. Therefore, a function can at last be assigned to the orphan “a” determinant, the first-discovered marker of HBV infection. The characterization of the AGL functions at viral entry may lead to novel approaches in the development of antivirals against HBV.Hepatitis B virus (HBV) causes acute and chronic infections in humans; such infections are often associated with severe liver diseases, including cirrhosis and hepatocellular carcinoma (10). To date, it is estimated that approximately 350 millions individuals worldwide suffer from chronic infection despite the availability of an effective vaccine for more than 25 years. Remarkably, the development of a vaccine soon after the HBV discovery was, at least in part, the consequence of a very peculiar feature that is unique to members of the Hepadnaviridae family: viral envelope proteins are produced in quantities far exceeding the amounts required for assembly of HBV virions (6) and, owing to their capacity for autoassembly, the vast majority are secreted as subviral particles. Besides the practical consequences in the original vaccine development, in nature, the phenomenon of HBV envelope protein overexpression has provided a helper function to the hepatitis delta virus (HDV) (29). The HBV envelope proteins assist in packaging the HDV ribonucleoprotein (RNP) in case of HBV-HDV coinfection, thereby ensuring spreading of the satellite HDV. As a result, the coats of HBV and HDV particles are similar, consisting of cell-derived lipids and the HBV envelope proteins—large, middle, and small—bearing the HBV surface antigen (HBsAg) and referred to as L-HBsAg, M-HBsAg, and S-HBsAg, respectively (4, 14).The HBsAg includes an immunodominant determinant common to all HBV strains, referred to as “a,” and several mutually exclusive subtype-specific determinants referred to as “d”/“y” and “w”/“r” (21). The “a” determinant is defined by a specific conformation of the antigenic loop (AGL) polypeptide present at the surface of subviral, HBV, or HDV particles. The AGL itself resides between the transmembrane domain II (residues 80 to 100) and the hydrophobic carboxyl terminus (residues 165 to 226) of the envelope proteins S domain (see Fig. ​Fig.1).1). It is the “a” determinant that elicits the most effective neutralizing antibody response upon vaccination or infection (32). Surprisingly, a function in the HBV life cycle had never been assigned to the “a” determinant, the first identified HBV marker, until the recent demonstration of its involvement in HDV entry (2, 15). More precisely, it was shown that the AGL cysteine residues were critical for both the structure of the “a” determinant and HDV infectivity (2).Open in a separate windowFIG. 1.Schematic representation of the HBV envelope protein AGL. (A) The topology of the L-, M-, and S-HBsAg proteins (L, M, and S, respectively) is represented. The determinants of viral entry, pre-S1 and AGL, are indicated in red. The M-HBsAg protein, represented in gray, is dispensable for infectivity. Open boxes represent transmembrane regions in the S domain. (B) Alignment of the AGL amino acids sequences (positions 101 to 172 in the S domain) of HBV (genotype D, ayw3 phenotype), WMHBV, and WHV. The GenBank sequence numbers of the isolates are as follows: {"type":"entrez-nucleotide","attrs":{"text":"J02203","term_id":"329640","term_text":"J02203"}}J02203 (HBV), {"type":"entrez-nucleotide","attrs":{"text":"AY226578","term_id":"29501369","term_text":"AY226578"}}AY226578 (WMHBV), and {"type":"entrez-nucleotide","attrs":{"text":"NC_004107","term_id":"22256030","term_text":"NC_004107"}}NC_004107 (WHV). HBV amino acid residues important for infectivity (the present study) are indicated in blue. A hyphen denotes amino acid identity with the HBV sequence.It is now well established that both HBV and HDV entry rely on the pre-S1 domain of L-HBsAg as the primary infectivity determinant that is likely to promote attachment to a specific receptor at the surface of human hepatocytes (11). The AGL determinant could thus fulfill complementary functions for attachment, uptake, or particle disassembly after entry (2, 15).In the present study, the AGL infectivity determinant was mapped and confirmed to be closely related to the “a” determinant. Moreover, its essential function at viral entry was demonstrated in the HBV model.     

Prolonged Grief Disorder: Psychometric Validation of Criteria Proposed for DSM-V and ICD-11

Background

Bereavement is a universal experience, and its association with excess morbidity and mortality is well established. Nevertheless, grief becomes a serious health concern for a relative few. For such individuals, intense grief persists, is distressing and disabling, and may meet criteria as a distinct mental disorder. At present, grief is not recognized as a mental disorder in the DSM-IV or ICD-10. The goal of this study was to determine the psychometric validity of criteria for prolonged grief disorder (PGD) to enhance the detection and potential treatment of bereaved individuals at heightened risk of persistent distress and dysfunction.

Methods and Findings

A total of 291 bereaved respondents were interviewed three times, grouped as 0–6, 6–12, and 12–24 mo post-loss. Item response theory (IRT) analyses derived the most informative, unbiased PGD symptoms. Combinatoric analyses identified the most sensitive and specific PGD algorithm that was then tested to evaluate its psychometric validity. Criteria require reactions to a significant loss that involve the experience of yearning (e.g., physical or emotional suffering as a result of the desired, but unfulfilled, reunion with the deceased) and at least five of the following nine symptoms experienced at least daily or to a disabling degree: feeling emotionally numb, stunned, or that life is meaningless; experiencing mistrust; bitterness over the loss; difficulty accepting the loss; identity confusion; avoidance of the reality of the loss; or difficulty moving on with life. Symptoms must be present at sufficiently high levels at least six mo from the death and be associated with functional impairment.

Conclusions

The criteria set for PGD appear able to identify bereaved persons at heightened risk for enduring distress and dysfunction. The results support the psychometric validity of the criteria for PGD that we propose for inclusion in DSM-V and ICD-11. Please see later in the article for Editors'' Summary     

Molecular Evolution of the Two-Component System BvgAS Involved in Virulence Regulation in Bordetella

The whooping cough agent Bordetella pertussis is closely related to Bordetella bronchiseptica, which is responsible for chronic respiratory infections in various mammals and is occasionally found in humans, and to Bordetella parapertussis, one lineage of which causes mild whooping cough in humans and the other ovine respiratory infections. All three species produce similar sets of virulence factors that are co-regulated by the two-component system BvgAS. We characterized the molecular diversity of BvgAS in Bordetella by sequencing the two genes from a large number of diverse isolates. The response regulator BvgA is virtually invariant, indicating strong functional constraints. In contrast, the multi-domain sensor kinase BvgS has evolved into two different types. The pertussis type is found in B. pertussis and in a lineage of essentially human-associated B. bronchiseptica, while the bronchiseptica type is associated with the majority of B. bronchiseptica and both ovine and human B. parapertussis. BvgS is monomorphic in B. pertussis, suggesting optimal adaptation or a recent population bottleneck. The degree of diversity of the bronchiseptica type BvgS is markedly different between domains, indicating distinct evolutionary pressures. Thus, absolute conservation of the putative solute-binding cavities of the two periplasmic Venus Fly Trap (VFT) domains suggests that common signals are perceived in all three species, while the external surfaces of these domains vary more extensively. Co-evolution of the surfaces of the two VFT domains in each type and domain swapping experiments indicate that signal transduction in the periplasmic region may be type-specific. The two distinct evolutionary solutions for BvgS confirm that B. pertussis has emerged from a specific B. bronchiseptica lineage. The invariant regions of BvgS point to essential parts for its molecular mechanism, while the variable regions may indicate adaptations to different lifestyles. The repertoire of BvgS sequences will pave the way for functional analyses of this prototypic system.     

Association and Mutation Analyses of 16p11.2 Autism Candidate Genes

Background

Autism is a complex childhood neurodevelopmental disorder with a strong genetic basis. Microdeletion or duplication of a ∼500–700-kb genomic rearrangement on 16p11.2 that contains 24 genes represents the second most frequent chromosomal disorder associated with autism. The role of common and rare 16p11.2 sequence variants in autism etiology is unknown.

Methodology/Principal Findings

To identify common 16p11.2 variants with a potential role in autism, we performed association studies using existing data generated from three microarray platforms: Affymetrix 5.0 (777 families), Illumina 550 K (943 families), and Affymetrix 500 K (60 families). No common variants were identified that were significantly associated with autism. To look for rare variants, we performed resequencing of coding and promoter regions for eight candidate genes selected based on their known expression patterns and functions. In total, we identified 26 novel variants in autism: 13 exonic (nine non-synonymous, three synonymous, and one untranslated region) and 13 promoter variants. We found a significant association between autism and a coding variant in the seizure-related gene SEZ6L2 (12/1106 autism vs. 3/1161 controls; p = 0.018). Sez6l2 expression in mouse embryos was restricted to the spinal cord and brain. SEZ6L2 expression in human fetal brain was highest in post-mitotic cortical layers, hippocampus, amygdala, and thalamus. Association analysis of SEZ6L2 in an independent sample set failed to replicate our initial findings.

Conclusions/Significance

We have identified sequence variation in at least one candidate gene in 16p11.2 that may represent a novel genetic risk factor for autism. However, further studies are required to substantiate these preliminary findings.     

The Potential Distance of Highly Pathogenic Avian Influenza Virus Dispersal by Mallard, Common Teal and Eurasian Pochard

Waterbirds represent the major natural reservoir for low pathogenic (LP) avian influenza viruses (AIV). Among the wide diversity of subtypes that have been described, two of them (H5 and H7) may become highly pathogenic (HP) after their introduction into domestic bird populations and cause severe outbreaks, as is the case for HP H5N1 in South-Eastern Asia. Recent experimental studies demonstrated that HP H5N1 AIV infection in ducks does not necessarily have significant pathological effects. These results suggest that wild migratory ducks may asymptomatically carry HP AIV and potentially spread viruses over large geographical distances. In this study, we investigated the potential spreading distance of HP AIV by common teal (Anas crecca), mallard (A. platyrhynchos), and Eurasian pochard (Aythya ferina). Based on capture-mark-recapture method, we characterized their wintering movements from a western Mediterranean wetland (Camargue, South of France) and identified the potential distance and direction of virus dispersal. Such data may be crucial in determining higher-risk areas in the case of HP AIV infection detection in this major wintering quarter, and may serve as a valuable reference for virus outbreaks elsewhere.