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Mosquitoes transmit medically important human pathogens, including viruses like dengue virus and parasites such as Plasmodium spp., the causative agent of malaria. Mosquito microbiomes are critically important for the ability of mosquitoes to transmit disease-causing agents. However, while large collections of bacterial isolates and genomic data exist for vertebrate microbiomes, the vast majority of work in mosquitoes to date is based on 16S rRNA gene amplicon data that provides limited taxonomic resolution and no functional information. To address this gap and facilitate future studies using experimental microbiome manipulations, we generated a bacterial Mosquito-Associated Isolate Collection (MosAIC) consisting of 392 bacterial isolates with extensive metadata and high-quality draft genome assemblies that are publicly available, both isolates and sequence data, for use by the scientific community. MosAIC encompasses 142 species spanning 29 bacterial families, with members of the Enterobacteriaceae comprising 40% of the collection. Phylogenomic analysis of 3 genera, Enterobacter, Serratia, and Elizabethkingia, reveal lineages of mosquito-associated bacteria isolated from different mosquito species in multiple laboratories. Investigation into species' pangenomes further reveals clusters of genes specific to these lineages, which are of interest for future work to test for functions connected to mosquito host association. Altogether, we describe the generation of a physical collection of mosquito-associated bacterial isolates, their genomic data, and analyses of selected groups in context of genome data from closely related isolates, providing a unique, highly valuable resource for research on bacterial colonisation and adaptation within mosquito hosts. Future efforts will expand the collection to include broader geographic and host species representation, especially from individuals collected from field populations, as well as other mosquito-associated microbes, including fungi, archaea, and protozoa., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Foo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Bacteria shape interactions between hosts and fungal pathogens. In some cases, bacteria associated with fungi are essential for pathogen virulence. In other systems, host-associated microbiomes confer resistance against fungal pathogens. We studied an aphid-specific entomopathogenic fungus called Pandora neoaphidis in the context of both host and pathogen microbiomes. Aphids host several species of heritable bacteria, some of which confer resistance against Pandora . We first found that spores that emerged from aphids that harbored protective bacteria were less virulent against subsequent hosts and did not grow on plate media. We then used 16S amplicon sequencing to study the bacterial microbiome of fungal mycelia and spores during plate culturing and host infection. We found that the bacterial community is remarkably stable in culture despite dramatic changes in pathogen virulence. Last, we used an experimentally transformed symbiont of aphids to show that Pandora can acquire host-associated bacteria during infection. Our results uncover new roles for bacteria in the dynamics of aphid-pathogen interactions and illustrate the importance of the broader microbiological context in studies of fungal pathogenesis., Importance: Entomopathogenic fungi play important roles in the population dynamics of many insect species. Understanding the factors shaping entomopathogen virulence is critical for agricultural management and for the use of fungi in pest biocontrol. We show that heritable bacteria in aphids, which confer protection to their hosts against fungal entomopathogens, influence virulence against subsequent hosts. Aphids reproduce asexually and are typically surrounded by genetically identical offspring, and thus these effects likely shape the dynamics of fungal disease in aphid populations. Furthermore, fungal entomopathogens are known to rapidly lose virulence in lab culture, complicating their laboratory use. We show that this phenomenon is not driven by changes in the associated bacterial microbiome. These results contribute to our broader understanding of the aphid model system and shed light on the biology of the Entomophthorales-an important but understudied group of fungi., Competing Interests: The authors declare no conflict of interest.

Host genetic variation plays an important role in the structure and function of heritable microbial communities. Recent studies have shown that insects use immune mechanisms to regulate heritable symbionts. Here we test the hypothesis that variation in symbiont density among hosts is linked to intraspecific differences in the immune response to harboring symbionts. We show that pea aphids (Acyrthosiphon pisum) harboring the bacterial endosymbiont Regiella insecticola (but not all other species of symbionts) downregulate expression of key immune genes. We then functionally link immune expression with symbiont density using RNAi. The pea aphid species complex is comprised of multiple reproductively-isolated host plant-adapted populations. These 'biotypes' have distinct patterns of symbiont infections: for example, aphids from the Trifolium biotype are strongly associated with Regiella. Using RNAseq, we compare patterns of gene expression in response to Regiella in aphid genotypes from multiple biotypes, and we show that Trifolium aphids experience no downregulation of immune gene expression while hosting Regiella and harbor symbionts at lower densities. Using F1 hybrids between two biotypes, we find that symbiont density and immune gene expression are both intermediate in hybrids. We propose that in this system, Regiella symbionts are suppressing aphid immune mechanisms to increase their density, but that some hosts have adapted to prevent immune suppression in order to control symbiont numbers. This work therefore suggests that antagonistic coevolution can play a role in host-microbe interactions even when symbionts are transmitted vertically and provide a clear benefit to their hosts. The specific immune mechanisms that we find are downregulated in the presence of Regiella have been previously shown to combat pathogens in aphids, and thus this work also highlights the immune system's complex dual role in interacting with both beneficial and harmful microbes., Competing Interests: The authors have declared that no competing interests exist.

Evolutionary arms races are broadly prevalent among organisms including bacteria, which have evolved defensive strategies against various attackers. A common microbial aggression mechanism is the type VI secretion system (T6SS), a contact-dependent bacterial weapon used to deliver toxic effector proteins into adjacent target cells. Sibling cells constitutively express immunity proteins that neutralize effectors. However, less is known about factors that protect non-sibling bacteria from T6SS attacks independently of cognate immunity proteins. In this study, we observe that human Escherichia coli commensal strains sensitive to T6SS attacks from Vibrio cholerae are protected when co-cultured with glucose. We confirm that glucose does not impair V. cholerae T6SS activity. Instead, we find that cells lacking the cAMP receptor protein (CRP), which regulates expression of hundreds of genes in response to glucose, survive significantly better against V. cholerae T6SS attacks even in the absence of glucose. Finally, we show that the glucose-mediated T6SS protection varies with different targets and killers. Our findings highlight the first example of an extracellular small molecule modulating a genetically controlled response for protection against T6SS attacks. This discovery may have major implications for microbial interactions during pathogen-host colonization and survival of bacteria in environmental communities.

Alternaria alternata is a fungal allergen associated with severe asthma and asthma exacerbations. Similarly to other asthma-associated allergens, Alternaria secretes a serine-like trypsin protease(s) that is thought to act through the G protein-coupled receptor protease-activated receptor-2 (PAR 2 ) to induce asthma symptoms. However, specific mechanisms underlying Alternaria-induced PAR 2 activation and signaling remain ill-defined. We sought to determine whether Alternaria-induced PAR 2 signaling contributed to asthma symptoms via a PAR 2 /β-arrestin signaling axis, identify the protease activity responsible for PAR 2 signaling, and determine whether protease activity was sufficient for Alternaria-induced asthma symptoms in animal models. We initially used in vitro models to demonstrate Alternaria-induced PAR 2 /β-arrestin-2 signaling. Alternaria filtrates were then used to sensitize and challenge wild-type, PAR 2 -/- and β-arrestin-2 -/- mice in vivo. Intranasal administration of Alternaria filtrate resulted in a protease-dependent increase of airway inflammation and mucin production in wild-type but not PAR 2 -/- or β-arrestin-2 -/- mice. Protease was isolated from Alternaria preparations, and select in vitro and in vivo experiments were repeated to evaluate sufficiency of the isolated Alternaria protease to induce asthma phenotype. Administration of a single isolated serine protease from Alternaria, Alternaria alkaline serine protease (AASP), was sufficient to fully activate PAR 2 signaling and induce β-arrestin-2 -/- -dependent eosinophil and lymphocyte recruitment in vivo. In conclusion, Alternaria filtrates induce airway inflammation and mucus hyperplasia largely via AASP using the PAR 2 /β-arrestin signaling axis. Thus, β-arrestin-biased PAR 2 antagonists represent novel therapeutic targets for treating aeroallergen-induced asthma.

Background: Proteinase-activated receptor 2 (PAR2 ) is a G protein-coupled receptor activated by trypsin-like serine proteinases. PAR2 activation has been associated with inflammation including allergic airway inflammation. We have also shown that PAR2 activation in the airways leads to allergic sensitization. The exact contribution of PAR2 in the development of eosinophilic inflammation and airway hyperresponsiveness (AHR) in sensitized individuals is not clear., Objective: To investigate whether functional inhibition of PAR2 during allergen challenge of allergic mice would inhibit allergen-induced AHR and inflammation in mouse models of asthma., Methods: Mice were sensitized and challenged with ovalbumin (OVA) or cockroach extract (CE). To investigate the role of PAR2 in the development of AHR and airway inflammation, we administered blocking anti-PAR2 antibodies, or a cell permeable peptide inhibitor of PAR2 signalling, pepducin, i.n. before allergen challenges and then assessed AHR and airway inflammation., Results: Administration of anti-PAR2 antibodies significantly inhibited OVA- and CE-induced AHR and airway inflammation. In particular, two anti-PAR2 antibodies, the monoclonal SAM-11 and polyclonal B5, inhibited AHR, airway eosinophilia, the increase of cytokines in the lung tissue and antigen-specific T cell proliferation, but had no effect on antigen-specific IgG and IgE levels. Pepducin was also effective in inhibiting AHR and airway inflammation in an OVA model of allergic airway inflammation., Conclusions and Clinical Relevance: Functional blockade of PAR2 in the airways during allergen challenge improves allergen-induced AHR and inflammation in mice. Therefore, topical PAR2 blockade in the airways, through anti-PAR2 antibodies or molecules that interrupt PAR2 signalling, has the potential to be used as a therapeutic option in allergic asthma., (© 2015 John Wiley & Sons Ltd.)

Purpose: To evaluate the feasibility of a respiratory-gated noncontrast magnetic resonance angiography (MRA) sequence for imaging the central veins of the chest., Materials and Methods: Eleven healthy subjects underwent MRA of the central veins of the chest with a respiratory-gated noncontrast (SPACE) sequence. Qualitative visualization and signal homogeneity of each central venous segment were scored by two radiologists on a scale of 1-4. Signal-to-noise and contrast-to-noise ratios (SNR and CNR) were also calculated. Retrospective review of our imaging database revealed 13 patients with suspected pathology of the central veins who underwent a clinical MRA examination using the SPACE sequence as well as reference standard central venous imaging with contrast-enhanced MRA or conventional venography., Results: In healthy subjects, all central venous segments demonstrated good to excellent venous visualization and homogeneity scores with the noncontrast SPACE sequence. The mean SNR and CNR of the central venous system were 192.7 and 175.0, respectively. In the 13 clinical examinations, the sensitivity and specificity for detection of stenosis or occlusions was 100% and 100% for reader 1 and 95% and 91% for reader 2, respectively., Conclusion: The respiratory-gated noncontrast SPACE sequence provided excellent imaging characteristics of the central veins in healthy subjects with promising diagnostic accuracy in patients with central venous pathology., (© 2013 Wiley Periodicals, Inc.)

Proteinase-Activated receptor-2 (PAR(2)), a G-protein-coupled Receptor, activated by serine proteinases, is reported to have both protective and proinflammatory effects in the airway. Given these opposing actions, both inhibitors and activators of PAR(2) have been proposed for treating asthma. PAR(2) can signal through two independent pathways: a β-arrestin-dependent one that promotes leukocyte migration, and a G-protein/Ca(2+) one that is required for prostaglandin E(2) (PGE(2)) production and bronchiolar smooth muscle relaxation. We hypothesized that the proinflammatory responses to PAR(2) activation are mediated by β-arrestins, whereas the protective effects are not. Using a mouse ovalbumin model for PAR(2)-modulated airway inflammation, we observed decreased leukocyte recruitment, cytokine production, and mucin production in β-arrestin-2(-/-) mice. In contrast, PAR(2)-mediated PGE(2) production, smooth muscle relaxation, and decreased baseline airway resistance (measures of putative PAR(2) "protective" effects) were independent of β-arrestin-2. Flow cytometry and cytospins reveal that lung eosinophil and CD4 T-cell infiltration, and production of IL-4, IL-6, IL-13, and TNFα, were enhanced in wild-type but not β-arrestin-2(-/-) mice. Using the forced oscillation technique to measure airway resistance reveals that PAR(2) activation protects against airway hyperresponsiveness by an unknown mechanism, possibly involving smooth muscle relaxation. Our data suggest that the PAR(2)-enhanced inflammatory process is β-arrestin-2 dependent, whereas the protective anticonstrictor effect of bronchial epithelial PAR(2) may be β-arrestin independent.

Hydroxyapatite (HA) nanoparticles are similar to bone apatite in size, phase composition, and crystal structure. When compared with micron-size HA particles, nano-HA possesses improved mechanical properties and superior bioactivity for promoting bone growth and regeneration. However, scaffolds fabricated from nano-HA alone cannot meet the mechanical requirements for direct-loading applications. A number of studies suggest that nanostructured composites may offer surface and/or chemical properties of native bone, and therefore represent ideal substrates to support bone regeneration. However, a common problem with nanohydroxyapatite (nano-HA)-polymer composites is the weak binding strength between the nano-HA filler and the polymer matrix since they are two different categories of materials and cannot form covalent bonds between them during the mixing process. Often, the mechanical strength of the composite is compromised due to the phase separation of the HA filler from the polymer matrix during the tissue repair process. To overcome this problem, an ultrathin degradable polymer film was grafted onto the surface of nano-HA using a radio-frequency plasma polymerization technology from acrylic acid monomers. The treated nano-HA powders are expected to bind to the polymer matrix via covalent bonds, thus enhancing the mechanical properties of the resultant composites. High-resolution transmission electron microscopy (HRTEM) experiments showed that an extremely thin polymer film (2 nm) was uniformly deposited on the surfaces of the nanoparticles. The HRTEM results were confirmed by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectroscopy (TOFSIMS). Tensile tests performed on the specimens revealed that the degradable coating had improved elastic and strength properties when compared with the nondegradable and uncoated controls. XPS and TOSIMS data revealed that more functional carboxyl groups were formed on degradable coatings than cross-linked nondegradable coatings. Cytocompatibility assay demonstrated that both the degradable and nondegradable coatings are cytocompatible.

Many serious adverse physiological changes occur during spaceflight. In the search for underlying mechanisms and possible new countermeasures, many experimental tools and methods have been developed to study microgravity caused physiological changes, ranging from in vitro bioreactor studies to spaceflight investigations. Recently, genomic and proteomic approaches have gained a lot of attention; after major scientific breakthroughs in the fields of genomics and proteomics, they are now widely accepted and used to understand biological processes. Understanding gene and/or protein expression is the key to unfolding the mechanisms behind microgravity-induced problems and, ultimately, finding effective countermeasures to spaceflight-induced alterations. Significant progress has been made in identifying the genes/proteins responsible for these changes. Although many of these genes and/or proteins were observed to be either upregulated or downregulated, however, no large-scale genomics and proteomics studies have been published so far. This review aims at summarizing the current status of microgravity-related genomics and proteomics studies and stimulating large-scale proteomics and genomics research activities.