Infection with Nannizziopsis guarroi and Ophidiomyces ophiodiicola in reptiles in Taiwan.

Fungal infection is an emerging threat to reptiles. The main pathogens are fungi of the genera Nannizziopsis, Paranannizziopsis and Ophidiomyces. The clinical symptoms range from mild skin lesions to the dissemination of internal organs and even death. Most of the reported cases are from Europe, North America and Australia. In this study, we report the Nannizziopsis guarroi infection in one captive inland bearded dragon (Pogona vitticeps), one captive green iguana (Iguana iguana) and Ophidiomyces ophiodiicola infection in one wild red-banded snake (Dinodon rufozonatum) and one wild Chinese cobra (Naja atra) in Taiwan. The infections were confirmed by the presence of fungal elements in the tissue. The pathogens were identified based on their morphological and DNA sequence characteristics. The susceptibility profiles of the fungal strains to nine antifungal drugs were obtained using broth microdilution methods. The presence of both fungal species in Asia highlights the urgent need for surveillance and close monitoring of reptile infections to prevent them from spreading and to the possible collapse of reptile populations in the wild.

Novel Blood Clot Retriever for Ischemic Stroke.

Stroke is the second leading cause of death in the world. Ischemic stroke, caused by the blockage of intracranial arteries, accounts for approximately 80% of strokes. Among this proportion, acute ischemic stroke, usually caused by the sudden formation of blood clots, can cause fatal blockages in arteries. We proposed a unique blood clot retriever for the treatment of acute ischemic stroke, and conducted a series of tasks, including design, computer simulation, prototyping, and bench testing, for the proof of concept. Unlike most blood clot retrievers used today, our novel design deviates from traditional stent-like blood clot retrievers and uses large closed cells, irregular spikes, and strut protrusions to achieve maximum entanglement for better retrieval performance. Experimental results showed that the retrieval rate of our blood clot retriever was 79%, which demonstrated the feasibility of our new design concept.

Respiratory chain components are required for peptidoglycan recognition protein-induced thiol depletion and killing in Bacillus subtilis and Escherichia coli.

Mammalian peptidoglycan recognition proteins (PGRPs or PGLYRPs) kill bacteria through induction of synergistic oxidative, thiol, and metal stress. Tn-seq screening of Bacillus subtilis transposon insertion library revealed that mutants in the shikimate pathway of chorismate synthesis had high survival following PGLYRP4 treatment. Deletion mutants for these genes had decreased amounts of menaquinone (MK), increased resistance to killing, and attenuated depletion of thiols following PGLYRP4 treatment. These effects were reversed by MK or reproduced by inhibiting MK synthesis. Deletion of cytochrome aa3-600 or NADH dehydrogenase (NDH) genes also increased B. subtilis resistance to PGLYRP4-induced killing and attenuated thiol depletion. PGLYRP4 treatment also inhibited B. subtilis respiration. Similarly in Escherichia coli, deletion of ubiquinone (UQ) synthesis, formate dehydrogenases (FDH), NDH-1, or cytochrome bd-I genes attenuated PGLYRP4-induced thiol depletion. PGLYRP4-induced low level of cytoplasmic membrane depolarization in B. subtilis and E. coli was likely not responsible for thiol depletion. Thus, our results show that the respiratory electron transport chain components, cytochrome aa3-600, MK, and NDH in B. subtilis, and cytochrome bd-I, UQ, FDH-O, and NDH-1 in E. coli, are required for both PGLYRP4-induced killing and thiol depletion and indicate conservation of the PGLYRP4-induced thiol depletion and killing mechanisms in Gram-positive and Gram-negative bacteria.

Comparisons of magnetic defects and coercive forces for Co/Si(100) and Co/rubrene/Si(100).

Spintronics can add new functionalities to electronic devices by utilizing the spin degree of freedom of electrons. Investigating magnetic defects is crucial for the performance of spintronics devices. However, the effects of magnetic defects that are introduced by the presence of organic materials on their magnetic properties remain unclear. Herein, we report on a novel method using rubrene combined with Kerr microscopy that enables quantitative and direct measurements of magnetic defect density. For Co/Si(100) at a magnetic field near the coercivity value, Kerr microscopy images show a dark image with some magnetic defects. Because of domain wall motion, small patches gradually change the contrast. These magnetic defects are immovable at different magnetic fields and serve as pinning sites for domain wall motion. Experimental evidence shows that coercive force can be reduced by controlling the magnetic defect density by introducing small amounts of rubrene into the films. Furthermore, direct quantitative measurements of magnetic defects show both a one-dimensional bowing of domain walls and strong defect-domain wall interactions in the films. Based on these findings, we propose a viable strategy for reducing the coercive force of Co/Si(100) by controlling the magnetic defect density and this new information promises to be valuable for future applications of spintronics devices.

Formate dehydrogenase, ubiquinone, and cytochrome bd-I are required for peptidoglycan recognition protein-induced oxidative stress and killing in Escherichia coli.

Mammalian Peptidoglycan Recognition Proteins (PGRPs) kill bacteria through induction of synergistic oxidative, thiol, and metal stress. PGRPs induce oxidative stress in bacteria through a block in the respiratory chain, which results in decreased respiration and incomplete reduction of oxygen (O2) to hydrogen peroxide (H2O2). In this study we identify the site of PGRP-induced generation of H2O2 in Escherichia coli. Tn-seq screening of E. coli Tn10 insertion library revealed that mutants in formate dehydrogenase (FDH) genes had the highest survival following PGRP treatment. Mutants lacking functional FDH-O had abolished PGRP-induced H2O2 production and the highest resistance to PGRP-induced killing, and formate enhanced PGRP-induced killing and H2O2 production in an FDH-dependent manner. Mutants in ubiquinone synthesis (but not menaquinone and demethylmenaquinone) and cytochrome bd-I (but not cytochromes bo3 and bd-II) also had completely abolished PGRP-induced H2O2 production and high resistance to PGRP-induced killing. Because electrons in the respiratory chain flow from dehydrogenases' substrates through quinones and then cytochromes to O2, these results imply that the site of PGRP-induced incomplete reduction of O2 to H2O2 is downstream from dehydrogenases and ubiquinone at the level of cytochrome bd-I, which results in oxidative stress. These results reveal several essential steps in PGRP-induced bacterial killing.

Variations of the elastic modulus perpendicular to the surface of rubrene bilayer films.

Investigations exploring the inherent mechanical properties of electronic materials have grown rapidly in recent years largely because they are important in developing flexible electronics, organic displays and sensors. However, our understanding of the mechanical properties of organic semiconductors with a thin-film form remains limited. We report herein on an investigation of the structures and related elastic moduli perpendicular to the surface of a rubrene thin film. A rubrene/Si(100) film typically has a cluster-type morphology mainly comprising crystalline nanodomains within the film. We propose a structural bilayer model that can be used to explain the layered nature or characteristics of the rubrene films. As the film thickness is increased, the enhancement in elastic modulus can be attributed to the presence of a soft surface layer on a hard underlayer. Based on four-point probe measurements, the bilayered nature of such materials can be used to characterize their electrical resistive behavior while interfacial roughness is sensitive to the transport paths of conduction electrons. This information is valuable for future applications of organic semiconductors in flexible devices.

Pathogenicity and transmission of a swine influenza A(H6N6) virus.

Subtype H6 influenza A viruses (IAVs) are commonly detected in wild birds and domestic poultry and can infect humans. In 2010, a H6N6 virus emerged in southern China, and since then, it has caused sporadic infections among swine. We show that this virus binds to α2,6-linked and α2,3-linked sialic acids. Mutations at residues 222 (alanine to valine) and 228 (glycine to serine) of the virus hemagglutinin (HA) affected its receptor-binding properties. Experiments showed that the virus has limited transmissibility between ferrets through direct contact or through inhalation of infectious aerosolized droplets. The internal genes of the influenza A(H1N1)pdm09 virus, which is prevalent in swine worldwide, increases the replication efficiency of H6N6 IAV in the lower respiratory tract of ferrets but not its transmissibility between ferrets. These findings suggest H6N6 swine IAV (SIV) currently poses a moderate risk to public health, but its evolution and spread should be closely monitored.

Association analyses of large-scale glycan microarray data reveal novel host-specific substructures in influenza A virus binding glycans.

Influenza A viruses can infect a wide variety of animal species and, occasionally, humans. Infection occurs through the binding formed by viral surface glycoprotein hemagglutinin and certain types of glycan receptors on host cell membranes. Studies have shown that the α2,3-linked sialic acid motif (SA2,3Gal) in avian, equine, and canine species; the α2,6-linked sialic acid motif (SA2,6Gal) in humans; and SA2,3Gal and SA2,6Gal in swine are responsible for the corresponding host tropisms. However, more detailed and refined substructures that determine host tropisms are still not clear. Thus, in this study, we applied association mining on a set of glycan microarray data for 211 influenza viruses from five host groups: humans, swine, canine, migratory waterfowl, and terrestrial birds. The results suggest that besides Neu5Acα2-6Galß, human-origin viruses could bind glycans with Neu5Acα2-8Neu5Acα2-8Neu5Ac and Neu5Gcα2-6Galß1-4GlcNAc substructures; Galß and GlcNAcß terminal substructures, without sialic acid branches, were associated with the binding of human-, swine-, and avian-origin viruses; sulfated Neu5Acα2-3 substructures were associated with the binding of human- and swine-origin viruses. Finally, through three-dimensional structure characterization, we revealed that the role of glycan chain shapes is more important than that of torsion angles or of overall structural similarities in virus host tropisms.

Functional characterization of the dguRABC locus for D-Glu and d-Gln utilization in Pseudomonas aeruginosa PAO1.

D-Glu, an essential component of peptidoglycans, can be utilized as a carbon and nitrogen source by Pseudomonas aeruginosa. DNA microarrays were employed to identify genes involved in D-Glu catabolism. Through gene knockout and growth phenotype analysis, the divergent dguR-dguABC (D-Glu utilization) gene cluster was shown to participate in D-Glu and D-Gln catabolism and regulation. Growth of the dguR and dguA mutants was abolished completely on D-Glu or retarded on D-Gln as the sole source of carbon and/or nitrogen. The dguA gene encoded a FAD-dependent D-amino acid dehydrogenase with d-Glu as its preferred substrate, and its promoter was specifically induced by exogenous D-Glu and D-Gln. The function of DguR as a transcriptional activator of the dguABC operon was demonstrated by promoter activity measurements in vivo and by mobility shift assays with purified His-tagged DguR in vitro. Although the DNA-binding activity of DguR did not require D-Glu, the presence of D-Glu, but not D-Gln, in the binding reaction was found to stabilize a preferred nucleoprotein complex. The presence of a putative DguR operator was revealed by in silica analysis of the dguR-dguA intergenic regions among Pseudomonas spp. and binding of DguR to a highly conserved 19 bp sequence motif was further demonstrated. The dguB gene encodes a putative enamine/imine deaminase of the RidA family, but its role in D-Glu catabolism remains to be determined. Whilst a lesion in dguC encoding a periplasmic solute binding protein only affected growth on D-Glu slightly, expression of the previously characterized AatJMQP transporter for acidic l-amino acid uptake was found inducible by D-Glu and essential for D-Glu utilization. In summary, the findings of this study supported DguA as a new member of the FAD-dependent d-amino acid dehydrogenase family, and DguR as a D-Glu sensor and transcriptional activator of the dguA promoter.

Stationary phases for the enrichment of glycoproteins and glycopeptides.

The analysis of protein glycosylation is important for biomedical and biopharmaceutical research. Recent advances in LC-MS analysis have enabled the identification of glycosylation sites, the characterisation of glycan structures and the identification and quantification of glycoproteins and glycopeptides. However, this type of analysis remains challenging due to the low abundance of glycopeptides in complex protein digests, the microheterogeneity at glycosylation sites, ion suppression effects and the competition for ionisation by co-eluting peptides. Specific sample preparation is necessary for comprehensive and site-specific glycosylation analyses using MS. Therefore, researchers continue to pursue new columns to broaden their applications. The current manuscript covers recent literature published from 2008 to 2013. The stationary phases containing various chemical bonding methods or ligands immobilisation strategies on solid supports that selectively enrich N-linked or sialylated N-glycopeptides are categorised with either physical or chemical modes of binding. These categories include lectin affinity, hydrophilic interactions, boronate affinity, titanium dioxide affinity, hydrazide chemistry and other separation techniques. This review should aid in better understanding the syntheses and physicochemical properties of each type of stationary phases for enriching glycoproteins and glycopeptides.

An internal hydrophobic helical domain of Bacillus subtilis enolase is essential but not sufficient as a non-cleavable signal for its secretion.

Many cytoplasmic proteins without a cleavable signal peptide, including enolase, are secreted during the stationary phase in Bacillus subtilis but the molecular mechanism is not yet clear. We previously identified a highly conserved embedded membrane domain in an internal hydrophobic α-helix of enolase that plays an important role in its secretion. In this study, we examined the role of the helix in more detail for the secretion of enolase. Altering this helix by mutations showed that many mutated forms in this domain were not secreted, some of which were not stable as a soluble form in the cytoplasm. On the other hand, mutations on the flanking regions of the helix or the conserved basic residues showed no deleterious effect. Bacillus enolase with the proper hydrophobic helical domain was also exported extracellularly in Escherichia coli, indicating that the requirement of the helix for the secretion of enolase is conserved in these species. GFP fusions with enolase regions showed that the hydrophobic helix domain itself was not sufficient to serve as a functional secretion signal; a minimal length of N-terminus 140 amino acids was required to mediate the secretion of the fused reporter GFP. We conclude that the internal hydrophobic helix of enolase is essential but is not sufficient as a signal for secretion; the intact long N-terminus including the hydrophobic helix domain is required to serve as a non-cleavable signal for the secretion of Bacillus enolase.

Time-related transcriptome analysis of B. subtilis 168 during growth with glucose.

Gene expression in Bacillus subtilis from late exponential to stationary phase was monitored by DNA microarrays with samples taken from the culture in LB broth with glucose supplement to prevent sporulation. Three major patterns of gene expression as revealed in this study were consistent to the expression profiling of PerR/Spx regulons and three major sigma factors-SigA, SigB, and SigW. Expression of most SigA-dependent house-keeping genes was significantly decreased and remained at low levels in the stationary phase. The sigB gene and additional genes of the SigB regulon for stress response exhibited a distinct pattern of transient induction with a peak in transition phase. The majority of induced genes after cessation of SigB-dependent surge were subjected to regulation by SigW, PerR, and Spx in response to oxidative stress. No induction of spo0A and skfA regulons supports the suppression of sporulation and cannibalism processes in the stationary phase by glucose supplement. In summary, these results depicted complicated strategies by cells to adapt changes from the fast growing exponential phase toward the stationary phase. The absence of programmed cell death and sporulation greatly facilitated data analysis and the identification of distinct expression patterns in the stationary phase of growth in B. subtilis.

Differential expression of secretion machinery during bacterial growth: SecY and SecF decrease while SecA increases during transition from exponential phase to stationary phase.

Transcription of many house-keeping genes, including secY and some other sec genes, decreases in the transition from the exponential phase to the stationary phase (feast to famine) in Bacillus subtilis. Unexpectedly and in contradiction to earlier reports, enhanced transcription was observed for another group of sec genes, including secA which codes for an essential ATPase for protein secretion. Consistent with the transcription data, the SecA protein of B. subtilis increases significantly in the stationary phase. Immunoblot analyses of Sec proteins during the transition in Escherichia coli also revealed the pronounced decreases of SecY and SecF and the increase of SecA, resulting in drastic increases of SecA/SecY and SecA/SecF ratios from exponential to stationary phases. The differential expression of Sec proteins in the stationary phase suggests the possibility of specific physiological functions.

Nonclassical protein secretion by Bacillus subtilis in the stationary phase is not due to cell lysis.

The carboxylesterase Est55 has been cloned and expressed in Bacillus subtilis strains. Est55, which lacks a classical, cleavable N-terminal signal sequence, was found to be secreted during the stationary phase of growth such that there is more Est55 in the medium than inside the cells. Several cytoplasmic proteins were also secreted in large amounts during late stationary phase, indicating that secretion in B. subtilis is not unique to Est55. These proteins, which all have defined cytoplasmic functions, include GroEL, DnaK, enolase, pyruvate dehydrogenase subunits PdhB and PdhD, and SodA. The release of Est55 and those proteins into the growth medium is not due to gross cell lysis, a conclusion that is supported by several lines of evidence: constant cell density and secretion in the presence of chloramphenicol, constant viability count, the absence of EF-Tu and SecA in the culture medium, and the lack of effect of autolysin-deficient mutants. The shedding of these proteins by membrane vesicles into the medium is minimal. More importantly, we have identified a hydrophobic α-helical domain within enolase that contributes to its secretion. Thus, upon the genetic deletion or replacement of a potential membrane-embedding domain, the secretion of plasmid gene-encoded mutant enolase is totally blocked, while the wild-type chromosomal enolase is secreted normally in the same cultures during the stationary phase, indicating differential specificity. We conclude that the secretion of Est55 and several cytoplasmic proteins without signal peptides in B. subtilis is a general phenomenon and is not a consequence of cell lysis or membrane shedding; instead, their secretion is through a process(es) in which protein domain structure plays a contributing factor.