Lipopolysaccharide O1 Antigen Contributes to the Virulence in Klebsiella pneumoniae Causing Pyogenic Liver Abscess

Klebsiella pneumoniae is the common cause of a global emerging infectious disease, community-acquired pyogenic liver abscess (PLA). Capsular polysaccharide (CPS) and lipopolysaccharide (LPS) are critical for this microorganism's ability to spread through the blood and to cause sepsis. While CPS type K1 is an important virulence factor in K. pneumoniae causing PLA, the role of LPS in PLA is not clear. Here, we characterize the role of LPS O antigen in the pathogenesis of K. pneumoniae causing PLA. NTUH-K2044 is a LPS O1 clinical strain; the presence of the O antigen was shown via the presence of 1,3-galactan in the LPS, and of sequences that align with the wb gene cluster, known to produce O-antigen. Serologic analysis of K. pneumoniae clinical isolates demonstrated that the O1 serotype was more prevalent in PLA strains than that in non-tissue-invasive strains (38/42 vs. 9/32, P<0.0001). O1 serotype isolates had a higher frequency of serum resistance, and mutation of the O1 antigen changed serum resistance in K. pneumoniae. A PLA-causing strain of CPS capsular type K2 and LPS serotype O1 (i.e., O1:K2 PLA strain) deleted for the O1 synthesizing genes was profoundly attenuated in virulence, as demonstrated in separate mouse models of septicemia and liver abscess. Immunization of mice with the K2044 magA-mutant (K1− O1) against LPS O1 provided protection against infection with an O1:K2 PLA strain, but not against infection with an O1:K1 PLA strain. Our findings indicate that the O1 antigen of PLA-associated K. pneumoniae contributes to virulence by conveying resistance to serum killing, promoting bacterial dissemination to and colonization of internal organs after the onset of bacteremia, and could be a useful vaccine candidate against infection by an O1:K2 PLA strain

Plant-RRBS, a bisulfite and next-generation sequencing-based methylome profiling method enriching for coverage of cytosine positions

Background: Cytosine methylation in plant genomes is important for the regulation of gene transcription and transposon activity. Genome-wide methylomes are studied upon mutation of the DNA methyltransferases, adaptation to environmental stresses or during development. However, from basic biology to breeding programs, there is a need to monitor multiple samples to determine transgenerational methylation inheritance or differential cytosine methylation. Methylome data obtained by sodium hydrogen sulfite (bisulfite)-conversion and next-generation sequencing (NGS) provide genome- wide information on cytosine methylation. However, a profiling method that detects cytosine methylation state dispersed over the genome would allow high-throughput analysis of multiple plant samples with distinct epigenetic signatures. We use specific restriction endonucleases to enrich for cytosine coverage in a bisulfite and NGS-based profiling method, which was compared to whole-genome bisulfite sequencing of the same plant material. Methods: We established an effective methylome profiling method in plants, termed plant-reduced representation bisulfite sequencing (plant-RRBS), using optimized double restriction endonuclease digestion, fragment end repair, adapter ligation, followed by bisulfite conversion, PCR amplification and NGS. We report a performant laboratory protocol and a straightforward bioinformatics data analysis pipeline for plant-RRBS, applicable for any reference-sequenced plant species. Results: As a proof of concept, methylome profiling was performed using an Oryza sativa ssp. indica pure breeding line and a derived epigenetically altered line (epiline). Plant-RRBS detects methylation levels at tens of millions of cytosine positions deduced from bisulfite conversion in multiple samples. To evaluate the method, the coverage of cytosine positions, the intra-line similarity and the differential cytosine methylation levels between the pure breeding line and the epiline were determined. Plant-RRBS reproducibly covers commonly up to one fourth of the cytosine positions in the rice genome when using MspI-DpnII within a group of five biological replicates of a line. The method predominantly detects cytosine methylation in putative promoter regions and not-annotated regions in rice. Conclusions: Plant-RRBS offers high-throughput and broad, genome- dispersed methylation detection by effective read number generation obtained from reproducibly covered genome fractions using optimized endonuclease combinations, facilitating comparative analyses of multi-sample studies for cytosine methylation and transgenerational stability in experimental material and plant breeding populations

Macrophage-derived human resistin is induced in multiple helminth infections and promotes inflammatory monocytes and increased parasite burden.

Parasitic helminth infections can be associated with lifelong morbidity such as immune-mediated organ failure. A better understanding of the host immune response to helminths could provide new avenues to promote parasite clearance and/or alleviate infection-associated morbidity. Murine resistin-like molecules (RELM) exhibit pleiotropic functions following helminth infection including modulating the host immune response; however, the relevance of human RELM proteins in helminth infection is unknown. To examine the function of human resistin (hResistin), we utilized transgenic mice expressing the human resistin gene (hRetnTg+). Following infection with the helminth Nippostrongylus brasiliensis (Nb), hResistin expression was significantly upregulated in infected tissue. Compared to control hRetnTg- mice, hRetnTg+ mice suffered from exacerbated Nb-induced inflammation characterized by weight loss and increased infiltration of inflammatory monocytes in the lung, along with elevated Nb egg burdens and delayed parasite expulsion. Genome-wide transcriptional profiling of the infected tissue revealed that hResistin promoted expression of proinflammatory cytokines and genes downstream of toll-like receptor signaling. Moreover, hResistin preferentially bound lung monocytes, and exogenous treatment of mice with recombinant hResistin promoted monocyte recruitment and proinflammatory cytokine expression. In human studies, increased serum resistin was associated with higher parasite load in individuals infected with soil-transmitted helminths or filarial nematode Wuchereria bancrofti, and was positively correlated with proinflammatory cytokines. Together, these studies identify human resistin as a detrimental factor induced by multiple helminth infections, where it promotes proinflammatory cytokines and impedes parasite clearance. Targeting the resistin/proinflammatory cytokine immune axis may provide new diagnostic or treatment strategies for helminth infection and associated immune-mediated pathology

MSH3 polymorphisms and protein levels affect CAG repeat instability in huntington's disease mice

Expansions of trinucleotide CAG/CTG repeats in somatic tissues are thought to contribute to ongoing disease progression through an affected individual's life with Huntington's disease or myotonic dystrophy. Broad ranges of repeat instability arise between individuals with expanded repeats, suggesting the existence of modifiers of repeat instability. Mice with expanded CAG/CTG repeats show variable levels of instability depending upon mouse strain. However, to date the genetic modifiers underlying these differences have not been identified. We show that in liver and striatum the R6/1 Huntington's disease (HD) (CAG)~100 transgene, when present in a congenic C57BL/6J (B6) background, incurred expansion-biased repeat mutations, whereas the repeat was stable in a congenic BALB/cByJ (CBy) background. Reciprocal congenic mice revealed the Msh3 gene as the determinant for the differences in repeat instability. Expansion bias was observed in congenic mice homozygous for the B6 Msh3 gene on a CBy background, while the CAG tract was stabilized in congenics homozygous for the CBy Msh3 gene on a B6 background. The CAG stabilization was as dramatic as genetic deficiency of Msh2. The B6 and CBy Msh3 genes had identical promoters but differed in coding regions and showed strikingly different protein levels. B6 MSH3 variant protein is highly expressed and associated with CAG expansions, while the CBy MSH3 variant protein is expressed at barely detectable levels, associating with CAG stability. The DHFR protein, which is divergently transcribed from a promoter shared by the Msh3 gene, did not show varied levels between mouse strains. Thus, naturally occurring MSH3 protein polymorphisms are modifiers of CAG repeat instability, likely through variable MSH3 protein stability. Since evidence supports that somatic CAG instability is a modifier and predictor of disease, our data are consistent with the hypothesis that variable levels of CAG instability associated with polymorphisms of DNA repair genes may have prognostic implications for various repeat-associated diseases

Demographic Diversity and Sustainable Fisheries

Fish species are diverse. For example, some exhibit early maturation while others delay maturation, some adopt semelparous reproductive strategies while others are iteroparous, and some are long-lived and others short-lived. The diversity is likely to have profound effects on fish population dynamics, which in turn has implications for fisheries management. In this study, a simple density-dependent stage-structured population model was used to investigate the effect of life history traits on sustainable yield, population resilience, and the coefficient of variation (CV) of the adult abundance. The study showed that semelparous fish can produce very high sustainable yields, near or above 50% of the carrying capacity, whereas long-lived iteroparous fish can produce very low sustainable yields, which are often much less than 10% of the carrying capacity. The difference is not because of different levels of sustainable fishing mortality rate, but because of difference in the sensitivity of the equilibrium abundance to fishing mortality. On the other hand, the resilience of fish stocks increases from delayed maturation to early maturation strategies but remains almost unchanged from semelparous to long-lived iteroparous. The CV of the adult abundance increases with increased fishing mortality, not because more individuals are recruited into the adult stage (as previous speculated), but because the mean abundance is more sensitive to fishing mortality than its standard deviation. The magnitudes of these effects vary depending on the life history strategies of the fish species involved. It is evident that any past high yield of long-lived iteroparous fish is a transient yield level, and future commercial fisheries should focus more on fish that are short-lived (including semelparous species) with high compensatory capacity

Mucosal Lipocalin 2 Has Pro-Inflammatory and Iron-Sequestering Effects in Response to Bacterial Enterobactin

Nasal colonization by both gram-positive and gram-negative pathogens induces expression of the innate immune protein lipocalin 2 (Lcn2). Lcn2 binds and sequesters the iron-scavenging siderophore enterobactin (Ent), preventing bacterial iron acquisition. In addition, Lcn2 bound to Ent induces release of IL-8 from cultured respiratory cells. As a countermeasure, pathogens of the Enterobacteriaceae family such as Klebsiella pneumoniae produce additional siderophores such as yersiniabactin (Ybt) and contain the iroA locus encoding an Ent glycosylase that prevents Lcn2 binding. Whereas the ability of Lcn2 to sequester iron is well described, the ability of Lcn2 to induce inflammation during infection is unknown. To study each potential effect of Lcn2 on colonization, we exploited K. pneumoniae mutants that are predicted to be susceptible to Lcn2-mediated iron sequestration (iroA ybtS mutant) or inflammation (iroA mutant), or to not interact with Lcn2 (entB mutant). During murine nasal colonization, the iroA ybtS double mutant was inhibited in an Lcn2-dependent manner, indicating that the iroA locus protects against Lcn2-mediated growth inhibition. Since the iroA single mutant was not inhibited, production of Ybt circumvents the iron sequestration effect of Lcn2 binding to Ent. However, colonization with the iroA mutant induced an increased influx of neutrophils compared to the entB mutant. This enhanced neutrophil response to Ent-producing K. pneumoniae was Lcn2-dependent. These findings suggest that Lcn2 has both pro-inflammatory and iron-sequestering effects along the respiratory mucosa in response to bacterial Ent. Therefore, Lcn2 may represent a novel mechanism of sensing microbial metabolism to modulate the host response appropriately

Altering the trajectory of early postnatal cortical development can lead to structural and behavioural features of autism

<p>Abstract</p> <p>Background</p> <p>Autism is a behaviourally defined neurodevelopmental disorder with unknown etiology. Recent studies in autistic children consistently point to neuropathological and functional abnormalities in the temporal association cortex (TeA) and its associated structures. It has been proposed that the trajectory of postnatal development in these regions may undergo accelerated maturational alterations that predominantly affect sensory recognition and social interaction. Indeed, the temporal association regions that are important for sensory recognition and social interaction are one of the last regions to mature suggesting a potential vulnerability to early maturation. However, direct evaluation of the emerging hypothesis that an altered time course of early postnatal development can lead to an ASD phenotype remains lacking.</p> <p>Results</p> <p>We used electrophysiological, histological, and behavioural techniques to investigate if the known neuronal maturational promoter valproate, similar to that in culture systems, can influence the normal developmental trajectory of TeA <it>in vivo</it>. Brain sections obtained from postnatal rat pups treated with VPA <it>in vivo </it>revealed that almost 40% of cortical cells in TeA prematurely exhibited adult-like intrinsic electrophysiological properties and that this was often associated with gross cortical hypertrophy and a reduced predisposition for social play behaviour.</p> <p>Conclusions</p> <p>The co-manifestation of these functional, structural and behavioural features suggests that alteration of the developmental time course in certain high-order cortical networks may play an important role in the neurophysiological basis of autism.</p

A dimensioning and tolerancing methodology for concurrent engineering applications II: comprehensive solution strategy

Dimensioning and tolerancing (D&T) is a multidisciplinary problem which requires the fulfillment of a large number of dimensional requirements. However, almost all of the currently available D&T tools are only intended for use by the designer. In addition, they typically provide solutions for the requirements one at time. This paper presents a methodology for determining the dimensional specifications of the component parts and sub-assemblies of a product by satisfying all of its requirements. The comprehensive solution strategy presented here includes: a strategy for separating D&T problems into groups, the determination of an optimum solution order for coupled functional equations, a generic tolerance allocation strategy, and strategies for solving different types of D&T problems. A number of commonly used cost minimization strategies, such as the use of standard parts, preferred sizes, preferred fits, and preferred tolerances, have also been incorporated into the proposed methodology. The methodology is interactive and intended for use in a concurrent engineering environment by members of a product development team

Ambient levels of volatile organic compounds in the vicinity of petrochemical industrial area of Yokohama, Japan

Urban ambient air concentrations of 39 aromatic (including benzene, toluene, and xylenes) and aliphatic volatile organic compounds (VOCs) were measured in Yokohama city, Japan. Yokohama city was selected as a case study to assess the amount of VOC released from Industrial area to characterize the ambient air quality with respect to VOC as well as to know the impact of petrochemical storage facilities on local air quality. For this purpose, ambient air samples were collected (from June 2007 to November 2008) at six selected locations which are designated as industrial, residential, or commercial areas. To find out the diurnal variations of VOC, hourly nighttime sampling was carried out for three nights at one of the industrial locations (Shiohama). Samples were analyzed using gas chromatographic system (GC-FID). Results show strong variation between day and nighttime concentrations and among the seasons. Aliphatic fractions were most abundant, suggesting petrochemical storage facilities as the major source of atmospheric hydrocarbons. High concentrations of benzene, toluene, ethyl benzene, and xylene (BTEX) were observed at industrial locations. BTEX showed strong diurnal variation which is attributed to change in meteorology. During our campaign, low ambient VOC concentrations were observed at the residential site