MetAMOS: a modular and open source metagenomic assembly and analysis pipeline

We describe MetAMOS, an open source and modular metagenomic assembly and analysis pipeline. MetAMOS represents an important step towards fully automated metagenomic analysis, starting with next-generation sequencing reads and producing genomic scaffolds, open-reading frames and taxonomic or functional annotations. MetAMOS can aid in reducing assembly errors, commonly encountered when assembling metagenomic samples, and improves taxonomic assignment accuracy while also reducing computational cost. MetAMOS can be downloaded from: https://github.com/treangen/MetAMOS.     

Macrophage Specific Caspase-1/11 Deficiency Protects against Cholesterol Crystallization and Hepatic Inflammation in Hyperlipidemic Mice

Background & Aims

While non-alcoholic steatohepatitis (NASH) is characterized by hepatic steatosis combined with inflammation, the mechanisms triggering hepatic inflammation are unknown. In Ldlr^-/- mice, we have previously shown that lysosomal cholesterol accumulation in Kupffer cells (KCs) correlates with hepatic inflammation and cholesterol crystallization. Previously, cholesterol crystals have been shown to induce the activation of inflammasomes. Inflammasomes are protein complexes that induce the processing and release of pro-inflammatory cytokines IL-1b and IL-18 via caspase-1 activation. Whereas caspase-1 activation is independent of caspase-11 in the canonical pathway of inflammasome activation, caspase-11 was found to trigger caspase-1-dependent IL-1b and IL-18 in response to non-canonical inflammasome activators. So far, it has not been investigated whether inflammasome activation stimulates the formation of cholesterol crystals. We hypothesized that inflammasome activation in KCs stimulates cholesterol crystallization, thereby leading to hepatic inflammation.

Methods

Ldlr ^-/- mice were transplanted (tp) with wild-type (Wt) or caspase-1/11^-/- (dKO) bone marrow and fed either regular chow or a high-fat, high-cholesterol (HFC) diet for 12 weeks. In vitro, bone marrow derived macrophages (BMDM) from wt or caspase-1/11^-/- mice were incubated with oxLDL for 24h and autophagy was assessed.

Results

In line with our hypothesis, caspase-1/11^-/--tp mice had less severe hepatic inflammation than Wt-tp animals, as evident from liver histology and gene expression analysis in isolated KCs. Mechanistically, KCs from caspase-1/11^-/--tp mice showed less cholesterol crystals, enhanced cholesterol efflux and increased autophagy. In wt BMDM, oxLDL incubation led to disturbed autophagy activity whereas BMDM from caspase-1/11^-/- mice had normal autophagy activity.

Conclusion

Altogether, these data suggest a vicious cycle whereby disturbed autophagy and decreased cholesterol efflux leads to newly formed cholesterol crystals and thereby maintain hepatic inflammation during NASH by further activating the inflammasome.     

Accurate and Rapid Identification of the Burkholderia pseudomallei Near-Neighbour,Burkholderia ubonensis,Using Real-Time PCR

Burkholderia ubonensis is an environmental bacterium belonging to the Burkholderia cepacia complex (Bcc), a group of genetically related organisms that are associated with opportunistic but generally nonfatal infections in healthy individuals. In contrast, the near-neighbour species Burkholderia pseudomallei causes melioidosis, a disease that can be fatal in up to 95% of cases if left untreated. B. ubonensis is frequently misidentified as B. pseudomallei from soil samples using selective culturing on Ashdown’s medium, reflecting both the shared environmental niche and morphological similarities of these species. Additionally, B. ubonensis shows potential as an important biocontrol agent in B. pseudomallei-endemic regions as certain strains possess antagonistic properties towards B. pseudomallei. Current methods for characterising B. ubonensis are laborious, time-consuming and costly, and as such this bacterium remains poorly studied. The aim of our study was to develop a rapid and inexpensive real-time PCR-based assay specific for B. ubonensis. We demonstrate that a novel B. ubonensis-specific assay, Bu550, accurately differentiates B. ubonensis from B. pseudomallei and other species that grow on selective Ashdown’s agar. We anticipate that Bu550 will catalyse research on B. ubonensis by enabling rapid identification of this organism from Ashdown’s-positive colonies that are not B. pseudomallei.     

Trends in Prevalence of Overweight and Obesity in Danish Infants,Children and Adolescents – Are We Still on a Plateau?

Background

After the worldwide steep increase in child and adolescent overweight and obesity during the last decades, there is now evidence of a levelling off in the prevalence in many countries in the Western world.

Aim

To examine whether there still is a plateau in the prevalence of overweight and obesity in Danish children and adolescents, or whether the prevalence is decreasing or rising again.

Methods

The trends in the prevalence rates were based on three data sets providing comparable repeated estimates: 1) the Danish Health Visitors Child Health Database (DHVCHD) with measurements on infant and childhood height and weight from 2002 to 2011 (n up to 39,984), 2) the Danish National Birth Cohort (DNBC) with maternal reports of measured infant and childhood height and weight from 1998 to 2010 (n up to 56,826) and 3) the Danish part of the Health Behaviour in School-aged Children survey (HBSC) with self-reported information on adolescent height and weight from the years 2002 to 2010 (n = 16,557). Overweight and obesity were categorized according to WHO growth standards. Trends were assessed by repeated point estimates and linear regression analyses providing regression coefficients for changes in per cent per year with 95% confidence intervals (CI).

Results

The prevalence rates of overweight and obesity for infants, children and adolescents showed a mixed pattern of decline, stability and increase (ranging from -1.10 through 0.29 per cent per year with CI’s from -3.10 through 2.37). Overall, there were no consistent statistically significant trends upwards or downwards, although some significant downward trends in childhood and adolescence were observed.

Conclusion

This study, based on data from 1998 through 2011, showed that the prevalence rates of overweight and obesity among Danish infants, children and adolescents were largely still on a plateau with tendencies for a decline among children and adolescents.     

As Facts and Chats Go Online,What Is Important for Adolescents with Type 1 Diabetes?

Background

Continued refinement of resources for patient information, education and support is needed. Considering the rapid development of new communication practices, the perspectives of young people themselves warrant more attention using a wide research focus. The purpose of this study was to understand information-seeking behaviours, Internet use and social networking online in adolescents with type 1 diabetes (T1DM). This applied to their everyday life, including the context of diabetes and their experiences and need of contact with T1DM peers.

Methodology/Principal Findings

Twenty-four adolescents aged 10–17 years with T1DM were recruited from a county hospital in the south-east of Sweden. Qualitative data were obtained using eight focus groups, wherein each participant engaged in a 60–90 minute video/audio-recorded session. The focus group data were transcribed and analysed using qualitative content analysis. Some demographic and medical information was also collected. The three main categories that were identified; Aspects of Security, Updating, and Plainness and their sub-categories gave significant information about how to enhance information retrieval and peer contacts related to T1DM. Regarding the persons'' information-seeking behaviour, Internet use, and use of social media some differences could be identified depending on gender and age.

Conclusions/Significance

Sensitivity and adaptation to users'' needs and expectations seem crucial in the development of future online resources for adolescents with T1DM. To start with, this could mean applying a wider range of already existing information and communication technologies. Health practitioners need to focus on the areas of security of information and communication, frequency of updating, and simplicity of design-less is more.     

CD14-Dependent Monocyte Isolation Enhances Phagocytosis of Listeria monocytogenes by Proinflammatory,GM-CSF-Derived Macrophages

Macrophages are an important line of defence against invading pathogens. Human macrophages derived by different methods were tested for their suitability as models to investigate Listeria monocytogenes (Lm) infection and compared to macrophage-like THP-1 cells. Human primary monocytes were isolated by either positive or negative immunomagnetic selection and differentiated in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF) into pro- or anti-inflammatory macrophages, respectively. Regardless of the isolation method, GM-CSF-derived macrophages (GM-Mφ) stained positive for CD206 and M-CSF-derived macrophages (M-Mφ) for CD163. THP-1 cells did not express CD206 or CD163 following incubation with PMA, M- or GM-CSF alone or in combination. Upon infection with Lm, all primary macrophages showed good survival at high multiplicities of infection whereas viability of THP-1 was severely reduced even at lower bacterial numbers. M-Mφ generally showed high phagocytosis of Lm. Strikingly, phagocytosis of Lm by GM-Mφ was markedly influenced by the method used for isolation of monocytes. GM-Mφ derived from negatively isolated monocytes showed low phagocytosis of Lm whereas GM-Mφ generated from positively selected monocytes displayed high phagocytosis of Lm. Moreover, incubation with CD14 antibody was sufficient to enhance phagocytosis of Lm by GM-Mφ generated from negatively isolated monocytes. By contrast, non-specific phagocytosis of latex beads by GM-Mφ was not influenced by treatment with CD14 antibody. Furthermore, phagocytosis of Lactococcus lactis, Escherichia coli, human cytomegalovirus and the protozoan parasite Leishmania major by GM-Mφ was not enhanced upon treatment with CD14 antibody indicating that this effect is specific for Lm. Based on these observations, we propose macrophages derived by ex vivo differentiation of negatively selected human primary monocytes as the most suitable model to study Lm infection of macrophages.     

Characterization and expression analysis of P5CS (Δ1-pyrroline-5-carboxylate synthase) gene in two distinct populations of the Atlantic Forest native species Eugenia uniflora L.

Molecular Biology Reports - Eugenia uniflora is an Atlantic Forest native species, occurring in contrasting edaphoclimatic environments. The identification of genes involved in response to abiotic...     

Caspase-1 deficiency in mice reduces intestinal triglyceride absorption and hepatic triglyceride secretion

Caspase-1 is known to activate the proinflammatory cytokines IL-1β and IL-18. Additionally, it can cleave other substrates, including proteins involved in metabolism. Recently, we showed that caspase-1 deficiency in mice strongly reduces high-fat diet-induced weight gain, at least partly caused by an increased energy production. Increased feces secretion by caspase-1-deficient mice suggests that lipid malabsorption possibly further reduces adipose tissue mass. In this study we investigated whether caspase-1 plays a role in triglyceride-(TG)-rich lipoprotein metabolism using caspase-1-deficient and wild-type mice. Caspase-1 deficiency reduced the postprandial TG response to an oral lipid load, whereas TG-derived fatty acid (FA) uptake by peripheral tissues was not affected, demonstrated by unaltered kinetics of [³H]TG-labeled very low-density lipoprotein (VLDL)-like emulsion particles. An oral gavage of [³H]TG-containing olive oil revealed that caspase-1 deficiency reduced TG absorption and subsequent uptake of TG-derived FA in liver, muscle, and adipose tissue. Similarly, despite an elevated hepatic TG content, caspase-1 deficiency reduced hepatic VLDL-TG production. Intestinal and hepatic gene expression analysis revealed that caspase-1 deficiency did not affect FA oxidation or FA uptake but rather reduced intracellular FA transport, thereby limiting lipid availability for the assembly and secretion of TG-rich lipoproteins. The current study reveals a novel function for caspase-1, or caspase-1-cleaved substrates, in controlling intestinal TG absorption and hepatic TG secretion.     

Reduced CCK signaling in obese-prone rats fed a high fat diet

Deficits in satiation signaling during obesogenic feeding have been proposed to play a role in hyperphagia and weight gain in animals prone to become obese. However, whether this impaired signaling is due to high fat (HF) feeding or to their obese phenotype is still unknown. Therefore, in the current study, we examined the effects of CCK-8 (0.5, 1.0, 2.0, and 4.0 μg/kg) on suppression of food intake of HF-fed obese prone (OP) and resistant (OR) rats. Additionally, we determined the role of endogenous CCK in lipid-induced satiation by measuring plasma CCK levels following a lipid gavage, and tested the effect of pretreatment with devazepide, a CCK-1R antagonist on intragastric lipid-induced satiation. Finally, we examined CCK-1R mRNA levels in the nodose ganglia. We show that OP rats have reduced feeding responses to the low doses of exogenous CCK-8 compared to OR rats. Furthermore, OP rats exhibit deficits in endogenous CCK signaling, as pretreatment with devazepide failed to abolish the reduction in food intake following lipid gavage. These effects were associated with reduced plasma CCK after intragastric lipid in OP but not OR rats. Furthermore, HF feeding resulted in downregulation of CCK-1Rs in the nodose ganglia of OP rats. Collectively, these results demonstrate that HF feeding leads to impairments in lipid-induced CCK satiation signaling in obese-prone rats, potentially contributing to hyperphagia and weight gain.