Carboxylesterase 1 Gene Duplication and mRNA Expression in Adipose Tissue Are Linked to Obesity and Metabolic Function

Context and Aims

Carboxylesterase 1 (CES1) appears to play an important role in the control of the metabolism of triglycerides and cholesterol in adipocytes and other cell types including hepatocytes. Therefore, it is relevant to gain insights into the genetic versus non-genetic mechanisms involved in the control of CES1 mRNA expression. Here, we investigated CES1 mRNA expression level in adipose tissue and its association with measures of adiposity and metabolic function in a population of elderly twins. Furthermore, the heritability of CES1 mRNA expression level in adipose tissue and the effect of CES1 gene duplication were assessed.

Methodology

A total of 295 monozygotic and dizygotic twin subjects (62–83 years) with (n = 48) or without (n = 247) type 2 diabetes mellitus were enrolled in the study. They were subjected to a standard oral glucose tolerance test and excision of abdominal subcutaneous fat biopsies during the fasting state. Levels of CES1 mRNA and copy number of the gene were assessed by quantitative PCR.

Results

CES1 mRNA expression level in adipose tissue was positively associated with body-mass index (P<0.001), homeostasis model assessment-insulin resistance (P = 0.003) and level of fasting glucose (P = 0.002), insulin (P = 0.006), and triglycerides (P = 0.003). The heritability for the expression of CES1 mRNA in adipose tissue was high. CES1 gene duplication was positively associated with insulin sensitivity (P = 0.05) as well as glucose tolerance (P = 0.03) and negatively associated with homeostasis model assessment-insulin resistance (P = 0.02). Duplication of CES1 was not linked to mRNA level of this gene (P = 0.63).

Conclusion

CES1 mRNA in adipose tissue appears to be under strong genetic control and was associated with measures of metabolic function raising the possibility of a potential role of this enzyme in the development of type 2 diabetes mellitus. Further studies are needed to understand the potential effect of CES1 gene duplication on adipocyte and whole-body metabolic functions.     

Natural Haemozoin Induces Expression and Release of Human Monocyte Tissue Inhibitor of Metalloproteinase-1

Recently matrix metalloproteinase-9 (MMP-9) and its endogenous inhibitor (tissue inhibitor of metalloproteinase-1, TIMP-1) have been implicated in complicated malaria. In vivo, mice with cerebral malaria (CM) display high levels of both MMP-9 and TIMP-1, and in human patients TIMP-1 serum levels directly correlate with disease severity. In vitro, natural haemozoin (nHZ, malarial pigment) enhances monocyte MMP-9 expression and release. The present study analyses the effects of nHZ on TIMP-1 regulation in human adherent monocytes. nHZ induced TIMP-1 mRNA expression and protein release, and promoted TNF-α, IL-1β, and MIP-1α/CCL3 production. Blocking antibodies or recombinant cytokines abrogated or mimicked nHZ effects on TIMP-1, respectively. p38 MAPK and NF-κB inhibitors blocked all nHZ effects on TIMP-1 and pro-inflammatory molecules. Still, total gelatinolytic activity was enhanced by nHZ despite TIMP-1 induction. Collectively, these data indicate that nHZ induces inflammation-mediated expression and release of human monocyte TIMP-1 through p38 MAPK- and NF-κB-dependent mechanisms. However, TIMP-1 induction is not sufficient to counterbalance nHZ-dependent MMP-9 enhancement. Future investigation on proteinase-independent functions of TIMP-1 (i.e. cell survival promotion and growth/differentiation inhibition) is needed to clarify the role of TIMP-1 in malaria pathogenesis.     

Molecular Mechanics Calculations of Proteins. Comparison of Different Energy Minimization Strategies

Abstract

A general strategy for performing energy minimization of proteins using the SYBYL molecular modelling program has been developed. The influence of several variables including energy minimization procedure, solvation, dielectric function and dielectric constant have been investigated in order to develop a general method, which is capable of producing high quality protein structures. Avian pancreatic polypeptide (APP) and bovine pancreatic phospholipase A2 (BP PLA2) were selected for the calculations, because high quality X-ray structures exist and because all classes of secondary structure are represented in the structures. The energy minimized structures were evaluated relative to the corresponding X-ray structures. The overall similarity was checked by calculating RMS distances for all atom positions. Backbone conformation was checked by Ramachandran plots and secondary structure elements evaluated by the length on hydrogen bonds. The dimensions of active site in BP PLA2 is very dependent on electrostatic interactions, due to the presence of the positively charged calcium ion. Thus, the distances between calcium and the calcium-coordinating groups were used as a quality index for this protein. Energy minimized structures of the trimeric PLA2 from Indian cobra (N.n.n. PLA2) were used for assessing the impact of protein-protein interactions. Based on the above mentioned criteria, it could be concluded that using the following conditions: Dielectric constant ? = 4 or 20; a distance dependent dielectric function and stepwise energy minimization, it is possible to reproduce X-ray structures very accurately without including explicit solvent molecules.     

Meta-Analysis of Quantification Methods Shows that Archaea and Bacteria Have Similar Abundances in the Subseafloor

There is no universally accepted method to quantify bacteria and archaea in seawater and marine sediments, and different methods have produced conflicting results with the same samples. To identify best practices, we compiled data from 65 studies, plus our own measurements, in which bacteria and archaea were quantified with fluorescent in situ hybridization (FISH), catalyzed reporter deposition FISH (CARD-FISH), polyribonucleotide FISH, or quantitative PCR (qPCR). To estimate efficiency, we defined “yield” to be the sum of bacteria and archaea counted by these techniques divided by the total number of cells. In seawater, the yield was high (median, 71%) and was similar for FISH, CARD-FISH, and polyribonucleotide FISH. In sediments, only measurements by CARD-FISH in which archaeal cells were permeabilized with proteinase K showed high yields (median, 84%). Therefore, the majority of cells in both environments appear to be alive, since they contain intact ribosomes. In sediments, the sum of bacterial and archaeal 16S rRNA gene qPCR counts was not closely related to cell counts, even after accounting for variations in copy numbers per genome. However, qPCR measurements were precise relative to other qPCR measurements made on the same samples. qPCR is therefore a reliable relative quantification method. Inconsistent results for the relative abundance of bacteria versus archaea in deep subsurface sediments were resolved by the removal of CARD-FISH measurements in which lysozyme was used to permeabilize archaeal cells and qPCR measurements which used ARCH516 as an archaeal primer or TaqMan probe. Data from best-practice methods showed that archaea and bacteria decreased as the depth in seawater and marine sediments increased, although archaea decreased more slowly.     

Comparison of 26 Sphingomonad Genomes Reveals Diverse Environmental Adaptations and Biodegradative Capabilities

Sphingomonads comprise a physiologically versatile group within the Alphaproteobacteria that includes strains of interest for biotechnology, human health, and environmental nutrient cycling. In this study, we compared 26 sphingomonad genome sequences to gain insight into their ecology, metabolic versatility, and environmental adaptations. Our multilocus phylogenetic and average amino acid identity (AAI) analyses confirm that Sphingomonas, Sphingobium, Sphingopyxis, and Novosphingobium are well-resolved monophyletic groups with the exception of Sphingomonas sp. strain SKA58, which we propose belongs to the genus Sphingobium. Our pan-genomic analysis of sphingomonads reveals numerous species-specific open reading frames (ORFs) but few signatures of genus-specific cores. The organization and coding potential of the sphingomonad genomes appear to be highly variable, and plasmid-mediated gene transfer and chromosome-plasmid recombination, together with prophage- and transposon-mediated rearrangements, appear to play prominent roles in the genome evolution of this group. We find that many of the sphingomonad genomes encode numerous oxygenases and glycoside hydrolases, which are likely responsible for their ability to degrade various recalcitrant aromatic compounds and polysaccharides, respectively. Many of these enzymes are encoded on megaplasmids, suggesting that they may be readily transferred between species. We also identified enzymes putatively used for the catabolism of sulfonate and nitroaromatic compounds in many of the genomes, suggesting that plant-based compounds or chemical contaminants may be sources of nitrogen and sulfur. Many of these sphingomonads appear to be adapted to oligotrophic environments, but several contain genomic features indicative of host associations. Our work provides a basis for understanding the ecological strategies employed by sphingomonads and their role in environmental nutrient cycling.     

Risk factors for <Emphasis Type="Italic">Coxiella burnetii</Emphasis> antibodies in bulk tank milk from Danish dairy herds

The aim was to identify risk factors associated with Coxiella burnetii antibody positivity in bulk tank milk (BTM) samples from 100 randomly selected Danish dairy cattle herds. Antibody levels were measured by an enzyme-linked immuno-sorbent assay. Before testing the herds, the farm managers were interviewed about hired labour, biosecurity, housing and herd health during the 12 months prior to the study. Variables considered important for C. burnetii antibody positivity in multivariable logistic regression analysis included the sharing of machines between farms (OR?=?3.6), human contacts (OR?=?4.2), artificial insemination by other people than artificial insemination technicians (OR?=?7.7), routine herd health contract with the veterinarian (OR?=?4.3) and hygiene precautions taken by veterinarians (OR?=?5). In addition, herd size, hired labour, trading of cattle between farms, quarantine and use of calving and disease pens also showed significant association in univariable analysis. This study demonstrates that strict biosecurity is important for the prevention of infections with C. burnetii.     

Unique Gene Expression and MR T2 Relaxometry Patterns Define Chronic Murine Dextran Sodium Sulphate Colitis as a Model for Connective Tissue Changes in Human Crohn’s Disease

Introduction

Chronically relapsing inflammation, tissue remodeling and fibrosis are hallmarks of inflammatory bowel diseases. The aim of this study was to investigate changes in connective tissue in a chronic murine model resulting from repeated cycles of dextran sodium sulphate (DSS) ingestion, to mimic the relapsing nature of the human disease.

Materials and Methods

C57BL/6 mice were exposed to DSS in drinking water for 1 week, followed by a recovery phase of 2 weeks. This cycle of exposure was repeated for up to 3 times (9 weeks in total). Colonic inflammation, fibrosis, extracellular matrix proteins and colonic gene expression were studied. In vivo MRI T ₂ relaxometry was studied as a potential non-invasive imaging tool to evaluate bowel wall inflammation and fibrosis.

Results

Repeated cycles of DSS resulted in a relapsing and remitting disease course, which induced a chronic segmental, transmural colitis after 2 and 3 cycles of DSS with clear induction of fibrosis and remodeling of the muscular layer. Tenascin expression mirrored its expression in Crohn’s colitis. Microarray data identified a gene expression profile different in chronic colitis from that in acute colitis. Additional recovery was associated with upregulation of unique genes, in particular keratins, pointing to activation of molecular pathways for healing and repair. In vivo MRI T₂ relaxometry of the colon showed a clear shift towards higher T₂ values in the acute stage and a gradual regression of T₂ values with increasing cycles of DSS.

Conclusions

Repeated cycles of DSS exposure induce fibrosis and connective tissue changes with typical features, as occurring in Crohn’s disease. Colonic gene expression analysis revealed unique expression profiles in chronic colitis compared to acute colitis and after additional recovery, pointing to potential new targets to intervene with the induction of fibrosis. In vivo T₂ relaxometry is a promising non-invasive assessment of inflammation and fibrosis.     

Why Has the Number of Scientific Retractions Increased?

Background

The number of retracted scientific publications has risen sharply, but it is unclear whether this reflects an increase in publication of flawed articles or an increase in the rate at which flawed articles are withdrawn.

Methods and Findings

We examined the interval between publication and retraction for 2,047 retracted articles indexed in PubMed. Time-to-retraction (from publication of article to publication of retraction) averaged 32.91 months. Among 714 retracted articles published in or before 2002, retraction required 49.82 months; among 1,333 retracted articles published after 2002, retraction required 23.82 months (p<0.0001). This suggests that journals are retracting papers more quickly than in the past, although recent articles requiring retraction may not have been recognized yet. To test the hypothesis that time-to-retraction is shorter for articles that receive careful scrutiny, time-to-retraction was correlated with journal impact factor (IF). Time-to-retraction was significantly shorter for high-IF journals, but only ∼1% of the variance in time-to-retraction was explained by increased scrutiny. The first article retracted for plagiarism was published in 1979 and the first for duplicate publication in 1990, showing that articles are now retracted for reasons not cited in the past. The proportional impact of authors with multiple retractions was greater in 1972–1992 than in the current era (p<0.001). From 1972–1992, 46.0% of retracted papers were written by authors with a single retraction; from 1993 to 2012, 63.1% of retracted papers were written by single-retraction authors (p<0.001).

Conclusions

The increase in retracted articles appears to reflect changes in the behavior of both authors and institutions. Lower barriers to publication of flawed articles are seen in the increase in number and proportion of retractions by authors with a single retraction. Lower barriers to retraction are apparent in an increase in retraction for “new” offenses such as plagiarism and a decrease in the time-to-retraction of flawed work.     

Glucose‐based microbial production of the hormone melatonin in yeast Saccharomyces cerevisiae

Melatonin is a natural mammalian hormone that plays an important role in regulating the circadian cycle in humans. It is a clinically effective drug exhibiting positive effects as a sleep aid and a powerful antioxidant used as a dietary supplement. Commercial melatonin production is predominantly performed by complex chemical synthesis. In this study, we demonstrate microbial production of melatonin and related compounds, such as serotonin and N‐acetylserotonin. We generated Saccharomyces cerevisiae strains that comprise heterologous genes encoding one or more variants of an L‐tryptophan hydroxylase, a 5‐hydroxy‐L‐tryptophan decarboxylase, a serotonin acetyltransferase, an acetylserotonin O‐methyltransferase, and means for providing the cofactor tetrahydrobiopterin via heterologous biosynthesis and recycling pathways. We thereby achieved de novo melatonin biosynthesis from glucose. We furthermore accomplished increased product titers by altering expression levels of selected pathway enzymes and boosting co‐factor supply. The final yeast strain produced melatonin at a titer of 14.50 ± 0.57 mg L^?1 in a 76h fermentation using simulated fed‐batch medium with glucose as sole carbon source. Our study lays the basis for further developing a yeast cell factory for biological production of melatonin.