Intestinal gluconeogenesis is a key factor for early metabolic changes after gastric bypass but not after gastric lap-band in mice

Unlike the adjustable gastric banding procedure (AGB), Roux-en-Y gastric bypass surgery (RYGBP) in humans has an intriguing effect: a rapid and substantial control of type 2 diabetes mellitus (T2DM). We performed gastric lap-band (GLB) and entero-gastro anastomosis (EGA) procedures in C57Bl6 mice that were fed a high-fat diet. The EGA procedure specifically reduced food intake and increased insulin sensitivity as measured by endogenous glucose production. Intestinal gluconeogenesis increased after the EGA procedure, but not after gastric banding. All EGA effects were abolished in GLUT-2 knockout mice and in mice with portal vein denervation. We thus provide mechanistic evidence that the beneficial effects of the EGA procedure on food intake and glucose homeostasis involve intestinal gluconeogenesis and its detection via a GLUT-2 and hepatoportal sensor pathway.     

Sinorhizobium meliloti rpoE2 is necessary for H2O2 stress resistance during the stationary growth phase

RpoE2 is an extracytoplasmic σ factor produced by Sinorhizobium meliloti during stationary growth phase. Its inactivation affected the synthesis of the superoxide dismutase, SodC, and catalase, KatC. The absence of SodC within the cell did not result in an increased sensitivity to extracellular superoxides. In contrast, the absence of KatC affected the resistance of S. meliloti to H₂O₂ during the stationary growth phase. A katC strain behaved as an rpoE2 strain during an H₂O₂ challenge, suggesting that the H₂O₂ sensitivity of the rpoE2 strain resulted only from the lack of KatC in this strain.     

The First Transmembrane Domain of the Hepatitis B Virus Large Envelope Protein Is Crucial for Infectivity

The early steps of the hepatitis B virus (HBV) life cycle are still poorly understood. Indeed, neither the virus receptor at the cell surface nor the mechanism by which nucleocapsids are delivered to the cytosol of infected cells has been identified. Extensive mutagenesis studies in pre-S1, pre-S2, and most of the S domain of envelope proteins revealed the presence of two regions essential for HBV infectivity: the 77 first residues of the pre-S1 domain and a conformational motif in the antigenic loop of the S domain. In addition, at the N-terminal extremity of the S domain, a putative fusion peptide, partially overlapping the first transmembrane (TM1) domain and preceded by a PEST sequence likely containing several proteolytic cleavage sites, was identified. Since no mutational analysis of these two motifs potentially implicated in the fusion process was performed, we decided to investigate the ability of viruses bearing contiguous deletions or substitutions in the putative fusion peptide and PEST sequence to infect HepaRG cells. By introducing the mutations either in the L and M proteins or in the S protein, we demonstrated the following: (i) that in the TM1 domain of the L protein, three hydrophobic clusters of four residues were necessary for infectivity; (ii) that the same clusters were critical for S protein expression; and, finally, (iii) that the PEST sequence was dispensable for both assembly and infection processes.The hepatitis B virus (HBV) is the main human pathogen responsible for severe hepatic diseases like cirrhosis and hepatocellular carcinoma. Even though infection can be prevented by immunization with an efficient vaccine, about 2 billion people have been infected worldwide, resulting in 350 million chronic carriers that are prone to develop liver diseases (56). Current treatments consist either of the use of interferon α, which modulates antiviral defenses and controls infection in 30 to 40% of cases, or of the use of viral polymerase inhibitors that allow a stronger response to treatment but require long-term utilization and frequently lead to the outcome of resistant viruses (34, 55). A better understanding of the virus life cycle, and particularly of the mechanism by which the virus enters the cell, could provide background for therapeutics that inhibit the early steps of infection, as recently illustrated with the HBV pre-S1-derived entry inhibitor (25, 45).HBV belongs to the Hepadnaviridae family whose members infect different species. All viruses of this family share common properties. The capsid containing a partially double-stranded circular DNA genome is surrounded by a lipid envelope, in which two (in avihepadnaviruses infecting birds) or three (in orthohepadnaviruses infecting mammals) envelope proteins are embedded. A single open reading frame bearing several translation initiation sites encodes these surface proteins. Thus, the HBV envelope contains three proteins: S, M, and L that share the same C-terminal extremity corresponding to the small S protein that is crucial for virus assembly (7, 8, 46) and infectivity (1, 31, 53). These proteins are synthesized in the endoplasmic reticulum (ER), assembled, and secreted as particles through the Golgi apparatus (15, 42). The current model for the transmembrane structure of the S domain implies the luminal exposition of both N- and C-terminal extremities and the presence of four transmembrane (TM) domains: the TM1 and TM2 domains, both necessary for cotranslational protein integration into the ER membrane, and the TM3 and TM4 domains, located in the C-terminal third of the S domain (for a review, see reference 6). Among the four predicted TM domains, only the TM2 domain has a defined position between amino acids 80 and 98 of the S domain. The exact localization of the TM1 domain is still unclear, probably because of the relatively low hydrophobicity of its sequence, which contains polar residues and two prolines. The M protein corresponds to the S protein extended by an N-terminal domain of 55 amino acids called pre-S2. Its presence is dispensable for both assembly and infectivity (20, 21, 37). Finally, the L protein corresponds to the M protein extended by an N-terminal domain of 108 amino acids called pre-S1 (genotype D). The pre-S1 and pre-S2 domains of the L protein can be present either at the inner face of viral particles (on the cytoplasmic side of the ER), playing a crucial role in virus assembly (5, 8, 10, 11, 46), or on the outer face (on the luminal side of the ER), available for the interaction with target cells and necessary for viral infectivity (4, 14, 36). The pre-S translocation is independent from the M and S proteins and is driven by the L protein TM2 domain (33). Finally, HBV surface proteins are not only incorporated into virion envelopes but also spontaneously bud from ER-Golgi intermediate compartment membranes (30, 43) to form empty subviral particles (SVPs) that are released from the cell by secretion (8, 40).One approach to decipher viral entry is to interfere with the function of envelope proteins. Thus, by a mutagenesis approach, two envelope protein domains crucial for HBV infectivity have already been identified: (i) the 77 first amino acids of the pre-S1 domain (4, 36) including the myristic acid at the N-terminal extremity (9, 27) and (ii) possibly a cysteine motif in the luminal loop of the S domain (1, 31). In addition, a putative fusion peptide has been identified at the N-terminal extremity of the S domain due to its sequence homology with other viral fusion peptides (50). This sequence, either N-terminal in the S protein or internal in the L and M proteins, is conserved among the Hepadnaviridae family and shares common structural and functional properties with other fusion peptides (49, 50). Finally, a PEST sequence likely containing several proteolytic cleavage sites has been identified in the L and M proteins upstream of the TM1 domain (39). A cleavage within this sequence could activate the fusion peptide.In this study, we investigated whether the putative fusion peptide and the PEST sequence were necessary for the infection process. For this purpose, we constructed a set of mutant viruses bearing contiguous deletions in these regions and determined their infectivity using an in vitro infection model based on HepaRG cells (28). The introduction of mutations either in the L and M proteins or in only the S protein allowed us to demonstrate that, in the TM1 domain of L protein, three hydrophobic clusters not essential for viral assembly were crucial for HBV infectivity while their presence in the S protein was critical for envelope protein expression. In addition, we showed that the PEST sequence was clearly dispensable for both assembly and infection processes.     

Biomarker discovery in asthma‐related inflammation and remodeling

Asthma is a complex inflammatory disease of airways. A network of reciprocal interactions between inflammatory cells, peptidic mediators, extracellular matrix components, and proteases is thought to be involved in the installation and maintenance of asthma‐related airway inflammation and remodeling. To date, new proteic mediators displaying significant activity in the pathophysiology of asthma are still to be unveiled. The main objective of this study was to uncover potential target proteins by using surface‐enhanced laser desorption/ionization‐time of flight‐mass spectrometry (SELDI‐TOF‐MS) on lung samples from mouse models of allergen‐induced airway inflammation and remodeling. In this model, we pointed out several protein or peptide peaks that were preferentially expressed in diseased mice as compared to controls. We report the identification of different five proteins: found inflammatory zone 1 or RELMα (FIZZ‐1), calcyclin (S100A6), clara cell secretory protein 10 (CC10), Ubiquitin, and Histone H4.     

Standing variation and new mutations both contribute to a fast response to selection for flowering time in maize inbreds

Background  

In order to investigate the rate and limits of the response to selection from highly inbred genetic material and evaluate the respective contribution of standing variation and new mutations, we conducted a divergent selection experiment from maize inbred lines in open-field conditions during 7 years. Two maize commercial seed lots considered as inbred lines, F252 and MBS847, constituted two biological replicates of the experiment. In each replicate, we derived an Early and a Late population by selecting and selfing the earliest and the latest individuals, respectively, to produce the next generation.     

Two Years after Molecular Diagnosis of Familial Hypercholesterolemia: Majority on Cholesterol-Lowering Treatment but a Minority Reaches Treatment Goal

Background

The risk of premature cardiovascular disease in patients with familial hypercholesterolemia (FH) can be profoundly reduced by cholesterol-lowering therapy, and current guidelines for FH advocate ambitious low-density lipoprotein cholesterol (LDL-C) goals. In the present study, we determined whether these goals are reflected in current clinical practice once FH has been diagnosed.

Methodology/Principal Findings

In 2008, we sent questionnaires to all subjects (aged 18–65 years) who were molecularly diagnosed with FH in the year 2006 through the screening program in the Netherlands. Of these 1062 subjects, 781 completed the questionnaire (46% males; mean age: 42±12 years; mean LDL-C at molecular diagnosis (baseline): 4.1±1.3 mmol/L). The number of persons that used cholesterol-lowering therapy increased from 397 (51%) at baseline to 636 (81%) after diagnosis. Mean treated LDL-C levels decreased significantly to 3.2±1.1 mmol/L two years after diagnosis. Only 22% achieved the LDL-C target level of ≤2.5 mmol/L.

Conclusions/Significance

The proportion of patients using cholesterol-lowering medication was significantly increased after FH diagnosis through genetic cascade screening. The attained LDL-C levels were lower than those reported in previous surveys on medication use in FH, which could reflect the effect of more stringent lipid target levels. However, only a minority of the medication users reached the LDL-C target.     

Development of fluorescent substrates for microsomal epoxide hydrolase and application to inhibition studies

The microsomal epoxide hydrolase (mEH) plays a significant role in the metabolism of numerous xenobiotics. In addition, it has a potential role in sexual development and bile acid transport, and it is associated with a number of diseases such as emphysema, spontaneous abortion, eclampsia, and several forms of cancer. Toward developing chemical tools to study the biological role of mEH, we designed and synthesized a series of absorbent and fluorescent substrates. The highest activity for both rat and human mEH was obtained with the fluorescent substrate cyano(6-methoxy-naphthalen-2-yl)methyl glycidyl carbonate (11). An in vitro inhibition assay using this substrate ranked a series of known inhibitors similarly to the assay that used radioactive cis-stilbene oxide but with a greater discrimination between inhibitors. These results demonstrate that the new fluorescence-based assay is a useful tool for the discovery of structure–activity relationships among mEH inhibitors. Furthermore, this substrate could also be used for the screening chemical library with high accuracy and with a Z′ value of approximately 0.7. This new assay permits a significant decrease in labor and cost and also offers the advantage of a continuous readout. However, it should not be used with crude enzyme preparations due to interfering reactions.