Prevention and reversal of hepatic steatosis with a high-protein diet in mice

The hallmark of NAFLD is steatosis of unknown etiology. We tested the effect of a high-protein (HP)² diet on diet-induced steatosis in male C57BL/6 mice with and without pre-existing fatty liver. Mice were fed all combinations of semisynthetic low-fat (LF) or high-fat (HF) and low-protein (LP) or HP diets for 3 weeks. To control for reduced energy intake by HF/HP-fed mice, a pair-fed HF/LP group was included. Reversibility of pre-existing steatosis was investigated by sequentially feeding HF/LP and HF/HP diets. HP-containing diets decreased hepatic lipids to ~ 40% of corresponding LP-containing diets, were more efficient in this respect than reducing energy intake to 80%, and reversed pre-existing diet-induced steatosis. Compared to LP-containing diets, mice fed HP-containing diets showed increased mitochondrial oxidative capacity (elevated Pgc1α, mAco, and Cpt1 mRNAs, complex-V protein, and decreased plasma free and short-chain acyl-carnitines, and [C₀]/[C₁₆ + C₁₈] carnitine ratio); increased gluconeogenesis and pyruvate cycling (increased PCK1 protein and fed plasma–glucose concentration without increased G6pase mRNA); reduced fatty-acid desaturation (decreased Scd1 expression and [C_16:1n ? 7]/[C_16:0] ratio) and increased long-chain PUFA elongation; a selective increase in plasma branched-chain amino acids; a decrease in cell stress (reduced phosphorylated eIF2α, and Fgf21 and Chop expression); and a trend toward less inflammation (lower Mcp1 and Cd11b expression and less phosphorylated NFκB). Conclusion: HP diets prevent and reverse steatosis independently of fat and carbohydrate intake more efficiently than a 20% reduction in energy intake. The effect appears to result from fuel-generated, highly distributed small, synergistic increases in lipid and BCAA catabolism, and a decrease in cell stress.     

Insulin analog with additional disulfide bond has increased stability and preserved activity

Insulin is a key hormone controlling glucose homeostasis. All known vertebrate insulin analogs have a classical structure with three 100% conserved disulfide bonds that are essential for structural stability and thus the function of insulin. It might be hypothesized that an additional disulfide bond may enhance insulin structural stability which would be highly desirable in a pharmaceutical use. To address this hypothesis, we designed insulin with an additional interchain disulfide bond in positions A10/B4 based on Cα‐Cα distances, solvent exposure, and side‐chain orientation in human insulin (HI) structure. This insulin analog had increased affinity for the insulin receptor and apparently augmented glucodynamic potency in a normal rat model compared with HI. Addition of the disulfide bond also resulted in a 34.6°C increase in melting temperature and prevented insulin fibril formation under high physical stress even though the C‐terminus of the B‐chain thought to be directly involved in fibril formation was not modified. Importantly, this analog was capable of forming hexamer upon Zn addition as typical for wild‐type insulin and its crystal structure showed only minor deviations from the classical insulin structure. Furthermore, the additional disulfide bond prevented this insulin analog from adopting the R‐state conformation and thus showing that the R‐state conformation is not a prerequisite for binding to insulin receptor as previously suggested. In summary, this is the first example of an insulin analog featuring a fourth disulfide bond with increased structural stability and retained function.     

Development of new α-amylases for raw starch hydrolysis

This paper describes the discovery of a new 4 domain α-amylase from Anoxybacillus contaminans which very efficiently hydrolyses raw starch granules. Compared to traditional starch liquefying α-amylases, this new 4 domain α-amylase contains a starch binding domain. The presence of this starch-binding domain enables the enzyme to efficiently hydrolyse starch at a temperature below the gelatinisation temperature. At a reaction temperature of 60°C and in combination with a glucoamylase from Aspergillusniger it was possible to liquefy 99% of the starch obtaining a DX value of 95%.

Furthermore, we describe how the current HFCS process can be turned into a low temperature simultaneous liquefaction and saccharification process by using this new 4 domain α-amylase in combination with a glucoamylase.     

Evaluation of the INS-1 832/13 Cell Line as a Beta-Cell Based Screening System to Assess Pollutant Effects on Beta-Cell Function

Environmental pollutants have recently emerged as potential risk factors for metabolic diseases, urging systematic investigation of pollutant effects on metabolic disease processes. To enable risk assessment of these so-called metabolic disruptors the use of stable, robust and well-defined cell based screening systems has recently been encouraged. Since beta-cell (dys)functionality is central in diabetes pathophysiology, the need to develop beta-cell based pollutant screening systems is evident. In this context, the present research evaluated the strengths and weaknesses of the INS-1 832/13 pancreatic beta-cell line as diabetogenic pollutant screening system with a focus on beta-cell function. After optimization of exposure conditions, positive (exendin-4, glibenclamide) and negative (diazoxide) control compounds for acute insulin secretion responses were tested and those with the most profound effects were selected to allow potency estimations and ranking of pollutants. This was followed by a first explorative screening of acute bisphenol A and bis(2-ethylhexyl)phthalate effects. The same approach was applied for chronic exposures, focusing primarily on evaluation of acknowledged chronic stimulators (diazoxide, T0901317, exendin-4) or inhibitors (glibenclamide) of insulin secretion responses to select the most responsive ones for use as control compounds in a chronic pollutant testing framework. Our results showed that INS-1 832/13 cells responded conform previous observations regarding acute effects of control compounds on insulin secretion, while bisphenol A and bis(2-ethylhexyl)phthalate had limited acute effects. Furthermore, chronic exposure to known beta-cell reactive compounds resulted in deviating insulin secretion and insulin content profiles compared to previous reports. In conclusion, this INS-1 subclone appears to lack certain characteristics needed to respond appropriately to acute pollutant exposure or long term exposure to known beta-cell reactive compounds and thus seems to be, in our setting, inadequate as a diabetogenic pollutant screening system.     

Immunogenicity of Live Attenuated B. pertussis BPZE1 Producing the Universal Influenza Vaccine Candidate M2e

Background

Intranasal delivery of vaccines directed against respiratory pathogens is an attractive alternative to parenteral administration. However, using this delivery route for inactivated vaccines usually requires the use of potent mucosal adjuvants, and no such adjuvant has yet been approved for human use.

Methodology/Principal Findings

We have developed a live attenuated Bordetella pertussis vaccine, called BPZE1, and show here that it can be used to present the universal influenza virus epitope M2e to the mouse respiratory tract to prime for protective immunity against viral challenge. Three copies of M2e were genetically fused to the N-terminal domain of filamentous hemagglutinin (FHA) and produced in recombinant BPZE1 derivatives in the presence or absence of endogenous full-length FHA. Only in the absence of FHA intranasal administration of the recombinant BPZE1 derivative induced antibody responses to M2e and effectively primed BALB/c mice for protection against influenza virus-induced mortality and reduced the viral load after challenge. Strong M2e-specific antibody responses and protection were observed after a single nasal administration with the recombinant BPZE1 derivative, followed by a single administration of M2e linked to a virus-like particle without adjuvant, whereas priming alone with the vaccine strain did not protect.

Conclusions/Significance

Using recombinant FHA-3M2e-producing BPZE1 derivatives for priming and the universal influenza M2e peptide linked to virus-like particles for boosting may constitute a promising approach for needle-free and adjuvant-free nasal vaccination against influenza.     

Reference values for height, weight and body mass index of German born Turkish children

Turkish children and adolescents born in Northern Europe grow different from native Northern European children, but reference values for height, weight and BMI for these children do not exist. With this study, we intend to provide growth standards for German born Turkish children. Data were obtained from 797 Turkish children and adolescents born in Germany age 0-25.8 years (males), respectively 0-18.3 years (females). We generated synthetic reference values for height, weight, and BMI. The results show that Turkish children and adolescents are heavier after the age of 6 years, and that they remain short after puberty. Eighteen year old Turkish men, and 15-year-old Turkish women are shorter (males 175.2 cm vs. 180.4 cm, p < 0.05; females 159.3 cm vs. 165.0 cm, p < 0.05), and heavier than Germans. Six out of 53 young Turkish men and 9 out of 100 young Turkish women were obese. Twelve out of 53 young Turkish men (23%) and 18 out of 100 young Turkish women (18%) have fallen below the 3rd centile for height. It can be concluded that growth of Turkish children and adolescents born in Germany significantly differs from native children. Reference LMS values for body height, weight and BMI of German born Turkish boys and girls are presented.     

Conjugative transfer facilitates stable maintenance of IncP-1 plasmid pKJK5 in Escherichia coli cells colonizing the gastrointestinal tract of the germfree rat

Quantitative determination of IncP-1 plasmid loss from Escherichia coli cells colonizing the gastrointestinal tracts of germfree rats was achieved by flow cytometry. Results show that the plasmid's ability to conjugate counteracts plasmid loss and is thus an important mechanism for the stable maintenance of IncP-1 plasmids within the gastrointestinal environment.     

Recombinant expression and characterization of a reducing-end xylose-releasing exo-oligoxylanase from Bifidobacterium adolescentis

The family 8 glycoside hydrolase (RexA) from Bifidobacterium adolescentis was expressed in Escherichia coli. The recombinant enzyme was characterized as a reducing-end xylose-releasing exo-oligoxylanase. Apart from giving insights into this new class of enzymes, knowledge of the RexA enzyme helps to postulate a mechanism for the B. adolescentis breakdown of prebiotic xylooligosaccharides.     

Targeted molecular engineering of a family 11 endoxylanase to decrease its sensitivity towards Triticum aestivum endoxylanase inhibitor types

The Bacillus subtilis endoxylanase XynA (BSXY) is frequently used to improve the functionality of arabinoxylan-containing material in cereal based industries. The presence of endogenous Triticum aestivum xylanase inhibitors (TAXI-I and TAXI-II) in wheat is a real concern as they have a direct negative impact on the efficiency of this enzyme. Here, we used the recently determined structure of the complex between TAXI-I and an endoxylanase of Aspergillus niger to develop inhibitor-insensitive BSXY variants by site-directed mutagenesis of strategically chosen amino acids. We either induced steric hindrance to reject the inhibitors or interrupted key interactions with the inhibitors in the endoxylanase substrate-binding groove. The first strategy was successfully applied to position G12 where G12W combined inhibition insensitivity with unharmed catalytic performance. Variants from the second strategy showed altered inhibitor sensitivities concomitant with changes in enzyme activities and allowed to gain insight in the binding-mode of both TAXI-I and TAXI-II with BSXY.