Characterization of the proteins of bacterial strain isolated from contaminated site involved in heavy metal resistance--a proteomic approach

The present study describes response of a bacterial strain isolated from a polluted river to heavy metal toxicity. The bacterium was identified to be Klebsiella pneumoniae by biochemical tests using API 20E strips and 16S ribotyping. The isolate was studied for its tolerance to two heavy metals, i.e., cobalt (Co(2+)) and lead (Pb(2+)) by growing it in citrate mineral medium (CMM). Proteomic approach involving two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and mass spectrometry (MS) was used to identify the differentially expressed proteins under heavy metal stress. Two of the differentially expressed proteins were identified to be l-isoaspartate protein carboxymethyltransferase type II and DNA gyrase A. To our knowledge, this is for the first time that K. pneumoniae has been reported to be present in metal contaminated site and l-isoaspartate protein carboxymethyltransferase type II protein to be over expressed under heavy metal stress. The role of these proteins in metal tolerance is discussed.     

Rational design and construction of multi-copy biomanufacturing islands in mammalian cells

Cell line development is a critical step in the establishment of a biopharmaceutical manufacturing process. Current protocols rely on random transgene integration and amplification. Due to considerable variability in transgene integration profiles, this workflow results in laborious screening campaigns before stable producers can be identified. Alternative approaches for transgene dosage increase and integration are therefore highly desirable. In this study, we present a novel strategy for the rapid design, construction, and genomic integration of engineered multiple-copy gene constructs consisting of up to 10 gene expression cassettes. Key to this strategy is the diversification, at the sequence level, of the individual gene cassettes without altering their protein products. We show a computational workflow for coding and regulatory sequence diversification and optimization followed by experimental assembly of up to nine gene copies and a sentinel reporter on a contiguous scaffold. Transient transfections in CHO cells indicates that protein expression increases with the gene copy number on the scaffold. Further, we stably integrate these cassettes into a pre-validated genomic locus. Altogether, our findings point to the feasibility of engineering a fully mapped multi-copy recombinant protein ‘production island’ in a mammalian cell line with greatly reduced screening effort, improved stability, and predictable product titers.     

Measurement of sarcolemmal vesicle orientation by beta-adrenergic receptor binding.

To assess the orientation (inside-out vs. outside-out) of purified cardiac sarcolemmal vesicles, we developed a new method utilizing the known outward-facing binding site of the beta-adrenergic receptor. We compared the binding of the lipid-insoluble ligand 3H-CGP-12177, which binds to beta-adrenergic receptors on outside-out sarcolemmal vesicles only, to the binding of the lipid soluble ligand 125I-iodocyanopindolol, which binds to beta-adrenergic receptors in sarcolemmal vesicles of either orientation. The ratio of CGP to ICYP binding is equal to the fraction of outside-out sarcolemmal vesicles. Sidedness measurements by beta-adrenergic receptor-binding showed similar mean values but less scatter than sidedness assessments by measurement of 3H-ouabain-binding or Na+,K(+)-ATPase activity in the presence or absence of membrane permeabilizing agents.     

Zoonotic risk factors associated with seroprevalence of Ebola virus GP antibodies in the absence of diagnosed Ebola virus disease in the Democratic Republic of Congo

BackgroundEbola virus (EBOV) is a zoonotic filovirus spread through exposure to infected bodily fluids of a human or animal. Though EBOV is capable of causing severe disease, referred to as Ebola Virus Disease (EVD), individuals who have never been diagnosed with confirmed, probable or suspected EVD can have detectable EBOV antigen-specific antibodies in their blood. This study aims to identify risk factors associated with detectable antibody levels in the absence of an EVD diagnosis.MethodologyData was collected from September 2015 to August 2017 from 1,366 consenting individuals across four study sites in the DRC (Boende, Kabondo-Dianda, Kikwit, and Yambuku). Seroreactivity was determined to EBOV GP IgG using Zaire Ebola Virus Glycoprotein (EBOV GP antigen) ELISA kits (Alpha Diagnostic International, Inc.) in Kinshasa, DRC; any result above 4.7 units/mL was considered seroreactive. Among the respondents, 113 (8.3%) were considered seroreactive. Several zoonotic exposures were associated with EBOV seroreactivity after controlling for age, sex, healthcare worker status, location, and history of contact with an EVD case, namely: ever having contact with bats, ever having contact with rodents, and ever eating non-human primate meat. Contact with monkeys or non-human primates was not associated with seroreactivity.ConclusionsThis analysis suggests that some zoonotic exposures that have been linked to EVD outbreaks can also be associated with EBOV GP seroreactivity in the absence of diagnosed EVD. Future investigations should seek to clarify the relationships between zoonotic exposures, seroreactivity, asymptomatic infection, and EVD.     

Lack of acyl-CoA:diacylglycerol acyltransferase 1 reduces intestinal cholesterol absorption and attenuates atherosclerosis in apolipoprotein E knockout mice

Triacylglycerols (TG) are the major storage molecules of metabolic energy and fatty acids in several tissues. The final step in TG biosynthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. Lack of whole body DGAT1 is associated with reduced lipid-induced inflammation. Since one major component of atherosclerosis is chronic inflammation we hypothesized that DGAT1 deficiency might ameliorate atherosclerotic lesion development. We therefore crossbred Apolipoprotein E-deficient (ApoE(-/-)) mice with Dgat1(-/-) mice. ApoE(-/-) and ApoE(-/-)Dgat1(-/-) mice were fed Western-type diet (WTD) for 9weeks and thereafter examined for plaque formation. The mean atherosclerotic lesion area was substantially reduced in ApoE(-/-)Dgat1(-/-) compared with ApoE(-/-) mice in en face and aortic valve section analyses. The reduced lesion size was associated with decreased cholesterol uptake and absorption by the intestine, reduced plasma TG and cholesterol concentrations and increased cholesterol efflux from macrophages. The expression of adhesion molecules was reduced in aortas of ApoE(-/-)Dgat1(-/-) mice, which might be the reason for less migration capacities of monocytes and macrophages and the observed decreased amount of macrophages within the plaques. From our results we conclude that the lack of DGAT1 is atheroprotective, implicating an additional application of DGAT1 inhibitors with regard to maintaining cholesterol homeostasis and attenuating atherosclerosis.     

Ecological momentary assessment of obesogenic eating behavior: combining person-specific and environmental predictors

Obesity has been promoted by a food environment that encourages excessive caloric intake. An understanding of how the food environment contributes to obesogenic eating behavior in different types of individuals may facilitate healthy weight control efforts. In this study, Ecological Momentary Assessment (EMA) via palmtop computers was used to collect real-time information about participants' environment and eating patterns to predict overeating (i.e., greater than usual intake during routine meals/snacks, and eating outside of a participant's normal routine) that could lead to weight gain. Thirty-nine women (BMI = 21.6 ± 1.8; age = 20.1 ± 2.0 years; 61% white) of normal weight (BMI 18.5-25) completed the Three Factor Eating Questionnaire and the Power of Food Scale (PFS), and carried a palmtop computer for 7-10 days, which prompted them to answer questions about eating events, including a count of the types of good tasting high-calorie foods that were available. None of the self-report measures predicted overeating, but BMI interacted with the number of palatable foods available to predict overeating (P = 0.035). Compared to leaner individuals who reported a relatively low frequency of overeating regardless of the availability of palatable food, the probability of overeating among heavier individuals was very low in the absence of palatable food, but quickly increased in proportion to the number of palatable foods available. Our findings suggest that the eating behavior of those with higher relative weights is susceptible to the presence of palatable foods in the environment. Individuals practicing weight control may benefit from limiting their exposure to good tasting high-calorie food in their immediate environment.     

Permeation of Insulin,Calcitonin and Exenatide across Caco-2 Monolayers: Measurement Using a Rapid, 3-Day System

Objectives

Caco-2 monolayers are one of the most widely used in vitro models for prediction of intestinal permeability of therapeutic molecules. However, the conventional Caco-2 monolayer model has several drawbacks including labor-intensive culture process, unphysiological growth conditions, lack of reproducibility and limited throughput. Here, we report on the use of 3-day Caco-2 monolayers for assessing permeability of polypeptide drugs.

Methods

The 3-day monolayers were grown in a commercially available transwell set-up, which facilitates rapid development of the Caco-2 monolayers in an intestinal epithelial differentiation mimicking environment. This set-up included use of serum-free medium of defined composition with supplements such as butyric acid, hormones, growth factors, and other metabolites, reported to regulate the differentiation of intestinal epithelial cells in vivo. We measured permeability of 3 different therapeutic polypeptides; insulin, calcitonin, and exenatide across the monolayer.

Results

Preliminary validation of the monolayer was carried out by confirming dose-dependent permeation of FITC-insulin and sulforhodamine-B. Transport of insulin, calcitonin, and exenatide measured at different loading concentrations suggests that the permeability values obtained with 3-day cultures resemble more closely the values obtained with ex vivo models compared to permeability values obtained with conventional 21-day cultures.

Conclusions

Short-term 3-day Caco-2 monolayers provide new opportunities for developing reproducible and high-throughput models for screening of therapeutic macromolecules for oral absorption.