Optimization of codon composition and regulatory elements for expression of human insulin like growth factor-1 in transgenic chloroplasts and evaluation of structural identity and function

Background  

Transgenic chloroplasts are potential bioreactors for recombinant protein production, especially for achievement of high levels of protein expression and proper folding. Production of therapeutic proteins in leaves provides transgene containment by elimination of reproductive structures. Therefore, in this study, human Insulin like Growth Factor-1 is expressed in transgenic chloroplasts for evaluation of structural identity and function.     

The MAM (meprin/A5-protein/PTPmu) domain is a homophilic binding site promoting the lateral dimerization of receptor-like protein-tyrosine phosphatase mu

The MAM (meprin/A5-protein/PTPmu) domain is present in numerous proteins with diverse functions. PTPmu belongs to the MAM-containing subclass of protein-tyrosine phosphatases (PTP) able to promote cell-to-cell adhesion. Here we provide experimental evidence that the MAM domain is a homophilic binding site of PTPmu. We demonstrate that the MAM domain forms oligomers in solution and binds to the PTPmu ectodomain at the cell surface. The presence of two disulfide bridges in the MAM molecule was evidenced and their integrity was found to be essential for MAM homophilic interaction. Our data also indicate that PTPmu ectodomain forms oligomers and mediates the cellular adhesion, even in the absence of MAM domain homophilic binding. Reciprocally, MAM is able to interact homophilically in the absence of ectodomain trans binding. The MAM domain therefore contains independent cis and trans interaction sites and we predict that its main role is to promote lateral dimerization of PTPmu at the cell surface. This finding contributes to the understanding of the signal transduction mechanism in MAM-containing PTPs.     

Prospective Analysis of the Association of a Common Variant of FTO (rs9939609) with Adiposity in Children: Results of the IDEFICS Study

Objectives

We investigated cross-sectionally and longitudinally the relationship between FTO rs9939609 and obesity-related characteristics in the European children of the IDEFICS project and the interaction of this variant with a lifestyle intervention.

Population and Methods

A cohort of 16224 children (2–9 years) was recruited into a population-based survey (T0) from eight European countries. A second survey (T1) reassessed the children two years later. A random sample of 4405 children was extracted for genetic studies. 3168 children were re-examined two years later. Half of them underwent a lifestyle intervention program. The FTO rs9939609 was genotyped. Weight, height, waist circumference, triceps and subscapular skinfolds were measured at T0 and T1.

Results

At T0, the risk A allele of rs9939609 was significantly associated with higher values of body mass index (BMI), waist circumference and skinfolds (age, sex, and country-adjusted p-values: all p<0.001) and with a statistically significant increased risk of overweight/obesity.Over the two year follow-up, no interaction between genotype and intervention was observed. The A allele was associated to a significantly higher increase in all the anthropometric variables examined at T0 independently from the study group (intervention versus control) (p-values: all p<0.002, adjusted for age, sex, country, intervention/control study group, T0 values, and individual time interval between T0 and T1). Over the two-year follow–up, 210 new cases of overweight/obesity occurred. A statistically significant higher incidence of overweight/obesity was associated to the A allele [OR_A = 1.95, 95% CI = (1.29; 2.97)].

Conclusions

We confirmed the association between the FTO rs9939609 and body mass and overweight/obesity risk in European children. The main finding of the study is that the A allele carriers present higher increase of body mass and central adiposity over time and higher risk of developing overweight/obesity during growth, independently from intervention measures.     

Sex differences in mortality: results from a population-based study of 12 longitudinal cohorts

BACKGROUND:Women generally have longer life expectancy than men but have higher levels of disability and morbidity. Few studies have identified factors that explain higher mortality in men. The aim of this study was to identify potential factors contributing to sex differences in mortality at older age and to investigate variation across countries.METHODS:This study included participants age ≥ 50 yr from 28 countries in 12 cohort studies of the Ageing Trajectories of Health: Longitudinal Opportunities and Synergies (ATHLOS) consortium. Using a 2-step individual participant data meta-analysis framework, we applied Cox proportional hazards modelling to investigate the association between sex and mortality across different countries. We included socioeconomic (education, wealth), lifestyle (smoking, alcohol consumption), social (marital status, living alone) and health factors (cardiovascular disease, diabetes, mental disorders) as covariates or interaction terms with sex to test whether these factors contributed to the mortality gap between men and women.RESULTS:The study included 179 044 individuals. Men had 60% higher mortality risk than women after adjustment for age (pooled hazard ratio [HR] 1.6; 95% confidence interval 1.5–1.7), yet the effect sizes varied across countries (I² = 71.5%, HR range 1.1–2.4). Only smoking and cardiovascular diseases substantially attenuated the effect size (by about 22%).INTERPRETATION:Lifestyle and health factors may partially account for excess mortality in men compared with women, but residual variation remains unaccounted for. Variation in the effect sizes across countries may indicate contextual factors contributing to gender inequality in specific settings.

Life expectancy has increased over the last 6 decades in many societies around the world.¹ Women generally have longer life expectancy than men, yet have higher levels of disability and morbidity.^2,3 Male:female mortality ratios increased from the beginning of the 19th century and slightly decreased over the last 3 decades.^4,5 It has been suggested that the biological differences between the sexes, including genetics and hormones, provide stronger resilience to disadvantageous situations for women than men.⁶ However, biological sex is related to gender, a construct that also incorporates cultural and social differences between men and women. Although some studies suggest that the recent reduction in the male:female mortality ratio is likely a result of improvements in men’s health, lifestyle or occupational environments, others attribute it to women’s changing societal roles and increasing mortality from diseases such as lung cancer, which have traditionally affected mostly men.^3,7–9 Many studies have examined the potential impact of social, behavioural and biological factors on sex differences in mortality,^10,11 but few have been able to investigate potential variation across countries. Different cultural traditions, historical contexts, and economic and societal development may influence gender experiences in different countries, and thus variably affect the health status of men and women.We aimed to identify factors that may explain the difference in mortality risk between men and women at older age and to investigate potential variation across countries, using the harmonized data set of 12 cohort studies from the Ageing Trajectories of Health: Longitudinal Opportunities and Synergies (ATHLOS) consortium.¹²     

Spin-label study of the relation between enzymatic activity and lipid-protein organization in reconstituted cytochrome c oxidase.

Lipid-depleted cytochrome c oxidase (EC 1.9.3.1) containing less than 20 microgram lipids per milligram protein was reconstituted with pure phospholipids of well-defined chemical structure and fatty acid composition without using detergents and (or) sonication. For the maximal restoration of electron transport activity, lipid-depleted cytochrome c oxidase required acidic phospholipds such as phosphatidylglycerol or phosphatidylserine or lysophospholipids such as lysophosphatidylcholine or lysophosphatidic acid, but no specific phospholipid fatty acid composition was necessary. The organization of the lipid environment of the reconstituted cytochrome c oxidase, having a well-defined lipid composition, morphology, and a high specific activity, was examined by electron spin resonance spectroscopy using 2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl (16-doxyl stearic acid) and 16-doxyl stearic acid - containing phosphatidylglycerol. The presence of boundary lipid was established in both lamellar and micellar organizations of reconstituted cytochrome c oxidase and was not necessarily related to the enzymatic activity of the complex. Our results have established that aside from structural considerations, the boundary lipid, at least in the reconstituted cytochrome c oxidase, is a necessary but not sufficient condition for the enzymatic expression of cytochrome c oxidase.     

Abiotic synthesis of some polysaccharide-like and polypeptide-like structures in cold plasma.

Starting from methane, ammonia and water vapors polypeptide-like and polysaccharide-like structures have been synthesized in cold plasma conditions. By gel filtration analysis of the raw product eight fractions of different molecular weight have been separated. The partial chemical nature of each fraction and of the hydrolysis products has been determined using elementary analysis, IR spectroscopy, paper, thin-layer and gas-chromatography. It is noteworthy that higher proportions of H2O in the feed composition increase the amount of sugar and polysaccharide-like compounds in the reaction product.     

Comparative Genomic and Morphological Analyses of Listeria Phages Isolated from Farm Environments

The genus Listeria is ubiquitous in the environment and includes the globally important food-borne pathogen Listeria monocytogenes. While the genomic diversity of Listeria has been well studied, considerably less is known about the genomic and morphological diversity of Listeria bacteriophages. In this study, we sequenced and analyzed the genomes of 14 Listeria phages isolated mostly from New York dairy farm environments as well as one related Enterococcus faecalis phage to obtain information on genome characteristics and diversity. We also examined 12 of the phages by electron microscopy to characterize their morphology. These Listeria phages, based on gene orthology and morphology, together with previously sequenced Listeria phages could be classified into five orthoclusters, including one novel orthocluster. One orthocluster (orthocluster I) consists of large-genome (∼135-kb) myoviruses belonging to the genus “Twort-like viruses,” three orthoclusters (orthoclusters II to IV) contain small-genome (36- to 43-kb) siphoviruses with icosahedral heads, and the novel orthocluster V contains medium-sized-genome (∼66-kb) siphoviruses with elongated heads. A novel orthocluster (orthocluster VI) of E. faecalis phages, with medium-sized genomes (∼56 kb), was identified, which grouped together and shares morphological features with the novel Listeria phage orthocluster V. This new group of phages (i.e., orthoclusters V and VI) is composed of putative lytic phages that may prove to be useful in phage-based applications for biocontrol, detection, and therapeutic purposes.     

Calcium Uptake and Protein Phosphorylation in Myenteric Neurons, Like the Release of Vasoactive Intestinal Polypeptide and Acetylcholine, Are Frequency Dependent

The mechanism of the electrical-to-chemical decoding involved in the preferential release of the transmitters acetylcholine and vasoactive intestinal polypeptide (VIP) by electrical field stimulation at low (5 Hz) and high (50 Hz) frequencies was studied in superfused myenteric neurons. The stimulation-induced uptake of 45Ca2+ accompanying high frequency stimulation was markedly reduced by 10 microM nifedipine, a specific blocker of L-type voltage-sensitive Ca2+ channels (VSCCs), as was also the preferential high-frequency release of VIP. By contrast, the 45Ca2+ uptake during low-frequency stimulation was somewhat lower per pulse, and neither this uptake nor the preferential release of acetylcholine occurring at this frequency was significantly reduced by nifedipine. These findings suggest that the release of acetylcholine and VIP involve different VSCCs. The pattern of in vitro protein thiophosphorylation in tissue extracts of differentially stimulated myenteric neurons involved polypeptides of 205, 173, 86, 73, 57, 54, 46, 32, 28, and 24 kDa and was also markedly stimulus and nifedipine dependent. This suggests that different phosphoproteins are involved during the frequency-dependent activation of the different Ca2+ channels and exocytotic mechanisms.