Cloning and constitutive expression of Deschampsia antarctica Cu/Zn superoxide dismutase in Pichia pastoris

<p>Abstract</p> <p>Background</p> <p><it>Deschampsia antarctica </it>shows tolerance to extreme environmental factors such as low temperature, high light intensity and an increasing UV radiation as result of the Antarctic ozone layer thinning. It is very likely that the survival of this species is due to the expression of genes that enable it to tolerate high levels of oxidative stress. On that account, we planned to clone the <it>D. antarctica </it>Cu/ZnSOD gene into <it>Pichia pastoris </it>and to characterize the heterologous protein.</p> <p>Findings</p> <p>The Copper/Zinc superoxide dismutase (Cu/ZnSOD) gene, <it>SOD </it>gene, was isolated from a <it>D. antarctica </it>by cDNA library screening. This <it>SOD </it>gene was cloned in the expression vector pGAPZαA and successfully integrated into the genome of the yeast <it>P. pastoris </it>SMD1168H. A constitutive expression system for the expression of the recombinant SOD protein was used. The recombinant protein was secreted into the YPD culture medium as a glycosylated protein with a 32 mg/l expression yield. The purified recombinant protein possesses a specific activity of 440 U/mg.</p> <p>Conclusion</p> <p><it>D. antarctica </it>Cu/ZnSOD recombinant protein was expressed in a constitutive system, and purified in a single step by means of an affinity column. The recombinant SOD was secreted to the culture medium as a glycoprotein, corresponding to approximately 13% of the total secreted protein. The recombinant protein Cu/ZnSOD maintains 60% of its activity after incubation at 40°C for 30 minutes and it is stable (80% of activity) between -20°C and 20°C. The recombinant SOD described in this study can be used in various biotechnological applications.</p

Gait speed as a mediator of the effect of sarcopenia on dependency in activities of daily living

Background Sarcopenia in older adults is strongly associated with an increase in dependency in activities of daily living (ADL) and with a decline in gait speed. Interestingly, gait speed has been shown to independently predict mortality. In this context, our study aimed to explore the mediator role of gait speed on the relationship between sarcopenia and dependency in ADL. Methods A cross-sectional study was conducted in Colombia, 19 705 older adults with a mean age of 70 years, 55.6% women, 16.1% with sarcopenia, and 14.7% mild, moderate, or severe dependency in ADL, according to ‘SABE Survey 2015’. Sarcopenia was assessed by calf circumference and ADL dependence through the Barthel Index. Gait speed was measured over a distance of 3 m. The association between sarcopenia condition and gait speed and dependency level was analysed by linear regression adjusted by covariates. To examine whether gait speed mediated the association between sarcopenia and dependence components of physical function, simple mediation models were generated using ordinary least squares with the macro PROCESS version 3.2, adjusted for age, sex, and body mass index (BMI). Results Significant differences (P < 0.05) were found in gait speed and dependency in ADL between the sarcopenia and nonsarcopenia groups after adjusting for age, sex, and BMI. BMI was significantly higher in the non-sarcopenia group whereas dependency was significantly higher in the sarcopenia group (19.6% vs. 13.8%). Results from mediation model regression analysis indicated a significant and direct detrimental effect of sarcopenia on dependency in ADL (β = 0.05; P < 0.001), and a significant indirect effect of gait speed on the direct effect ( 0.009 to 0.004). Conclusions The negative effect of sarcopenia on functional dependence was mediated by the gait speed. Therefore, gait speed may positively influence the detrimental effect of sarcopenia for dependency, after adjusting for age, gender, and BMI. Consequently, physical exercise should be promoted and focused to circumvent the gait speed decline associated with age in older people with sarcopenia

Endothelial activation impairs the function of small extracellular vesicles

Small extracellular vesicles are nanosized vesicles (30–200 nm) that can ferry proteins, nucleic acids, and lipids between cells and therefore, have significant potential as biomarkers, drug delivery tools or therapeutic agents. SEVs of endothelial origin have been shown to -among other functions-reduce in vitro ischemia/reperfusion (I/R) injury in cardiomyocytes, but whether a pro-inflammatory state of the endothelium impairs the functionality of these SEVs remains to be elucidated. To test this, human umbilical vein endothelial cells cells were treated with TNF-α 10 ng/mL and the expression of the pro-inflammatory parameters VCAM-1, ICAM-1 and eNOS were determined by Western blot. SEVs were isolated from endothelial cells treated with or without TNF-α 10 ng/mL using size exclusion chromatography. The size and concentration of SEVs was measured by Nanoparticle Tracking Analysis. The expression of the surface marker CD81 was determined by immunoassay, whereas their morphology was assessed by electron microscopy. The function of endothelial SEVs was assessed by evaluating their cardioprotective effect in an ex vivo model of global I/R using isolated hearts from adult C57BL/6 mice. Treatment of HUVECs with TNF-α induced the expression of VCAM-1 and ICAM-1, whereas eNOS levels were decreased. TNF-α did not affect the production, size, morphology, or expression of CD81. SEVs significantly reduced the infarct size as compared with untreated mice hearts, but SEVs isolated from TNF-α treated cells were unable to achieve this effect. Therefore, a pro-inflammatory state induced by TNF-α does not alter the production of endothelial SEVs but impairs their function in the setting of I/R injury

Chagas Cardiomyopathy Manifestations and Trypanosoma cruzi Genotypes Circulating in Chronic Chagasic Patients

Chagas disease caused by Trypanosoma cruzi is a complex disease that is endemic and an important problem in public health in Latin America. The T. cruzi parasite is classified into six discrete taxonomic units (DTUs) based on the recently proposed nomenclature (TcI, TcII, TcIII, TcIV, TcV and TcVI). The discovery of genetic variability within TcI showed the presence of five genotypes (Ia, Ib, Ic, Id and Ie) related to the transmission cycle of Chagas disease. In Colombia, TcI is more prevalent but TcII has also been reported, as has mixed infection by both TcI and TcII in the same Chagasic patient. The objectives of this study were to determine the T. cruzi DTUs that are circulating in Colombian chronic Chagasic patients and to obtain more information about the molecular epidemiology of Chagas disease in Colombia. We also assessed the presence of electrocardiographic, radiologic and echocardiographic abnormalities with the purpose of correlating T. cruzi genetic variability and cardiac disease. Molecular characterization was performed in Colombian adult chronic Chagasic patients based on the intergenic region of the mini-exon gene, the 24Sα and 18S regions of rDNA and the variable region of satellite DNA, whereby the presence of T.cruzi I, II, III and IV was detected. In our population, mixed infections also occurred, with TcI-TcII, TcI-TcIII and TcI-TcIV, as well as the existence of the TcI genotypes showing the presence of genotypes Ia and Id. Patients infected with TcI demonstrated a higher prevalence of cardiac alterations than those infected with TcII. These results corroborate the predominance of TcI in Colombia and show the first report of TcIII and TcIV in Colombian Chagasic patients. Findings also indicate that Chagas cardiomyopathy manifestations are more correlated with TcI than with TcII in Colombia

COPD in never smokers: results from the population-based burden of obstructive lung disease study.

To access publisher full text version of this article. Please click on the hyperlink in Additional Links field.BACKGROUND: Never smokers comprise a substantial proportion of patients with COPD. Their characteristics and possible risk factors in this population are not yet well defined. METHODS: We analyzed data from 14 countries that participated in the international, population-based Burden of Obstructive Lung Disease (BOLD) study. Participants were aged ≥ 40 years and completed postbronchodilator spirometry testing plus questionnaires about respiratory symptoms, health status, and exposure to COPD risk factors. A diagnosis of COPD was based on the postbronchodilator FEV₁/FVC ratio, according to current GOLD (Global Initiative for Obstructive Lung Disease) guidelines. In addition to this, the lower limit of normal (LLN) was evaluated as an alternative threshold for the FEV₁/FVC ratio. RESULTS: Among 4,291 never smokers, 6.6% met criteria for mild (GOLD stage I) COPD, and 5.6% met criteria for moderate to very severe (GOLD stage II+) COPD. Although never smokers were less likely to have COPD and had less severe COPD than ever smokers, never smokers nonetheless comprised 23.3% (240/1,031) of those classified with GOLD stage II+ COPD. This proportion was similar, 20.5% (171/832), even when the LLN was used as a threshold for the FEV₁/FVC ratio. Predictors of COPD in never smokers include age, education, occupational exposure, childhood respiratory diseases, and BMI alterations. CONCLUSION: This multicenter international study confirms previous evidence that never smokers comprise a substantial proportion of individuals with COPD. Our data suggest that, in addition to increased age, a prior diagnosis of asthma and, among women, lower education levels are associated with an increased risk for COPD among never smokers.ALTANA Aventis AstraZeneca Boehringer-Ingleheim Chiesi GlaxoSmithKline Merck Novartis Pfizer Inc Schering-Plough Sunovion Pharmaceuticals Inc University of Kentucky Schering Plough Sepracor AstraZeneca, Spai

International lower limb collaborative (INTELLECT) study: a multicentre, international retrospective audit of lower extremity open fractures

Trauma remains a major cause of mortality and disability across the world1, with a higher burden in developing nations2. Open lower extremity injuries are devastating events from a physical3, mental health4, and socioeconomic5 standpoint. The potential sequelae, including risk of chronic infection and amputation, can lead to delayed recovery and major disability6. This international study aimed to describe global disparities, timely intervention, guideline-directed care, and economic aspects of open lower limb injuries