Overexpression, purification and characterization of the Aspergillus niger endoglucanase, EglA, in Pichia pastoris>/i>

Cellulases are industrially important hydrolytic enzymes applicable in the bioconversion of cellulosic biomass to simple sugars. In this work, an endoglucanase from Aspergillus niger ATCC 10574, EglA, was expressed in the methylotrophic yeast Pichia pastoris and the properties of the recombinant protein were characterized. The full length cDNA of eglA has been cloned into a pPICZαC expression vector and expressed extracellularly as a ~30 kDa recombinant protein in P. pastoris X-33. Pure EglA displayed optimum activity at 50°C and was stable between 30 and 55°C. The pH stability of this enzyme was shown to be in the range of pH 2.0 to 7.0 and optimum at pH 4.0. EglA showed the highest affinity toward β-glucan followed by carboxymethyl cellulose (CMC) with a specific activity of 63.83 and 9.47 U/mg, respectively. Very low or no detectable hydrolysis of cellobiose, laminarin, filter paper and avicel were observed. Metal ions such as Mn2+, Co2+, Zn2+, Mg2+, Ba2+, Fe2+, Ca2+ and K+ showed significant augmentation of endoglucanase activity, with manganese ions causing the highest increase in activity to about 2.7 fold when compared with the control assay, whereas Pd2+, Cu2+, SDS and EDTA showed inhibition of EglA activity.Key words: Cellulase, endoglucanase, recombinant, Aspergillus niger, Pichia pastoris

Evaluating the effects of increasing physical activity to optimize rehabilitation outcomes in hospitalized older adults (MOVE Trial): Study protocol for a randomized controlled trial

Background: Older adults who have received inpatient rehabilitation often have significant mobility disability at discharge. Physical activity levels in rehabilitation are also low. It is hypothesized that providing increased physical activity to older people receiving hospital-based rehabilitation will lead to better mobility outcomes at discharge. Methods/Design: A single blind, parallel-group, multisite randomized controlled trial with blinded assessment of outcome and intention-to-treat analysis. The cost effectiveness of the intervention will also be examined. Older people (age >60 years) undergoing inpatient rehabilitation to improve mobility will be recruited from geriatric rehabilitation units at two Australian hospitals. A computer-generated blocked stratified randomization sequence will be used to assign 198 participants in a 1:1 ratio to either an 'enhanced physical activity' (intervention) group or a 'usual care plus' (control) group for the duration of their inpatient stay. Participants will receive usual care and either spend time each week performing additional physical activities such as standing or walking (intervention group) or performing an equal amount of social activities that have minimal impact on mobility such as card and board games (control group). Self-selected gait speed will be measured using a 6-meter walk test at discharge (primary outcome) and 6 months follow-up (secondary outcome). The study is powered to detect a 0.1 m/sec increase in self-selected gait speed in the intervention group at discharge. Additional measures of mobility (Timed Up and Go, De Morton Mobility Index), function (Functional Independence Measure) and quality of life will be obtained as secondary outcomes at discharge and tertiary outcomes at 6 months follow-up. The trial commenced recruitment on 28 January 2014. Discussion: This study will evaluate the efficacy and cost effectiveness of increasing physical activity in older people during inpatient rehabilitation. These results will assist in the development of evidenced-based rehabilitation programs for this population. Trial registration: Australian New Zealand Clinical Trials Registry ACTRN12613000884707(Date of registration 08 August 2013); ClinicalTrials.gov Identifier NCT01910740(Date of registration 22 July 2013)

Oral treatment with a zinc complex of acetylsalicylic acid prevents diabetic cardiomyopathy in a rat model of type-2 diabetes: activation of the Akt pathway.

BACKGROUND: Type-2 diabetics have an increased risk of cardiomyopathy, and heart failure is a major cause of death among these patients. Growing evidence indicates that proinflammatory cytokines may induce the development of insulin resistance, and that anti-inflammatory medications may reverse this process. We investigated the effects of the oral administration of zinc and acetylsalicylic acid, in the form of bis(aspirinato)zinc(II)-complex Zn(ASA)2, on different aspects of cardiac damage in Zucker diabetic fatty (ZDF) rats, an experimental model of type-2 diabetic cardiomyopathy. METHODS: Nondiabetic control (ZL) and ZDF rats were treated orally with vehicle or Zn(ASA)2 for 24 days. At the age of 29-30 weeks, the electrical activities, left-ventricular functional parameters and left-ventricular wall thicknesses were assessed. Nitrotyrosine immunohistochemistry, TUNEL-assay, and hematoxylin-eosin staining were performed. The protein expression of the insulin-receptor and PI3K/AKT pathway were quantified by Western blot. RESULTS: Zn(ASA)2-treatment significantly decreased plasma glucose concentration in ZDF rats (39.0 +/- 3.6 vs 49.4 +/- 2.8 mM, P < 0.05) while serum insulin-levels were similar among the groups. Data from cardiac catheterization showed that Zn(ASA)2 normalized the increased left-ventricular diastolic stiffness (end-diastolic pressure-volume relationship: 0.064 +/- 0.008 vs 0.084 +/- 0.014 mmHg/microl; end-diastolic pressure: 6.5 +/- 0.6 vs 7.9 +/- 0.7 mmHg, P < 0.05). Furthermore, ECG-recordings revealed a restoration of prolonged QT-intervals (63 +/- 3 vs 83 +/- 4 ms, P < 0.05) with Zn(ASA)2. Left-ventricular wall thickness, assessed by echocardiography, did not differ among the groups. However histological examination revealed an increase in the cardiomyocytes' transverse cross-section area in ZDF compared to the ZL rats, which was significantly decreased after Zn(ASA)2-treatment. Additionally, a significant fibrotic remodeling was observed in the diabetic rats compared to ZL rats, and Zn(ASA)2-administered ZDF rats showed a similar collagen content as ZL animals. In diabetic hearts Zn(ASA)2 significantly decreased DNA-fragmentation, and nitro-oxidative stress, and up-regulated myocardial phosphorylated-AKT/AKT protein expression. Zn(ASA)2 reduced cardiomyocyte death in a cellular model of oxidative stress. Zn(ASA)2 had no effects on altered myocardial CD36, GLUT-4, and PI3K protein expression. CONCLUSIONS: We demonstrated that treatment of type-2 diabetic rats with Zn(ASA)2 reduced plasma glucose-levels and prevented diabetic cardiomyopathy. The increased myocardial AKT activation could, in part, help to explain the cardioprotective effects of Zn(ASA)2. The oral administration of Zn(ASA)2 may have therapeutic potential, aiming to prevent/treat cardiac complications in type-2 diabetic patients

Circum-Mediterranean cultural heritage and medicial plant uses in traditional animal healthcare: a field survey in eight selected areas within the RUBIA project

During the years 2003¿2005, a comparative ethnobotanical field survey was conducted on remedies used in traditional animal healthcare in eight Mediterranean areas. The study sites were selected within the EU-funded RUBIA project, and were as follows: the upper Kelmend Province of Albania; the Capannori area in Eastern Tuscany and the Bagnocavallo area of Romagna, Italy; Cercle de Ouezanne, Morocco; Sierra de Aracena y Picos de Aroche Natural Park in the province of Huelva, Spain; the St. Catherine area of the Sinai Peninsula, Egypt; Eastern and Western Crete, Greece; the Paphos and Larnaca areas of Cyprus; and the Mitidja area of Algeria. One hundred and thirty-six veterinary preparations and 110 plant taxa were recorded in the survey, with Asteraceae and Lamiaceae being the most quoted botanical families. For certain plant species the survey uncovered veterinary phytotherapeutical indications that were very uncommon, and to our knowledge never recorded before. These include Anabasis articulata (Chenopodiaceae), Cardopatium corymbosum (Asteraceae), Lilium martagon (Liliaceae), Dorycnium rectum (Fabaceae), Oenanthe pimpinelloides (Apiaceae), Origanum floribundum (Lamiaceae), Tuberaria lignosa (Cistaceae), and Dittrichia graveolens (Asteraceae). These phytotherapeutical indications are briefly discussed in this report, taking into account modern phytopharmacology and phytochemistry. The percentage of overall botanical veterinary taxa recorded in all the study areas was extremely low (8%), however when all taxa belonging to the same botanical genus are considered, this portion increases to 17%. Nevertheless, very few plant uses were found to be part of a presumed "Mediterranean" cultural heritage in veterinary practices, which raises critical questions about the concept of Mediterraneanism in ethnobotany and suggests that further discussion is required. Nearly the half of the recorded veterinary plant uses for mammals uncovered in this survey have also been recorded in the same areas in human folk medicine, suggesting a strong link between human and veterinary medical practices, and perhaps also suggesting the adaptive origins of a few medical practices. Since most of the recorded data concern remedies for treating cattle, sheep, goats, and camels, it would be interesting to test a few of the recorded phytotherapeuticals in the future, to see if they are indeed able to improve animal healthcare in breeding environments, or to raise the quality of dairy and meat products in the absence of classical, industrial, veterinary pharmaceuticals

Travel burden and clinical presentation of retinoblastoma: analysis of 1024 patients from 43 African countries and 518 patients from 40 European countries

BACKGROUND: The travel distance from home to a treatment centre, which may impact the stage at diagnosis, has not been investigated for retinoblastoma, the most common childhood eye cancer. We aimed to investigate the travel burden and its impact on clinical presentation in a large sample of patients with retinoblastoma from Africa and Europe. METHODS: A cross-sectional analysis including 518 treatment-naïve patients with retinoblastoma residing in 40 European countries and 1024 treatment-naïve patients with retinoblastoma residing in 43 African countries. RESULTS: Capture rate was 42.2% of expected patients from Africa and 108.8% from Europe. African patients were older (95% CI -12.4 to -5.4, p<0.001), had fewer cases of familial retinoblastoma (95% CI 2.0 to 5.3, p<0.001) and presented with more advanced disease (95% CI 6.0 to 9.8, p<0.001); 43.4% and 15.4% of Africans had extraocular retinoblastoma and distant metastasis at the time of diagnosis, respectively, compared to 2.9% and 1.0% of the Europeans. To reach a retinoblastoma centre, European patients travelled 421.8 km compared to Africans who travelled 185.7 km (p<0.001). On regression analysis, lower-national income level, African residence and older age (p<0.001), but not travel distance (p=0.19), were risk factors for advanced disease. CONCLUSIONS: Fewer than half the expected number of patients with retinoblastoma presented to African referral centres in 2017, suggesting poor awareness or other barriers to access. Despite the relatively shorter distance travelled by African patients, they presented with later-stage disease. Health education about retinoblastoma is needed for carers and health workers in Africa in order to increase capture rate and promote early referral

Characterisation of DOG-1 expression in salivary gland tumours and comparison with myoepithelial markers

DOG1 is an established diagnostic marker for gastrointestinal stromal tumours (GIST), but has been reported in salivary gland tumours (SGT) as an acinar and intercalated duct marker. However, its specificity and distribution is not well established. The aim of this study was to evaluate the diagnostic utility of DOG-1 expression in SGT in addition to comparing it with myoepithelial markers. Normal salivary tissue and SGT (n = 184) were examined for expression of DOG1 and a range of myoepithelial markers. SGT included: acinic cell carcinoma (ACC, n = 15), secretory carcinoma (SC, n = 9), pleomorphic adenoma (PA, n = 49), carcinoma ex-PA (Ca ex-PA, n = 11), adenoid cystic carcinoma (AdCC, n = 20), polymorphous adenocarcinoma (PAC, n = 6), myoepithelioma (n = 6), myoepithelial carcinoma (MC, n = 2), basal cell adenoma (BCA, n = 14), canalicular adenoma (CA, n = 19), mucoepidermoid carcinoma (MEC, n = 11), oncocytoma (n = 2), adenocarcinoma NOS (AdNOS, n = 4), basal cell adenocarcinoma (BCAC, n = 2), salivary duct carcinoma (SDC, n = 3) and papillary cystadenocarcinoma (PCAC, n = 1). Normal acini and ACC (14/15) showed strong luminal DOG1 staining; SC were largely negative with only focal expression in 3/9 cases. Luminal staining was seen in PA (14/49), PAC (4/6), Ca ex-PA (4/11) and AdCC (6/20). 8/11 MEC showed luminal and/or mucous cell staining. No staining was seen in myoepithelioma, MC, CA, adNOS and BCAC. BCA showed strong staining of myoepithelial cells in some cases (5/14). Variable myoepithelial DOG1 staining was seen in PA, Ca ex PA, BCA, SDC and PCAC which was not as consistent as myoepithelial markers such as calponin, p63 and αSMA. Absence of DOG1 can differentiate ACC from SC, but staining is variable in PA, PLGA and Ca ex-PA. Myoepithelial staining in some tumours but not in normal gland suggests a wider distribution in SGT than originally envisaged