Enzyme assay, cloning and sequencing of novel β-glucosidase gene from Aspergillus niger f321 (unidentified Nigerian strain)

β-glucosidase is a cellulase enzyme under intense investigation for its potential role in cellulose degradation for the generation of fermentable sugars used in biofuels production. Ten catalytic sites have been identified that are conserved in β- glucosidases from a range of prokaryotes and eukaryotes. NCBI Primer BLAST was used in this study to design primers that successfully clone a partial β-glucosidase gene from an uncharacterised Nigerian strain of the filamentous fungi Aspergillus niger F321 (A. niger F321). Two β-glucosidase genes from A. niger F321 denoted as ANRA12.6 and ANRA12.9 were amplified from genomic DNA using PCR techniques and the amplicons gave estimated PCR products of 1,190 bp and 1,950 bp respectively. Subsequent cloning into E. coli produced positive results for blue/white screening of transformed colonies while the colony PCR of their pDNA gave estimated sizes of 860 bp and 1,600 bp respectively. DNA sequencing confirmed that the chosen A. niger F321 partial β-glucosidase sequences had been successfully cloned. Bioinformatics studies also suggested that the cloned β-glucosidases share some characteristics with their bacterial counterparts. The findings in this study highlight the increasing need for more information on β-glucosidase structure and function.Keywords: Aspergillus niger, β-glucosidase, cellulase, PCR, sequencing, Bioinformatic

Mitochondrial apurinic/apyrimidinic endonuclease 1 enhances mtDNA repair contributing to cell proliferation and mitochondrial integrity in early stages of hepatocellular carcinoma

Background: Hepatocellular carcinoma (HCC) is the leading cause of primary liver cancers. Surveillance of individuals at specific risk of developing HCC, early diagnostic markers, and new therapeutic approaches are essential to obtain a reduction in disease-related mortality. Apurinic/apyrimidinic endonuclease 1 (APE1) expression levels and its cytoplasmic localization have been reported to correlate with a lower degree of differentiation and shorter survival rate. The aim of this study is to fully investigate, for the first time, the role of the mitochondrial form of APE1 in HCC. Methods: As a study model, we analyzed samples from a cohort of patients diagnosed with HCC who underwent surgical resection. Mitochondrial APE1 content, expression levels of the mitochondrial import protein Mia40, and mtDNA damage of tumor tissue and distal non-tumor liver of each patient were analyzed. In parallel, we generated a stable HeLa clone for inducible silencing of endogenous APE1 and re-expression of the recombinant shRNA resistant mitochondrially targeted APE1 form (MTS-APE1). We evaluated mtDNA damage, cell growth, and mitochondrial respiration. Results: APE1's cytoplasmic positivity in Grades 1 and 2 HCC patients showed a significantly higher expression of mitochondrial APE1, which accounted for lower levels of mtDNA damage observed in the tumor tissue with respect to the distal area. In the contrast, the cytoplasmic positivity in Grade 3 was not associated with APE1's mitochondrial accumulation even when accounting for the higher number of mtDNA lesions measured. Loss of APE1 expression negatively affected mitochondrial respiration, cell viability, and proliferation as well as levels of mtDNA damage. Remarkably, the phenotype was efficiently rescued in MTS-APE1 clone, where APE1 is present only within the mitochondrial matrix. Conclusions: Our study confirms the prominent role of the mitochondrial form of APE1 in the early stages of HCC development and the relevance of the non-nuclear fraction of APE1 in the disease progression. We have also confirmed overexpression of Mia40 and the role of the MIA pathway in the APE1 import process. Based on our data, inhibition of the APE1 transport by blocking the MIA pathway could represent a new therapeutic approach for reducing mitochondrial metabolism by preventing the efficient repair of mtDNA

Precipitation of the ordered α2 phase in a near-α titanium alloy

Precipitate evolution in a near-α alloy was studied using transmission electron microscopy (TEM) and correlative atom probe tomography (APT) after ageing at 550-700 for times up to 28 days. It is found that precipitation occurs much faster and is more prolific in samples heat treated at higher temperatures. Particles were spherical after ageing at 550 °C, while after ageing at 700 °C they become ellipsoids with the major axis lying close to the [0001] direction. At longer ageing times, the α2 precipitates were found to contain greater amounts of Sn + Si, indicating that Sn and Si are stronger Ti3(Al,Sn,Si) formers than Al

Improving survival of probiotic bacteria using bacterial poly-γ-glutamic acid

A major hurdle in producing a useful probiotic food product is bacterial survival during storage and ingestion. The aim of this study was to test the effect of γ-PGA immobilisation on the survival of probiotic bacteria when stored in acidic fruit juice. Fruit juices provide an alternative means of probiotic delivery, especially to lactose intolerant individuals. In addition, the survival of γ-PGA-immobilised cells in simulated gastric juice was also assessed. Bifidobacteria strains (B. longum, B. breve), immobilised on 2.5 % γ-PGA, survived significantly better (P < 0.05) in orange and pomegranate juice for 39 and 11 days respectively, compared to free cells. However, cells survived significantly better (P < 0.05) when stored in orange juice compared to pomegranate juice. Moreover, both strains, when protected with 2.5 % γ-PGA, survived in simulated gastric juice (pH 2.0) with a marginal reduction (<0.47 log CFU/ml) or no significant reduction in viable cells after four hours, whereas free cells died within two hours. In conclusion, this research indicates that γ-PGA can be used to protect Bifidobacteria cells in fruit juice, and could also help improve the survival of cells as they pass through the harsh conditions of the gastrointestinal tract (GIT). Following our previous report on the use of γ-PGA as a cryoprotectant for probiotic bacteria, this research further suggests that γ-PGA could be used to improve probiotic survival during the various stages of preparation, storage and ingestion of probiotic cells

Synthesis of Silver Nanoparticles Using Curcumin-Cyclodextrins Loaded into Bacterial Cellulose-Based Hydrogels for Wound Dressing Applications.

Chronic wounds are often recalcitrant to treatment because of high microbial bioburden and the problem of microbial resistance. Silver is a broad-spectrum natural antimicrobial agent with wide applications extending to proprietary wound dressings. Recently, silver nanoparticles have attracted attention in wound management. In the current study, the green synthesis of nanoparticles was accomplished using a natural reducing agent, curcumin, which is a natural polyphenolic compound that is well-known as a wound-healing agent. The hydrophobicity of curcumin was overcome by its microencapsulation in cyclodextrins. This study demonstrates the production, characterization of silver nanoparticles using aqueous curcumin:hydroxypropyl-β-cyclodextrin complex and loading them into bacterial cellulose hydrogel with moist wound-healing properties. These silver nanoparticle-loaded bacterial cellulose hydrogels were characterized for wound-management applications. In addition to high cytocompatibility, these novel dressings exhibited antimicrobial activity against three common wound-infecting pathogenic microbes , , and

Molecular dynamics simulations of oxide memristors: thermal effects

We have extended our recent molecular-dynamic simulations of memristors to include the effect of thermal inhomogeneities on mobile ionic species appearing during operation of the device. Simulations show a competition between an attractive short-ranged interaction between oxygen vacancies and an enhanced local temperature in creating/destroying the conducting oxygen channels. Such a competition would strongly affect the performance of the memristive devices.Comment: submit/0169777; 6 pages, 4 figure