A simple 2-step purification process of α-amylase from Bacillus subtilis: Optimization by response surface methodology.

Purification of extracellular α-amylase from Bacillus subtilis was carried out via fractional precipitation by acetone and ion exchange chromatography. These steps provide fast precipitation as well as purification of α-amylase to improve enzyme purity, activity and stability. Compared with two-phase methods in which the yield was less than 1, this method resulted in a yield of more than 3. Moreover, 95% of acetone was recovered that enhanced the economy of the downstream process. Using the data provided by 2D electrophoresis, purification was done by a single step ion exchange chromatography. The enzyme exhibited a molecular mass (SDS-PAGE) of 50KD and the pI of 5. Maximum "yield" and "purification fold" were achieved through optimization of operation parameters such as volume and flowrate of loaded protein using response surface methodology (RSM). 0.5ml of loaded protein at a flow rate of 0.5 ml/min was purified as 48 folds and achieved a specific activity of 524 U/mg.

Extraction, Purification, and Characterization of Trypsin Obtained from the Digestive System of Yellowfin Seabream (Acanthopagrus latus).

The development of the marine aquaculture industry has led to the generation of significant amounts of fish wastes. Marine farm wastes exert adverse effects on the surrounding area of the cages. On the other hand, wastes of fish and other aquatic animals are regarded as major sources of valuable natural bioactive compounds, including enzymes, proteins, bioactive peptides, oil, amino acids, collagen, gelatin, calcium, biopolymers, and water-soluble minerals. To investigate the potential of marine fish waste, the whole digestive system of yellowfin seabream (Acanthopagrus latus) was extracted for extraction and identification of trypsin enzyme. Fish (179.93±93.67 g; 184±28.17 cm) were caught from the Persian Gulf and stored at -20 °C. Yellowfin seabream were dissected and their whole digestive systems were removed. Samples were thoroughly washed with distilled water and purified through defatting using acetone and ammonium sulfate precipitation. The following issues were assessed: the total and specific activity of trypsin, protein determination, molecular weight, enzyme activity and stability in different pH values and temperatures. The obtained results indicated that specific activity and protein content of trypsin enzyme were 4.4 U and 3.4 mg/ml, respectively. The molecular weight of 23 kDa was reported for trypsin using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) method. Maximum activity and stability of trypsin were observed at 60°C and 45°C, respectively. Trypsin demonstrated maximum activity and stability at a pH value of 8.0. In general, the results of the current study suggested that trypsin extracted from the digestive system of yellowfin seabream has considerable potential for industrial applications, such as the food industry, owing to its characteristics and stability under alkaline conditions.

The role of microRNAs in different types of thyroid carcinoma: a comprehensive analysis to find new miRNA supplementary therapies.

The most common endocrine malignancy is thyroid cancer, and researchers have made a great deal of progress in deciphering its molecular mechanisms in the recent years. Many of molecular changes observed in thyroid cancer can be used as biomarkers for diagnosis, prognosis, and therapeutic targets for treatment. MicroRNAs (miRNAs) are important parts in biological and metabolic pathways such as regulation of developmental stages, signal transduction, cell maintenance, and differentiation. Therefore, their dysregulation can expose individuals to malignancies. It has been proved that miRNA expression is dysregulated in different types of tumors, like thyroid cancers, and can be the cause of tumor initiation and progression. In this paper, we have reviewed the available data on miRNA dysregulation in different thyroid tumors including papillary, follicular, anaplastic, and medullary thyroid carcinomas aiming to introduce the last updates in miRNAs-thyroid cancer relation.

The effect of miR-340 over-expression on cell-cycle-related genes in triple-negative breast cancer cells.

Breast cancer is a heterogeneous disease, and among all types, triple-negative breast cancer (TNBC) is characterised by high risk of recurrence. The discovery of microRNAs (miRNA) has opened the door for targeted therapy of TNBC. miR-340 down-regulation and sub-G1-accumulated cells in flowcytometry were observed in metastatic TNBC cells (data in publication), leading us to investigate the potential tumour suppressive role of this miRNA on cell-cycle-related genes. A lentiviral vector containing miR-340 was applied to over-express miR-340 in TNBC cell line, MDA-MB-231. Then, the expression of some cell-cycle-regulating genes including cyclin A2 (cyclin A2), Cyclin-dependent kinases 2 (CDK2), cyclin-dependent kinase inhibitors (P16, P18 and P27), Retinoblastoma (RB) and transcription factors (SMAD 4, SOX2 and SOX17) was investigated using quantitative RT-PCR. The results showed a decline in the expression of SOX2, P16 and P27 after miR-340 over-expression, whereas we observed an increase in the expression of cyclin A2, CDK2, SOX17, P18, SMAD 4 and RB. The over-expression of tumour suppressor genes such as RB and SOX17 and down-regulation of an oncogene such as SOX2 were in accordance to the inhibitory role of miR-340 that causes blockage of breast cancer metastasis which should be further investigated.

Molecular beacon probes-base multiplex NASBA Real-time for detection of HIV-1 and HCV.

BACKGROUND AND OBJECTIVES: Developed in 1991, nucleic acid sequence-based amplification (NASBA) has been introduced as a rapid molecular diagnostic technique, where it has been shown to give quicker results than PCR, and it can also be more sensitive. This paper describes the development of a molecular beacon-based multiplex NASBA assay for simultaneous detection of HIV-1 and HCV in plasma samples. MATERIALS AND METHODS: A well-conserved region in the HIV-1 pol gene and 5'-NCR of HCV genome were used for primers and molecular beacon design. The performance features of HCV/HIV-1 multiplex NASBA assay including analytical sensitivity and specificity, clinical sensitivity and clinical specificity were evaluated. RESULTS: The analysis of scalar concentrations of the samples indicated that the limit of quantification of the assay was <1000 copies/ml for HIV-1 and <500 copies/ml for HCV with 95% confidence interval. Multiplex NASBA assay showed a 98% sensitivity and 100% specificity. The analytical specificity study with BLAST software demonstrated that the primers do not attach to any other sequences except for that of HIV-1 or HCV. The primers and molecular beacon probes detected all HCV genotypes and all major variants of HIV-1. CONCLUSION: This method may represent a relatively inexpensive isothermal method for detection of HIV-1/HCV co-infection in monitoring of patients.

Design and development of an in-house multiplex RT-PCR assay for simultaneous detection of HIV-1 and HCV in plasma samples.

BACKGROUND AND OBJECTIVES: HIV-1 and HCV infections are life threatening problems in patients who receive blood products. Serological methods have proven useful in detecting these infections, but there are setbacks that make it challenging to detect these infectious agents. By the advent of Nucleic Acid Testing (NAT) methods, especially in multiplex format, more precise detection is possible. MATERIALS AND METHODS: We have developed a multiplex RT-PCR assay for simultaneous detection of HIV-1 and HCV. Primers were designed for highly conserved region of genome of each virus. Using these primers and standard plasmids, we determined the limit of detection, clinical and analytical specificity and sensitivity of the assay. Monoplex and multiplex RT-PCR were performed. RESULTS: Analytical sensitivity was considered to be 100 and 200 copies/ml for HIV-1 and HCV, respectively. High concentration of one virus had no significant effect on the detection of the other one with low concentration. By analysis of 40 samples, clinical sensitivity of the assay was determined to be 97.5%. Using different viral and human genome samples, the specificity of the assay was evaluated to be 100%. CONCLUSIONS: The aim of this study was to develop a reliable, rapid and cost effective method to detect HIV-1 and HCV simultaneously. Results showed that this simple and rapid method is perfectly capable of detecting two viruses in clinical samples.

Homocysteine levels in patients with risk factors for atherosclerosis.

BACKGROUND: Abundant epidemiological evidence has demonstrated that the presence of mild to moderate hyperhomocysteinemia is an independent risk factor for atherosclerosis in the coronary, cerebral, and peripheral vasculature, and for vascular disease, including coronary disease. It has been demonstrated that plasma total homocysteine level is a strong predictor of mortality in patients with angiographically confirmed coronary artery disease. HYPOTHESIS: The study was undertaken to determine the extent of homocysteine levels in patients without documented coronary artery disease, but with at least one risk factor for atherosclerosis. METHODS: Fasting blood samples were collected prospectively from 160 consecutive patients (50 women and 110 men, mean age 65+/-7 years) who had at least one risk factor for atherosclerosis, but had no documented coronary artery disease. Homocysteine levels were measured by an immunoassay method. RESULTS: Of the patients studied, 78 (48.75%) with at least one risk factor for atherosclerosis had high homocysteine levels; 62 patients had mild hyperhomocysteinemia (15-30 micromol/l); and 16 patients had moderate hyperhomocysteinemia (30-100 micromol/l). CONCLUSIONS: Our data suggest that hyperhomocysteinemia is highly prevalent in patients with risk factors for atherosclerosis. Homocysteine level (an independent convertible risk factor to atherosclerosis) should be measured routinely in patients with risk factors for atherosclerosis and treated appropriately.

Quantification of root surface plaque using a new 3-D laser scanning method.

There are no published reports in the literature objectively quantifying thickness of plaque on teeth. The aim of this study was to quantify plaque on a tooth surface and assess if this quantification correlates with a clinical index of plaque from each of 51 patients. Patients were instructed not to perform any oral hygiene on the day of the assessment. The Silness and Löe plaque index was scored and replicas were scanned using a co-ordinate measuring machine (CMM) and laser scanning probe. A replica was obtained from this surface before and after toothbrushing. Plaque adjacent to the gingival margin had a mean thickness of 0.106+/-0.118 mm (mean+/-SD) whilst mean plaque thickness 250 microm from the gingival margin was 0.053+/-0.052 mm (mean+/-SD). There was a significant correlation between the plaque index and the plaque thickness (p < or = 0.002). The finding that plaque is present in the greatest amount adjacent to the gingival margin supports a previously reported hypothesis that primary root carious lesions (PRCL's) may initiate adjacent to the gingival margin. This method quantifies plaque thickness on exposed root surfaces which correlates with the plaque index as well as illustrating how the morphological characteristics of teeth, gingivae and plaque can be studied in vivo from replicas recorded.

Trace-element analysis by charged-particle-induced reactions as a tool for physicians and chemists.

The object of this article is to apprise physicians and chemists of nuclear analytical techniques and, in particular, of ion beam analysis (PIXE and PIGE) for the purpose of application to the clinical diagnostic method.The feasibility of the technique, sampling, and sample preparation for trace element analysis in biological and biomedical samples has been described previously (1-3). Analysis data from normal human blood samples and biomedical samples by ion beam reactions have been compared at the end.Emphasis will be placed on the use of the analytical technique on determination of the range of trace and toxic elements in human blood samples.