Biochemical characterization of an alkaline surfactant-stable keratinase from a new keratinase producer,Bacillus zhangzhouensis

A new keratinase producer, Bacillus sp. BK111, isolated from a poultry feather was identified as Bacillus zhangzhouensis, which is the first report for its keratinolytic activity. The keratinase production was optimized, followed by the enzyme purification and characterization using biochemical assays. A 2.34-fold increase was observed in the enzyme production under optimized conditions. The enzyme was characterized as a serine protease with 42 kDa molecular weight, stable in a wide range of temperature and pH with maximum keratinolytic activity at 60 °C and pH 9.5. The enzyme had a wide range of different substrates with the best performance on the feather meal substrate. Metal ions of Ca²⁺, K⁺, Na⁺ and Mn²⁺ enhanced the enzyme activity. The enzyme showed a great deal of stability in the presence of ethanol, methanol, acetone, 2-propanol, dimethyl sulfoxide, Tween-80 and Triton X-100. Dithiothreitol (DTT), as a reducing agent, caused a twofold increase in keratinolytic activity. The half-life of the enzyme at optimum temperature was calculated to be 125 min and the ratio of keratinolytic:caseinolytic for the enzyme was 0.8. Our results showed the remarkable features of the enzyme that make it suitable for biotechnological usages.

     

Genetic assessment of the internal transcribed spacer region (ITS1.2) in Mangifera indica L. landraces

Mango (Mangifera indica) is one of the most important tropical fruits in the world. Twenty-two genotypes of native mangoes from different regions of southern Iran (Hormozgan and Kerman) were collected and analyzed for the ribosomal genes. GC content was found to be 55.5%. Fu and Li’s D* test statistic (0.437), Fu and Li’s F* test statistic (0.500) and Tajima’s D (1.801) were positive and nonsignificant. A total of 769 positions were identified (319 with insertion or deletion including 250 polymorphic and 69 monomorphic loci; 450 loci without any insertion or deletion including 35 Singletons and 22 haplotypes). Nucleotide diversity of 0.309 and a high genetic differentiation including Chi square of 79.8; P value of 0.3605 and df value of 76 was observed among mango genotypes studied. The numerical value of the ratio dN/dS (0.45) indicated a pure selection in the examined gene and the absence of any key changes. Cluster analysis differentiated the mango used in this research (M. indica L.) into two genotypes but could not differentiate their geographical locations. The results of this study indicated that a high genetic distance exists between HajiGholam (Manojan) and Arbabi (Rodan) genotypes and showed higher genetic diversity in mango of Rodan region. Results of present study suggested that for successful breeding, the genotypes of Rodan region mango especially Arbabi mango can be used as a gene donor and ITS can be a suitable tool for genetic evaluations of inter and intra species.     

Comparison of PCR-RFLP with 21-plex PCR and rDNA Sequencing for Identification of Clinical Yeast Isolates

Non-albicans Candida species and other rare yeasts have emerged as major opportunistic pathogens in fungal infections. Identification of opportunistic yeasts in developing countries is mainly performed by phenotypic assay, which are time-consuming and prone to errors. The aim of the present study was to evaluate PCR-RFLP as a routinely used identification technique for the most clinically important Candida species in Iran and make a comparison with a novel multiplex PCR, called 21-plex PCR. One hundred and seventy-three yeast isolates from clinical sources were selected and identified with sequence analysis of the D1/D2 domains of rDNA (LSU rDNA) sequencing as the gold standard method. The results were compared with those obtained by PCR-RFLP using MspI restriction enzyme and the 21-plex PCR. PCR-RFLP correctly identified 93.4% of common pathogenic Candida species (C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and P. kudriavsevii (=?C. krusei)) and was able to identify 45.5% of isolates of the uncommon yeast species compared to the D1/D2 rDNA sequencing. Compared with PCR-RFLP, all common Candida species and 72.7% of uncommon yeast species were correctly identified by the 21-plex PCR. The application of the 21-plex PCR assay as a non-sequence-based molecular method for the identification of common and rare yeasts can reduce turnaround time and costs for the identification of clinically important yeasts and can be applied in resource-limited settings.

     

Dental stem cell banking: Techniques and protocols

Dental tissue-derived stem cells (DSCs) provide an easy, accessible, relatively noninvasive promising source of adult stem cells (ASCs), which brought encouraging prospective for their clinical applications. DSCs provide a perfect opportunity to apply for a patient's own ASC, which poses a low risk of immune rejection. However, problems associated with the long-term culture of stem cells, including loss of proliferation and differentiation capacities, senescence, genetic instability, and the possibility of microbial contamination, make cell banking necessary. With the rapid development of advanced cryopreservation technology, various international DSC banks have been established for both research and clinical applications around the world. However, few studies have been published that provide step-by-step guidance on DSCs isolation and banking methods. The purpose of this review is to present protocols and technical details for all steps of cryopreserved DSCs, from donor selection, isolation, cryopreservation, to characterization and quality control. Here, the emphasis is on presenting practical principles in accordance with the available valid guidelines.     

Retinoic acid and/or progesterone differentiate mouse induced pluripotent stem cells into male germ cells in vitro

Numerous reagents were employed for differentiating induced pluripotent stem cells (iPSCs) into male germ cells; however, the induction procedure was ineffective. The aim of this study was to improve the in vitro differentiation of mice iPSCs (miPSCs) into male germ cells with retinoic acid (RA) and progesterone (P). miPSCs were differentiated to embryoid bodies (EBs) in suspension with RA with or without progesterone for 0, 4, and 7 days. Then, the expression of certain genes at different stages of male germ cell development including Ddx4 (pre meiosis), Stra8 (meiosis), AKAP3 (post meiosis), and Mvh protein was examined in RNA and/or protein levels by real-time polymerase chain reaction or flow cytometry, respectively. The Stra8 gene expression increased in the RA groups on all days. But, expression of this gene declined in RA + P groups. In addition, an increased expression of Ddx4 gene was observed on day 0 in the P group. Also, a significant upregulation was observed in the expression of AKAP3 gene in the RA + P group on days 0 and 4. However, gene expression decreased in P and RA groups on day 7. The expression of Mvh protein significantly increased in the RA group on day 7. The Mvh expression was also enhanced in the P group on day 4, but it decreased on day 7, while this protein upregulated on day 0 and 7 in the RA + P group. The miPSCs have the capacity for in vitro differentiation into male germ cells by RA and/or progesterone. However, the effects of these inducers depend on the type of combination and an effective time.     

Production of ascorbic acid,total protein,callus and root in vitro of non-heading Chinese cabbage by tissue culture

Molecular Biology Reports - The objective of the present work was the selection of cultivar, suitable medium and explant type for callus, root production, ascorbic acid, total ascorbic acid,...