Live cell imaging of low- and non-repetitive chromosome loci using CRISPR-Cas9.

Imaging chromatin dynamics is crucial to understand genome organization and its role in transcriptional regulation. Recently, the RNA-guidable feature of CRISPR-Cas9 has been utilized for imaging of chromatin within live cells. However, these methods are mostly applicable to highly repetitive regions, whereas imaging regions with low or no repeats remains as a challenge. To address this challenge, we design single-guide RNAs (sgRNAs) integrated with up to 16 MS2 binding motifs to enable robust fluorescent signal amplification. These engineered sgRNAs enable multicolour labelling of low-repeat-containing regions using a single sgRNA and of non-repetitive regions with as few as four unique sgRNAs. We achieve tracking of native chromatin loci throughout the cell cycle and determine differential positioning of transcriptionally active and inactive regions in the nucleus. These results demonstrate the feasibility of our approach to monitor the position and dynamics of both repetitive and non-repetitive genomic regions in live cells

Designing of a novel dextransucrase efficient in acceptor reactions

Dextransucrase is produced by Leuconostoc, Streptococcus and Lactobacillus Species. The enzyme synthesizes dextran and acceptor products some of which act as prebiotics that are increasingly used in such industries as food, medicine, and cosmetics. B-512F Leuconostoc mesenteroides dextransucrase (DSR-S) is the preferred enzyme in commercial production of dextran and prebiotics. In the present work, a novel dextransucrase which is efficient in prebiotics production was designed. The enzyme was produced at optimal conditions in Escherichia coli by truncation and fusion to glutathione S-transferase (GST) in the gene from Leuconostoc mesenteroides B-512 FMC. The novel enzyme (MW: 119 kDa) was active and carried out dextran biosynthesis and acceptor reactions effectively. The novel dextransucrase (fTDSR-S) was produced by truncating signal, variable, and the glucan-binding regions in the gene and fusion of gst gene at the 50 end. fTDSR-S was characterized in detail and compared to the DSR-S. Truncation and fusion resulted in an increase in fTDSR-S biosynthesis in E. coli BL21 (DE3) by 35 fold. fTDSR-S leads to production of dextran as well as increased acceptor reactions. Due to GST fusion, it was possible to immobilize fTDSR-S covalently onto Eupergit C successfully. It was also found that the size of the active site of dextransucrase is 49 amino acids shorter than that reported previously in the literature. (C) 2014 Elsevier Ltd. All rights reserved

A novel method for covalent immobilization of dextransucrase

B-512F dextransucrase is an industrial enzyme used in commercial production of dextran and prebiotics. Several researchers have studied the covalent immobilization of this enzyme with little success due to dextran masking the reactive groups on the enzyme and inactivation of the enzyme during immobilization. A novel dextransucrase was designed and produced successfully to eliminate problems faced in covalent immobilization. In production of the novel enzyme, B-512F dextransucrase was truncated at N and C terminals and fused to glutathione S-transferase. The novel dextransucrase was fully active and carried out dextran biosynthesis and acceptor reactions effectively. The novel enzyme was immobilized covalently onto Eupergit C 250L, giving rise to 100% immobilization and 83.3% activity yields. Immobilized enzyme was used successfully for the production of acceptor products and low molecular weight dextran. The immobilized enzyme showed no decrease in activity for 15 batch reactions and retained its initial activity at storage (4 degrees C) for 35 days. Optimal conditions were not affected by the immobilization. The kinetic parameters for the free and immobilized enzyme were determined. The acceptor reactions using fructose, glucose, maltose, and lactose were also studied. The novel method developed could also be used in immobilization of some other biomolecules. (C) 2012 Elsevier B.V. All rights reserved

Histopathological results of adenoidectomy and/or tonsillectomy: 14 years of experience

The necessity of routine pathologic examinations is still debatable considering the cost and the increase in workload. With a very wide spectrum of patients, this study aims to examine the cases collected in our hospital archive for the past 14 years retrospectively and to contribute to the literature on a subject that is still controversial. The results of adenoidectomy and/or tonsillectomy specimens of 5.658 patients who underwent adenoidectomy and/or tonsillectomy under general anaesthesia were evaluated. The number of patients who underwent only adenoidectomy was 2.057 (36.3%), whereas the number of patients who underwent only tonsillectomy was 978 (17.2%). Adenoidectomy together with tonsillectomy was performed on the remaining 2.623 (46.5%) patients. A total number of 11.882 specimens from 5.658 were evaluated histopathologically. The results showed that 2.846 (50.3%) patients had lymphoid hyperplasia, 1.651 (29.1%) had chronic inflammation, 1.012 (17.8%) had coexistence of lymphoid hyperplasia and chronic inflammation, 113 (1.99%) had Actinomyces granules, nine (0.15%) had squamous papilloma, three (0.05%) had epidermoid cyst, two (0.03%) had Aspergillus sphericules and 1 (0.01%) had foreign object reaction. None of the patients who underwent tonsillectomy without a pre-diagnosis of malignancy were diagnosed with cancer. However, a 12-year-old patient without a pre-diagnosis of malignancy was reported to have undifferentiated nasopharyngeal cancer (1/4.680, 0.021%). It's possible that routine pathological examination, especially on patients under the age of 10, is not necessary when there is no finding that raises suspicion for malignancy. [Med-Science 2023; 12(4.000): 1062-5

Biliary stenting in difficult common bile duct stones: a single tertiary center experience

Conclusion: For CBD stones that cannot be removed by standard methods, temporary plastic stenting is an alternative method

Evaluation of intestinal damage biomarkers in calves with atresia coli

Intestinal obstruction such as atresia coli causes pathophysiological changes in gastrointestinal tissue due to the rise of intra-abdominal pressure. The aim of this study is to determine the intestinal damage with intestinal biomarkers in calves with atresia coli