A novel repeat sequence-based PCR (rep-PCR) using specific repeat sequences of Mycobacterium intracellulare as a DNA fingerprinting

Repetitive sequence-based PCR (rep-PCR) is a potential epidemiological technique that can provide high-throughput genotype fingerprints of heterogeneous Mycobacterium strains rapidly. Previously published rep-PCR primers, which are based on nucleotide sequences of Gram-negative bacteria may have low specificity for mycobacteria. Moreover, it was difficult to ensure the continuity of the study after the commercial rep-PCR kit was discontinued. Here, we designed a novel rep-PCR for Mycobacterium intracellulare, a major cause of nontuberculous mycobacterial pulmonary disease with frequent recurrence. We screened the 7,645 repeat sequences for 200 fragments from the genome of M. intracellulare ATCC 13950 in silico, finally generating five primers with more than 90% identity for a total of 226 loci in the genome. The five primers could make different band patterns depending on the genome of three different M. intracellulare strains using an in silico test. The novel rep-PCR with the five primers was conducted using 34 bacterial samples of 7 species containing 25 M. intracellulare clinical isolates, compared with previous published rep-PCRs. This shows distinguished patterns depending on species and blotting assay for 6 species implied the sequence specificity of the five primers. The Designed rep-PCR had a 95–98% of similarity value in the reproducibility test and showed 7 groups of fingerprints in M. intracellulare strains. Designed rep-PCR had a correlation value of 0.814 with VNTR, reference epidemiological method. This study provides a promising genotype fingerprinting method for tracing the recurrence of heterogeneous M. intracellulare

Alpha-2-Macroglobulin as a New Promising Biomarker Improving the Diagnostic Sensitivity of Bovine Paratuberculosis

Johne's disease (JD) is a chronic granulomatous enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP), which induces persistent diarrhea and cachexia. JD causes huge economic losses to the dairy industry due to reduced milk production and premature culling. Infected animals excrete MAP via feces during the prolonged subclinical stage without exhibiting any clinical signs. Therefore, accurate detection of subclinical stage animals is crucial for successful eradication of JD in the herd. In the current study, we analyzed serum samples of MAP-infected and non-infected cattle to identify potential biomarker candidates. First, we identified 12 differentially expressed serum proteins in subclinical and clinical shedder groups compared to the healthy control group. Second, we conducted ELISA for three selected biomarkers (alpha-2-macroglobulin (A2M), alpha-1-beta glycoprotein, and transthyretin) and compared their diagnostic performance with that of two commercial ELISA diagnostic kits. Serum A2M levels were significantly higher in the MAP-exposed, subclinical shedder, subclinical non-shedder, and clinical shedder groups than in the healthy control group, suggesting its possible use as a diagnostic biomarker for MAP infection. Furthermore, A2M demonstrated a sensitivity of 90.4%, and a specificity of 100% while the two commercial ELISA kits demonstrated a sensitivity of 67.83 and 73.04% and a specificity of 100%, respectively. In conclusion, our results suggest that measuring A2M by ELISA can be used as a diagnostic tool to detect MAP infection, considerably improving the detection rate of subclinical shedders and MAP-exposed animals that are undetectable using current diagnostic tools

A Deep Neural Network for Simultaneous Estimation of b Jet Energy and Resolution

We describe a method to obtain point and dispersion estimates for the energies of jets arising from b quarks produced in proton-proton collisions at an energy of s = 13 TeV at the CERN LHC. The algorithm is trained on a large sample of simulated b jets and validated on data recorded by the CMS detector in 2017 corresponding to an integrated luminosity of 41 fb - 1 . A multivariate regression algorithm based on a deep feed-forward neural network employs jet composition and shape information, and the properties of reconstructed secondary vertices associated with the jet. The results of the algorithm are used to improve the sensitivity of analyses that make use of b jets in the final state, such as the observation of Higgs boson decay to b b ¯

Performance comparison of photodynamic antimicrobial chemotherapy with visible-light-activated organic dyes: Rose bengal, crystal violet, methylene blue, and toluidine blue O

This study evaluated the photobiocidal performance of four widely distributed visible-light-activated (VLA) dyes against two bacteria (Staphylococcus epidermidis and Escherichia coli) and two bacteriophages (phages MS2 and phi 6): rose bengal (RB), crystal violet, methylene blue, and toluidine blue O (TBO). The photobiocidal performance of each dye depended on the relationship between the type of dye and microorganism. Gram-negative E. coli and the non-enveloped structure of phage MS2 showed more resistance to the photobiocidal reaction than Gram-positive S. epidermidis and the enveloped structure of phage phi 6. RB had the highest potential to yield reactive oxygen species. However, the photobiocidal performance of RB was dependent on the magnitude of the surface charge of the microorganisms; for example, anionic RB induced a negative surface charge and thus electrical repulsion. On the other hand, the photobiocidal performance of TBO was observed to be less affected by the microorganism type. The comparative results presented in our study have significant implications for selecting photodynamic antimicrobial chemotherapy (PACT) dyes suitable for specific situations and purposes. Furthermore, they contribute to the advancement of PACT-related technologies by enhancing their applicability and scalability

Molecular cloning of an Arabidopsis cDNA encoding a dynamin-like protein that is localized to plastids

Dynamin-related proteins are high molecular weight GTPase proteins found in a variety of eukaryotic cells from yeast to human. They are involved in diverse biological processes that include endocytosis in animal cells and vacuolar protein sorting in yeast. We isolated a new gene, ADL2, that encodes a dynamin-like protein in Arabidopsis. The ADL2 cDNA is 2.68 kb in size and has an open reading frame for 809 amino acid residues with a calculated molecular mass of 90 kDa. Sequence analysis of ADL2 revealed a high degree of amino acid sequence similarity to other members of the dynamin superfamily. Among those members ADL2 was most closely related to Dnm1p of yeast and thus appears to be a member of the Vps1p subfamily. Expression studies showed that the ADL2 gene is widely expressed in various tissues with highest expression in flower tissues. In vivo targeting experiments showed that ADL2:smGFP fusion protein is localized to chloroplasts in soybean photoautroph cells. In addition experiments with deletion constructs revealed that the N-terminal 35 amino acid residues were sufficient to direct the smGFP into chloroplasts in tobacco protoplasts when expressed as a fusion protein.X1148sciescopu

Identification of Nontuberculous Mycobacteria in Patients with Pulmonary Diseases in Gyeongnam, Korea, Using Multiplex PCR and Multigene Sequence-Based Analysis

Background. Nontuberculous mycobacteria (NTM) are widely present in environments, such as soil and water, and have recently been recognized as important pathogenic bacteria. The incidence of NTM-related infections is steadily increasing. As the diagnosis and treatment of NTM infection should be distinguished from tuberculosis, and the treatment should be specific to the species of NTM acquired, accurate species identification is required. Methods. In this study, two-step multiplex PCR (mPCR) and multigene sequence-based analysis were used to accurately identify NTM species in 320 clinical isolates from Gyeongsang National University Hospital (GNUH). In particular, major mycobacterial strains with a high isolation frequency as well as coinfections with multiple species were diagnosed through two-step mPCR. Multigene sequencing was performed to accurately identify other NTM species not detected by mPCR. Variable regions of the genes 16S rRNA, rpoB, hsp65, and 16S-23S rRNA internal transcribed spacer were included in the analysis. Results. Two-step mPCR identified 234 (73.1%) cases of M. intracellulare, 26 (8.1%) cases of M. avium subsp. avium, and 13 (4.1%) cases of M. avium subsp. hominissuis infection. Additionally, 9 (2.8%) M. fortuitum, 9 (2.8%) M. massiliense, 2 (0.6%) M. abscessus, and 4 (1.2%) M. kansasii isolates were identified. Coinfection was identified in 7 (2.2%) samples. The sixteen samples not classified by two-step mPCR included 6 (1.9%) cases of M. chimaera, 4 (1.3%) M. gordonae, 1 (0.3%) M. colombiense, 1 (0.3%) M. mageritense, and 1 (0.3%) M. persicum identified by sequence analysis. Conclusions. The results of this study suggest a strategy for rapid detection and accurate identification of species using two-step mPCR and multigene sequence-based analysis. To the best of our knowledge, this study is the first to report the identification of NTM species isolated from patients in Gyeongnam/Korea

Influence of impedance phase angle on sound pressures and reverberation times in a rectangular room

In most room acoustic predictions, phase shift on reflection has been overlooked. This study aims to quantify the effects of the surface impedance phase angle of the boundary surfaces on room acoustic conditions. As a preliminary attempt, a medium-sized rectangular room is simulated by a phased beam tracing model, after verifying it numerically against boundary element simulations. First, the absorption characteristic of the boundary surfaces varies uniformly from 0.2 to 0.8, but with various impedance phase angles. Second, typical non-uniform cases having hard walls and floor, but with an absorptive ceiling are investigated. The zero phase angle, which has commonly been assumed in practice, is regarded as reference and differences in the sound pressure level and early decay time from the reference are quantified. As expected, larger differences in the room acoustic parameters are found for larger impedance phase angles. Additionally, binaural impulse responses are compared in a listening test for the uniform absorption cases, revealing that non-zero impedance phase angle cases can be perceptually different from the reference condition in terms of reverberance perception. For the non-uniform settings, the change in the impedance phase angle of the ceiling does not affect the acoustic conditions significantly. © 2014 Acoustical Society of America