Comparative interactome analysis of α-arrestin families in human and Drosophila.

The α-arrestins form a large family of evolutionally conserved modulators that control diverse signaling pathways, including both G-protein-coupled receptor (GPCR)-mediated and non-GPCR-mediated pathways, across eukaryotes. However, unlike ß-arrestins, only a few α-arrestin targets and functions have been characterized. Here, using affinity purification and mass spectrometry, we constructed interactomes for 6 human and 12 Drosophila α-arrestins. The resulting high-confidence interactomes comprised 307 and 467 prey proteins in human and Drosophila, respectively. A comparative analysis of these interactomes predicted not only conserved binding partners, such as motor proteins, proteases, ubiquitin ligases, RNA splicing factors, and GTPase-activating proteins, but also those specific to mammals, such as histone modifiers and the subunits of V-type ATPase. Given the manifestation of the interaction between the human α-arrestin, TXNIP, and the histone-modifying enzymes, including HDAC2, we undertook a global analysis of transcription signals and chromatin structures that were affected by TXNIP knockdown. We found that TXNIP activated targets by blocking HDAC2 recruitment to targets, a result that was validated by chromatin immunoprecipitation assays. Additionally, the interactome for an uncharacterized human α-arrestin ARRDC5 uncovered multiple components in the V-type ATPase, which plays a key role in bone resorption by osteoclasts. Our study presents conserved and species-specific protein-protein interaction maps for α-arrestins, which provide a valuable resource for interrogating their cellular functions for both basic and clinical research.

The First Identification of Cryptosporidium parvum Virus-1 (CSpV1) in Hanwoo (Bos taurus coreanae) Calves in Korea.

Cryptosporidium is an obligate coccidian parasite that causes enteric diseases in bovine species. A double-stranded RNA virus associated with C. parvum oocysts, Cryptosporidium parvum virus-1 (CSpV1), has been characterized. However, the relationship between the abovementioned coccidian parasite and the virus has not been studied in the context of the known clinical outcomes. This study aimed to characterize the prevalence and molecular traits of CSpV1 in diarrheal feces of Hanwoo (Korean indigenous cattle) calves. Of the 140 fecal samples previously tested for C. parvum, which were obtained from Hanwoo calves aged 60 days, 70 tested positive and 70 tested negative. These samples were included in this study. By using the polymerase chain reaction (PCR) analysis targeting the RdRp gene of CSpV1, we detected CSpV1 in 28 samples (20.0%), with infection rates of 31.4% (22/70) in C. parvum-positive and 8.6% (6/70) in C. parvum-negative samples. CSpV1 samples detected in the same farm were clustered together. To the best of our knowledge, this is the first study to report the prevalence and molecular characteristics of CSpV1 in Hanwoo calves in the Republic of Korea, providing important insights into the relationship between C. parvum and CSpV1 in bovine hosts.

Development of a one-step reverse transcription-quantitative polymerase chain reaction assay for the detection of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) is an important disease that severely affects the swine industry and, therefore, warrants rapid and accurate diagnosis for its control. Despite the progress in developing diagnostic tools, including polymerase chain reaction (PCR)-based methods such as reverse transcription quantitative PCR (RT-qPCR) to diagnose PRRSV infection, its diagnosis at the genetic level is challenging because of its high genetic variability. Nevertheless, RT-qPCR is the easiest and fastest method for diagnosing PRRSV. Therefore, this study aimed to develop an RT-qPCR assay for rapid and accurate diagnosis of PRRSV by encompassing all publicly available PRRSV sequences. The developed assay using highly specific primers and probes could detect up to 10 copies of PRRSV-1 and -2 subtypes. Furthermore, a comparison of the performance of the developed assay with those of two commercial kits widely used in South Korea demonstrated the higher efficiency of the developed assay in detecting PRRSV infections in field samples. For PRRSV-1 detection, the developed assay showed a diagnostic agreement of 97.7% with the results of ORF5 sequencing, while for commercial kits, it showed 95.3% and 72.1% agreement. For PRRSV-2, the developed assay showed a diagnostic agreement of 97.7%, whereas the commercial kits showed 93% and 90.7% agreement. In conclusion, we developed an assay with higher accuracy than those of the tested commercial kits, which will contribute markedly to global PRRSV control.

Importance of differential identification of Mycobacterium tuberculosis strains for understanding differences in their prevalence, treatment efficacy, and vaccine development.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a serious global health problem in the 21st century because of its high mortality. Mtb is an extremely successful human-adapted pathogen that displays a multifactorial ability to control the host immune response and to evade killing by drugs, resulting in the breakdown of BCG vaccine-conferred anti-TB immunity and development of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mtb. Although genetic components of the genomes of the Mtb complex strains are highly conserved, showing over 99% similarity to other bacterial genera, recently accumulated evidence suggests that the genetic diversity of the Mtb complex strains has implications for treatment outcomes, development of MDR/XDR Mtb, BCG vaccine efficacy, transmissibility, and epidemiological outbreaks. Thus, new insights into the pathophysiological features of the Mtb complex strains are required for development of novel vaccines and for control of MDR/XDR Mtb infection, eventually leading to refinement of treatment regimens and the health care system. Many studies have focused on the differential identification of Mtb complex strains belonging to different lineages because of differences in their virulence and geographical dominance. In this review, we discuss the impact of differing genetic characteristics among Mtb complex strains on vaccine efficacy, treatment outcome, development of MDR/XDR Mtb strains, and epidemiological outbreaks by focusing on the best-adapted human Mtb lineages. We further explore the rationale for differential identification of Mtb strains for more effective control of TB in clinical and laboratory settings by scrutinizing current diagnostic methods.

Development of a One-Step Multiplex PCR Assay for Differential Detection of Major Mycobacterium Species.

The prevalence of tuberculosis continues to be high, and nontuberculous mycobacterial (NTM) infection has also emerged worldwide. Moreover, differential and accurate identification of mycobacteria to the species or subspecies level is an unmet clinical need. Here, we developed a one-step multiplex PCR assay using whole-genome analysis and bioinformatics to identify novel molecular targets. The aims of this assay were to (i) discriminate between the Mycobacterium tuberculosis complex (MTBC) and NTM using rv0577 or RD750, (ii) differentiate M. tuberculosis (M. tuberculosis) from MTBC using RD9, (iii) selectively identify the widespread M. tuberculosis Beijing genotype by targeting mtbk_20680, and (iv) simultaneously detect five clinically important NTM (M. avium, M. intracellulare, M. abscessus, M. massiliense, and M. kansasii) by targeting IS1311, DT1, mass_3210, and mkan_rs12360 An initial evaluation of the multiplex PCR assay using reference strains demonstrated 100% specificity for the targeted Mycobacterium species. Analytical sensitivity ranged from 1 to 10 pg for extracted DNA and was 103 and 104 CFU for pure cultures and nonhomogenized artificial sputum cultures, respectively, of the targeted species. The accuracy of the multiplex PCR assay was further evaluated using 55 reference strains and 94 mycobacterial clinical isolates. Spoligotyping, multilocus sequence analysis, and a commercial real-time PCR assay were employed as standard assays to evaluate the multiplex PCR assay with clinical M. tuberculosis and NTM isolates. The PCR assay displayed 100% identification agreement with the standard assays. Our multiplex PCR assay is a simple, convenient, and reliable technique for differential identification of MTBC, M. tuberculosis, M. tuberculosis Beijing genotype, and major NTM species.