Sputum processing method for lateral flow immunochromatographic assays to detect coronaviruses

Coronavirus causes an infectious disease in various species and crosses the species barriers leading to the outbreak of zoonotic diseases. Due to the respiratory diseases are mainly caused in humans and viruses are replicated and excreted through the respiratory tract, the nasal fluid and sputum are mainly used for diagnosis. Early diagnosis of coronavirus plays an important role in preventing its spread and is essential for quarantine policies. For rapid decision and prompt triage of infected host, the immunochromatographic assay (ICA) has been widely used for point of care testing. However, when the ICA is applied to an expectorated sputum in which antigens are present, the viscosity of sputum interferes with the migration of the antigens on the test strip. To overcome this limitation, it is necessary to use a mucolytic agent without affecting the antigens. In this study, we combined known mucolytic agents to lower the viscosity of sputum and applied that to alpha and beta coronavirus, porcine epidemic diarrhea virus (PEDV) and Middle East respiratory syndrome coronavirus (MERS-CoV), respectively, spiked in sputum to find optimal pretreatment conditions. The pretreatment method using tris(2-carboxyethyl)phosphine (TCEP) and BSA was suitable for ICA diagnosis of sputum samples spiked with PEDV and MERS-CoV. This sensitive assay for the detection of coronavirus in sputum provides an useful information for the diagnosis of pathogen in low respiratory tract.

유해 미세조류-미생물, 바이러스 분리, 배양 및 상호작용 연구

유해 미세조류-미생물, 바이러스 분리, 배양 및 상호작용 연구OBM001202

TSHZ2 is an EGF-regulated tumor suppressor that binds to the cytokinesis regulator PRC1 and inhibits metastasis

Unlike early transcriptional responses to mitogens, later events are less well-characterized. Here, we identified delayed down-regulated genes (DDGs) in mammary cells after prolonged treatment with epidermal growth factor (EGF). The expression of these DDGs was low in mammary tumors and correlated with prognosis. The proteins encoded by several DDGs directly bind to and inactivate oncoproteins and might therefore act as tumor suppressors. The transcription factor teashirt zinc finger homeobox 2 (TSHZ2) is encoded by a DDG, and we found that overexpression of TSHZ2 inhibited tumor growth and metastasis and accelerated mammary gland development in mice. Although the gene TSHZ2 localizes to a locus (20q13.2) that is frequently amplified in breast cancer, we found that hypermethylation of its promoter correlated with down-regulation of TSHZ2 expression in patients. Yeast two-hybrid screens and protein-fragment complementation assays in mammalian cells indicated that TSHZ2 nucleated a multiprotein complex containing PRC1/Ase1, cyclin B1, and additional proteins that regulate cytokinesis. TSHZ2 increased the inhibitory phosphorylation of PRC1, a key driver of mitosis, mediated by cyclin-dependent kinases. Furthermore, similar to the tumor suppressive transcription factor p53, TSHZ2 inhibited transcription from the PRC1 promoter. By recognizing DDGs as a distinct group in the transcriptional response to EGF, our findings uncover a group of tumor suppressors and reveal a role for TSHZ2 in cell cycle regulation.

Complete genome sequence of platycodon closterovirus 1, a novel putative member of the genus Closterovirus

A new member of the genus Closterovirus was detected in Platycodon grandiflorus using high-throughput RNA sequencing analysis. The complete genome sequence of this new virus isolate, tentatively named “platycodon closterovirus 1” (PlaCV1), comprises 16,771 nucleotides with nine predicted open reading frames (ORFs) having the typical closterovirus genome organization. PlaCV1 shares 37%？50% nucleotide sequence identity with other known closterovirus genome sequences. The putative RNA-dependent RNA polymerase (RdRp), heat shock protein 70-like protein (HSP70h), viral heat shock protein 90-like protein (HSP90h), minor coat protein (CPm), and coat protein (CP) show 47？68%, 39？66%, 24？52%, 21？57%, and 16？35% amino acid sequence identity, respectively, to homologous proteins in previously identified closteroviruses, suggesting that it represents a distinct, new species in the genus. Phylogenetic analysis of HSP70h sequences places PlaCV1 alongside other members of the genus Closterovirus in the family Closteroviridae. To our knowledge, this study is the first report of the complete genome sequence of PlaCV1 infecting P. grandiflorus in the Republic of Korea.

Characterization of terminal-ileal and colonic Crohn's disease in treatment-naive paediatric patients based on transcriptomic profile using logistic regression

Background: Inflammatory bowel disease (IBD) is a chronic and idiopathic inflammatory disorder of the gastrointestinal tract and comprises ulcerative colitis (UC) and Crohn's disease (CD). Crohn's disease can affect any part of the gastrointestinal tract, but mainly the terminal ileum and colon. In the present study, we aimed to characterize terminal-ileal CD (ICD) and colonic CD (CCD) at the molecular level, which might enable a more optimized approach for the clinical care and scientific research of CD. Methods: We analyzed differentially expressed genes in samples from 23 treatment-naive paediatric patients with CD and 25 non-IBD controls, and compared the data with previously published RNA-Seq data using multi-statistical tests and confidence intervals. We implemented functional profiling and proposed statistical methods for feature selection using a logistic regression model to identify genes that are highly associated in ICD or CCD. We also validated our final candidate genes in independent paediatric and adult cohorts. Results: We identified 550 genes specifically expressed in patients with CD compared with those in healthy controls (p < 0.05). Among these DEGs, 240 from patients with CCD were mainly involved in mitochondrial dysfunction, whereas 310 from patients with ICD were enriched in the ileum functions such as digestion, absorption, and metabolism. To choose the most effective gene set, we selected the most powerful genes (p-value ≤ 0.05, accuracy ≥ 0.8, and AUC ≥ 0.8) using logistic regression. Consequently, 33 genes were identified as useful for discriminating CD location; the accuracy and AUC were 0.86 and 0.83, respectively. We then validated the 33 genes with data from another independent paediatric cohort (accuracy = 0.93, AUC = 0.92) and adult cohort (accuracy = 0.88, AUC = 0.72). Conclusions: In summary, we identified DEGs that are specifically expressed in CCD and ICD compared with those in healthy controls and patients with UC. Based on the feature selection analysis, 33 genes were identified as useful for discriminating CCD and ICD with high accuracy and AUC, for not only paediatric patients but also independent cohorts. We propose that our approach and the final gene set are useful for the molecular classification of patients with CD, and it could be beneficial in treatments based on disease location.

Directed evolution of glycosyltransferase for enhanced efficiency of avermectin glucosylation

Avermectin, produced by Streptomyces avermitilis, is an active compound protective against nematodes, insects, and mites. However, its potential usage is limited by its low aqueous solubility. The uridine diphosphate (UDP)-glycosyltransferase (BLC) from Bacillus licheniformis synthesizes avermectin glycosides with improved water solubility and in vitro antinematodal activity. However, enzymatic glycosylation of avermectin by BLC is limited due to the low conversion rate of this reaction. Thus, improving BLC enzyme activity is necessary for mass production of avermectin glycosides for field application. In this study, the catalytic activity of BLC toward avermectin was enhanced via directed evolution. Three mutants from the BLC mutant library (R57H, V227A, and D252V) had specific glucosylation activity for avermectin 2.0-, 1.8-, and 1.5-fold higher, respectively, than wild-type BLC. Generation of combined mutations via site-directed mutagenesis led to even further enhancement of activity. The triple mutant, R57H/V227A/D252V, had the highest activity, 2.8-fold higher than that of wild-type BLC. The catalytic efficiencies (Kcat/Km) of the best mutant (R57H/V227A/D252V) toward the substrates avermectin and UDP-glucose were improved by 2.71- and 2.29-fold, respectively, compared to those of wild-type BLC. Structural modeling analysis revealed that the free energy of the mutants was - 1.1 to - 7.1 kcal/mol lower than that of wild-type BLC, which was correlated with their improved activity. KEY POINTS: ？ Directed evolution improved the glucosylation activity of BLC toward avermectin. ？ Combinatorial site-directed mutagenesis led to further enhanced activity. ？ The mutants exhibited lower free energy values than wild-type BLC.

출연(연) 4차인재 양성사업

출연(연) 4차인재 양성사업TGM091202

Interplay among conformation, intramolecular hydrogen bonds, and chameleonicity in the membrane permeability and cyclophilin A binding of Mmacrocyclic peptide cyclosporin O derivatives

A macrocyclic peptide scaffold with well-established structure-property relationship is desirable for tackling undruggable targets. Here, we adopted a natural macrocycle, cyclosporin O (CsO) and its derivatives (CP1-3), and evaluated the impact of conformation on membrane permeability, cyclophilin A (CypA) binding, and the pharmacokinetic (PK) profile. In nonpolar media, CsO showed a similar conformation to cyclosporin A (CsA), a well-known chameleonic macrocycle, but less chameleonic behavior in a polar environment. The weak chameleonicity of CsO resulted in decreased membrane permeability; however, the more rigid conformation of CsO was not detrimental to its PK profile. CsO exhibited a higher plasma concentration than CsA, which resulted from minimal CypA binding and lower accumulation in red blood cells and moderate oral bioavailability (F = 12%). Our study aids understanding of CsO, a macrocyclic peptide that is less explored than CsA but with greater potential for diversity generation and rational design.

Olfactory detection of toluene by detection rats for potential screening of lung cancer

Early detection is critical to successfully eradicating a variety of cancers, so the development of a new cancer primary screening system is essential. Herein, we report an animal nose sensor system for the potential primary screening of lung cancer. To establish this, we developed an odor discrimination training device based on operant conditioning paradigms for detection of toluene, an odor indicator component of lung cancer. The rats (N = 15) were trained to jump onto a floating ledge in response to toluene-spiked breath samples. Twelve rats among 15 trained rats reached performance criterion in 12 consecutive successful tests within a given set, or over 12 sets, with a success rate of over 90%. Through a total of 1934 tests, the trained rats (N = 3) showed excellent performance for toluene detection with 82% accuracy, 83% sensitivity, 81% specificity, 80% positive predictive value (PPV) and 83% negative predictive value (NPV). The animals also acquired considerable performance for odor discrimination even in rigorous tests, validating odor specificity. Since environmental and long-term stability are important factors that can influence the sensing results, the performance of the trained rats was studied under specified temperature (20, 25, and 30 °C) and humidity (30%, 45%, and 60% RH) conditions, and monitored over a period of 45 days. At given conditions of temperature and humidity, the animal sensors showed an average accuracy within a deviation range of ±10%, indicating the excellent environmental stability of the detection rats. Surprisingly, the trained rats did not differ in retention of last odor discrimination when tested 45 days after training, denoting that the rats’ memory for trained odor is still available over a long period of time. When taken together, these results indicate that our odor discrimination training system can be useful for non-invasive breath testing and potential primary screening of lung cancer.

Assessment of Erythrobacter species diversity through pan-genome analysis with newly isolated Erythrobacter sp. 3-20A1M

Erythrobacter species are extensively studied marine bacteria that produce various carotenoids. Due to their photoheterotrophic ability, it has been suggested that they play a crucial role in marine ecosystems. It is essential to identify the genome sequence and the genes of the species to predict their role in the marine ecosystem. In this study, we report the complete genome sequence of the marine bacterium Erythrobacter sp. 3-20A1M. The genome size was 3.1 Mbp and its GC content was 64.8%. In total, 2998 genetic features were annotated, of which 2882 were annotated as functional coding genes. Using the genetic information of Erythrobacter sp. 3-20A1M, we performed pangenome analysis with other Erythrobacter species. This revealed highly conserved secondary metabolite biosynthesis-related COG functions across Erythrobacter species. Through subsequent secondary metabolite biosynthetic gene cluster prediction and KEGG analysis, the carotenoid biosynthetic pathway was proven conserved in all Erythrobacter species, except for the spheroidene and spirilloxanthin pathways, which are only found in photosynthetic Erythrobacter species. The presence of virulence genes, especially the plant-algae cell wall degrading genes, revealed that Erythrobacter sp. 3-20A1M is a potential marine plant-algae scavenger.