A 2-Hydroxyisoquinoline-1,3-Dione Active-Site RNase H Inhibitor Binds in Multiple Modes to HIV-1 Reverse Transcriptase.

The RNase H (RNH) function of HIV-1 reverse transcriptase (RT) plays an essential part in the viral life cycle. We report the characterization of YLC2-155, a 2-hydroxyisoquinoline-1,3-dione (HID)-based active-site RNH inhibitor. YLC2-155 inhibits both polymerase (50% inhibitory concentration [IC50] = 2.6 µM) and RNH functions (IC50 = 0.65 µM) of RT but is more effective against RNH. X-ray crystallography, nuclear magnetic resonance (NMR) analysis, and molecular modeling were used to show that YLC2-155 binds at the RNH-active site in multiple conformations.

Development of an engineered bioluminescent reporter phage for the sensitive detection of viable Salmonella typhimurium.

Because foodborne illnesses continuously threaten public health, rapid and sensitive detection of pathogens in food has become an important issue. As an alternative to time-consuming and laborious conventional detection methods, a technique using recombinant reporter phages has been developed. Here, we developed an advanced bioluminescent reporter phage SPC32H-CDABE by inserting a bacterial luxCDABE operon into the Salmonella temperate phage SPC32H genome. Whole SPC32H genome sequencing enabled the selection of nonessential genes, which can be replaced with approximately 6-kb luxCDABE operon, which provides both luciferase (LuxAB) and its substrate, fatty aldehyde, as generated by fatty acid reductase (LuxCDE). Thus, the SPC32H-CDABE detection assay is simpler and more efficient compared to the luxAB-based assay because the substrate addition step is excluded. At least 20 CFU/mL of pure S. Typhimurium culture was detectable using SPC32H-CDABE within 2 h, and the signals increased proportionally to the number of cells contaminated in lettuce, sliced pork, and milk. These results thereby demonstrate that this phage successfully detects live Salmonella without appreciable interference from food components. Furthermore, the presented data suggest that SPC32H-CDABE represents a promising easy-to-use diagnostic tool for the detection of Salmonella contamination in food.

EGFR inhibits TNF-α-mediated pathway by phosphorylating TNFR1 at tyrosine 360 and 401.

Tumour necrosis factor receptor 1 (TNFR1) induces the nuclear factor kappa-B (NF-κB) signalling pathway and regulated cell death processes when TNF-α ligates with it. Although mechanisms regulating the downstream pathways of TNFR1 have been elucidated, the direct regulation of TNFR1 itself is not well known. In this study, we showed that the kinase domain of the epidermal growth factor receptor (EGFR) regulates NF-κB signalling and TNF-α-induced cell death by directly phosphorylating TNFR1 at Tyr 360 and 401 in its death domain. In contrast, EGFR inhibition by EGFR inhibitors, such as erlotinib and gefitinib, prevented their interaction. Once TNFR1 is phosphorylated, its death domain induces the suppression of the NF-κB pathways, complex II-mediated apoptosis, or necrosome-dependent necroptosis. Physiologically, in mouse models, EGF treatment mitigates TNF-α-dependent necroptotic skin inflammation induced by treatment with IAP and caspase inhibitors. Our study revealed a novel role for EGFR in directly regulating TNF-α-related pathways.

A Preliminary Evaluation of the Diagnostic Performance of a Smartphone-Based Machine Learning-Assisted System for Evaluation of Clinical Activity Score in Digital Images of Thyroid-Associated Orbitopathy.

Background: We previously developed a machine learning (ML)-assisted system for predicting the clinical activity score (CAS) in thyroid-associated orbitopathy (TAO) using digital facial images taken by a digital single-lens reflex camera in a studio setting. In this study, we aimed to apply this system to smartphones and detect active TAO (CAS ≥3) using facial images captured by smartphone cameras. We evaluated the performance of our system on various smartphone models and compared it with the performance of ophthalmologists with varying clinical experience. Methods: We applied the preexisting ML architecture to classify photos taken with smartphones (Galaxy S21 Ultra, iPhone 12 pro, iPhone 11, iPhone SE 2020, Galaxy M20, and Galaxy A21S). The performance was evaluated with smartphone-captured images from 100 patients with TAO. Three ophthalmology residents, three general ophthalmologists with <5 years of clinical experience, and three oculoplastic specialists independently interpreted the same set of images taken under a studio environment and compared their results with those generated by the smartphone-based ML-assisted system. Reference CAS was determined by a consensus of three oculoplastic specialists. Results: Active TAO (CAS ≥3) was identified in 28 patients. Smartphone model used in capturing facial images influenced active TAO detection performance (F1 score 0.59-0.72). The smartphone-based system showed 74.5% sensitivity, 84.8% specificity, and F1 score 0.70 on top three smartphones. On images from all six smartphones, average sensitivity, specificity, and F1 score were 71.4%, 81.6%, and 0.66, respectively. Ophthalmology residents' values were 69.1%, 55.1%, and 0.46. General ophthalmologists' values were 61.9%, 79.6%, and 0.55. Oculoplastic specialists' values were 73.8%, 90.7%, and 0.75. This smartphone-based ML-assisted system predicted CAS within 1 point of reference CAS in 90.7% using facial images from smartphones. Conclusions: Our smartphone-based ML-assisted system shows reasonable accuracy in detecting active TAO, comparable with oculoplastic specialists and outperforming residents and general ophthalmologists. It may enable reliable self-monitoring for disease activity, but confirmatory research is needed for clinical application.

Trends in Utilization of Visual Field Tests for Glaucoma Patients: A Nationwide Study Using the Korean Health Insurance Review and Assessment Database.

PURPOSE: To analyze 10-year trends in utilization of visual field tests for adult glaucoma or glaucoma-suspect patients using the Korean Health Insurance Review and Assessment data. METHODS: Health claims for the years 2010 to 2019, as recorded via Korea's Health Insurance Review and Assessment service, were accessed. We identified glaucoma patients using the glaucoma diagnostic codes H40 (glaucoma) and H42 (glaucoma in other diseases classified elsewhere). For verification of the glaucoma diagnosis, information on any antiglaucoma medication prescriptions and ocular surgery history also was obtained. Visual field testing data was isolated using procedural codes E6690 (kinetic perimetry) and E6691 (standard automated perimetry [SAP]) performed in tertiary hospitals. Any changes in visual field test utilization were identified using regression trend analysis. RESULTS: From 2010 to 2019, the total number of SAP procedures performed in tertiary hospitals for either glaucoma or glaucoma-suspect patients increased gradually from 93,459 to 216,433. With regard to kinetic perimetry examinations, the total number decreased gradually from 6,364 to 3,792. The yearly average SAP number per patient showed a slight increase, from 1.168 to 1.248 (ß = 0.008, R2 = 0.669, p = 0.004). Meanwhile, the yearly average number of kinetic perimeter examinations per patient showed a significant decrease, from 1.093 to 0.940 (ß = -0.013, R2 = 0.580, p = 0.010). CONCLUSIONS: Between 2010 and 2019, the yearly average number of SAP procedures performed per glaucoma or glaucoma-suspect patient increased in Korea. Meanwhile, the yearly average number of kinetic perimetry examinations per patient significantly decreased.

Functional and dynamic mitochondrial damage by chloromethylisothiazolinone/methylisothiazolinone (CMIT/MIT) mixture in brain endothelial cell lines and rat cerebrovascular endothelium.

The mixture of 5-chloro-2-methyl-4-isothiazolin-3-one (CMIT, chloromethylisothiazolinone) and 2-methyl-4-isothiazolin-3-one (MIT, methylisothiazolinone) is a commonly used biocide in consumer products. Despite the health issues related to its usage in cosmetics and humidifier disinfectants (HD), understanding its adverse outcome is still limited. Using in vitro cell lines and ex vivo rat models, we examined the effects of CMIT/MIT on the cellular redox homeostasis and energy metabolism in the brain microvascular endothelium, a highly restrictive interface between the bloodstream and brain. In murine bEND.3 and human hCMEC/D3, CMIT/MIT significantly amplified the mitochondrial-derived oxidative stress causing disruption of the mitochondrial membrane potential and oxidative phosphorylation at a sub-lethal concentration (1 µg/mL) or treatment duration (1 h). In addition, CMIT/MIT significantly increased a dynamic imbalance between mitochondrial fission and fusion, and endogenous pathological stressors significantly potentiated the CMIT/MIT-induced endothelial dysfunction. Notably, in the brain endothelium isolated from intravenously CMIT/MIT-administered rats, we observed significant mitochondrial damage and decreased tight junction protein. Taken together, we report that CMIT/MIT significantly impaired mitochondrial function and dynamics resulting in endothelial barrier dysfunction, giving an insight into the role of mitochondrial damage in CMIT/MIT-associated systemic health effects.

Necroptosis molecular mechanisms: Recent findings regarding novel necroptosis regulators.

Necroptosis is a form of programmed necrosis that is mediated by various cytokines and pattern recognition receptors (PRRs). Cells dying by necroptosis show necrotic phenotypes, including swelling and membrane rupture, and release damage-associated molecular patterns (DAMPs), inflammatory cytokines, and chemokines, thereby mediating extreme inflammatory responses. Studies on gene knockout or necroptosis-specific inhibitor treatment in animal models have provided extensive evidence regarding the important roles of necroptosis in inflammatory diseases. The necroptosis signaling pathway is primarily modulated by activation of receptor-interacting protein kinase 3 (RIPK3), which phosphorylates mixed-lineage kinase domain-like protein (MLKL), mediating MLKL oligomerization. In the necroptosis process, these proteins are fine-tuned by posttranslational regulation via phosphorylation, ubiquitination, glycosylation, and protein-protein interactions. Herein, we review recent findings on the molecular regulatory mechanisms of necroptosis.

Exploring Socio-Demographic Factors Affecting Psychological Symptoms in Humidifier Disinfectant Survivors.

This study aimed to compare the psychological symptoms of humidifier disinfectant survivors to the general population and explore socio-demographic factors influencing survivors' psychological symptoms. A one-way Multivariate Analysis of Covariance (MANCOVA) and a series of two-way MANCOVA were conducted with a sample of 228 humidifier disinfectant survivors and 228 controls. The results demonstrated that the survivor group displayed higher anxious/depressed symptoms, withdrawn symptoms, somatic complaints, thought problems, attention problems, aggressive behavior and rule-breaking behavior than the general group. Moreover, among the socio-demographic factors, the two-way interaction effects of group × family economic status and group × number of friends were found to be statistically significant. The limitations and implications of this study are discussed.

Sensitive detection of viable Escherichia coli O157:H7 from foods using a luciferase-reporter phage phiV10lux.

Escherichia coli O157:H7, a major foodborne pathogen, is a major public health concern associated with life-threatening diseases such as hemolytic uremic syndrome. To alleviate this burden, a sensitive and rapid system is required to detect this pathogen in various kinds of foods. Herein, we propose a phage-based pathogen detection method to replace laborious and time-consuming conventional methods. We engineered an E. coli O157:H7-specific phage phiV10 to rapidly and sensitively detect this notorious pathogen. The luxCDABE operon was introduced into the phiV10 genome and allowed the engineered phage phiV10lux to generate bioluminescence proportional to the number of viable E. coli O157:H7 cells without any substrate addition. The phage phiV10lux was able to detect at least 1CFU/ml of E. coli O157:H7 in a pure culture within 40min after 5h of pre-incubation. In artificially contaminated romaine lettuce, apple juice (pH3.51), and ground beef, the reporter phage could detect approximately 10CFU/cm2, 13CFU/ml, and 17CFU/g of E. coli O157:H7, respectively. Taken together, the constructed reporter phage phiV10lux could be applied as a powerful tool for rapid and sensitive detection of live E. coli O157:H7 in foods.

Decomposition of biological macromolecules by plasma generated with helium and oxygen.

In this study, we attempted to characterize the biomolecular effects of an atmospheric pressure cold plasma (APCP) system which utilizes helium/oxygen (He/O(2)). APCP using He/O(2) generates a low level of UV while generating reactive oxygen radicals which probably serve as the primary factor in sterilization; these reactive oxygen radicals have the advantage of being capable to access the interiors of the structures of microbial cells. The damaging effects of plasma exposure on polypeptides, DNA, and enzyme proteins in the cell were assessed using biochemical methods.

Characterization of monoclonal antibody specific to the Z39Ig protein, a member of immunoglobulin superfamily.

Z39Ig, a recently identified immunoglobulin (Ig) superfamily member, is localized in the pericentromeric region of human chromosome X and detectable in all human tissue, but it is predominantly expressed in fetal human tissues as well as in adult lungs and placenta. In the present study, we generated a monoclonal antibody against Z39Ig protein to investigate the immunological role of Z39Ig protein on various immune cells. The anti-Z39Ig mAb that we generated specifically bound to Z39Ig protein on human promonocytic THP-1 cells, monocytes isolated from human peripheral blood mononuclear cells (PBMC) and mature CD14(+) dendritic cells (DC) differentiated from umbilical-cord blood CD34(+) hematopoietic progenitor cells. In addition, a signal through the Z39Ig protein induced an obvious cell surface expression of HLA-DR on THP-1 cells mediated by MHC class II transactivator (CIITA). These data suggest that the Z39Ig protein might be a critical molecule to regulate an immune response mediated by phagocytosis and/or antigen presentation.

Evaluating the efficiency of an asbestos stabilizer on ceiling tiles and the characteristics of the released asbestos fibers.

The efficiency of asbestos stabilizers and their adaptability were evaluated by investigating the characteristics of asbestos fibers released from ceiling tiles. The impact of such variables as the wind speed or vibration conditions was also studied along with the asbestos stabilizers. The concentrations of the asbestos fibers released from damaged ceiling tiles treated with stabilizers decreased by 69.5-84.4% compared with those of untreated tiles for all variables, with a statistically significant difference (p<0.001). The effects of the environmental factors on the asbestos concentrations were analyzed through a multiple regression analysis. It was determined that the surface status of the ceiling tiles and stabilizers were the main factors affecting the concentration, and the reliability of these factors was estimated as 58.3%. The lengths of the chrysotile fibers released from the damaged ceiling tiles were in the range of 0.991-79.1 µm for the untreated tiles and 3.74-35.6 µm for the tiles treated with inorganic stabilizers. It was confirmed that inorganic stabilizers are more efficient for damaged ceiling tiles. The results of this study also show that the asbestos concentrations are greatly reduced after treating damaged ceiling tiles with a stabilizer.