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Tularemia, caused by the bacterium , poses health risks to humans and can spread through a variety of routes. It has also been classified as a Tier 1 Select agent by the CDC, highlighting its potential as a bioterrorism agent. Moreover, it is difficult to diagnose in a timely fashion, owing to the non-specific nature of tularemia infections. Rapid, sensitive, and accurate detection methods are required to reduce mortality rates. We aimed to develop antibodies directed against the outer membrane protein A of (FopA) for rapid and accurate diagnosis of tularemia. We used a baculovirus insect cell expression vector system to produce the FopA antigen and generate anti-FopA antibodies through immunization of BALB/c mice. We then employed hybridoma and phage display technologies to screen for antibodies that could recognize unique epitopes on FopA. Two monoclonal antibodies, 6B12 and 3C1, identified through phage display screening specifically bound to recombinant FopA in a dose-dependent manner. The binding affinity of the anti-FopA 6B12 and 3C1 antibodies was observed to have an equilibrium dissociation constant of 1.76 × 10-10 M and 1.32 × 10-9 M, respectively. These antibodies were used to develop a sandwich ELISA system for the diagnosis of tularemia. This assay was found to be highly specific and sensitive, with detection limits ranging from 0.062 ng/mL in PBS to 0.064 ng/mL in skim milk matrices. Our findings demonstrate the feasibility of a novel diagnostic approach for detecting based on targeting FopA, as opposed to existing tests that target the bacterial lipopolysaccharide.

Staphylococcal enterotoxin B (SEB) is a potent bacterial toxin that causes inflammatory stimulation and toxic shock, thus it is necessary to detect SEB in food and environmental samples. Here, we developed a sensitive immunodetection system using monoclonal antibodies (mAbs). Our study is the first to employ a baculovirus expression vector system (BEVS) to produce recombinant wild-type SEB. BEVS facilitated high-quantity and pure SEB production from suspension-cultured insect cells, and the SEB produced was characterized by mass spectrometry analysis. The SEB was stable at 4 °C for at least 2 years, maintaining its purity, and was further utilized for mouse immunization to generate mAbs. An optimal pair of mAbs non-competitive to SEB was selected for sandwich enzyme-linked immunosorbent assay-based immunodetection. The limit of detection of the immunodetection method was 0.38 ng/mL. Moreover, it displayed higher sensitivity in detecting SEB than commercially available immunodetection kits and retained detectability in various matrices and S. aureus culture supernatants. Thus, the results indicate that BEVS is useful for producing pure recombinant SEB with its natural immunogenic property in high yield, and that the developed immunodetection assay is reliable and sensitive for routine identification of SEB in various samples, including foods.

Obesity is a growing global epidemic that can cause serious adverse health consequences, including insulin resistance (IR) and nonalcoholic fatty liver disease (NAFLD). Obesity development can be attributed to energy imbalance and metabolic inflexibility. Here, we demonstrated that lack of Kelch-like protein 3 (KLHL3) mitigated the development of obesity, IR, and NAFLD by increasing energy expenditure. KLHL3 mutations in humans cause Gordon’s hypertension syndrome; however, the role of KLHL3 in obesity was previously unknown. We examined differences in obesity-related parameters between control and Klhl3 −/− mice. A significant decrease in body weight concomitant with fat mass loss and improved IR and NAFLD were observed in Klhl3 −/− mice fed a high-fat (HF) diet and aged. KLHL3 deficiency inhibited obesity, IR, and NAFLD by increasing energy expenditure with augmentation of O 2 consumption and CO 2 production. Delivering dominant-negative (DN) Klhl3 using adeno-associated virus into mice, thereby dominantly expressing DN-KLHL3 in the liver, ameliorated diet-induced obesity, IR, and NAFLD. Finally, adenoviral overexpression of DN-KLHL3, but not wild-type KLHL3, in hepatocytes revealed an energetic phenotype with an increase in the oxygen consumption rate. The present findings demonstrate a novel function of KLHL3 mutation in extrarenal tissues, such as the liver, and may provide a therapeutic target against obesity and obesity-related diseases. Obesity: A protein target for control Mice that are genetically engineered to lack a protein involved in regulating energy expenditure are protected against the onset of obesity and the related problems of insulin resistance and non-alcoholic fatty liver disease. Jeong-Ki Min, Jong-Gil Park and colleagues at the Korea Research Institute of Bioscience & Biotechnology in South Korea, Daejon, discovered that the beneficial effect of the lack of the protein, called KLHL3, was due to an increase in energy expenditure. Mutations in the gene for KLHL3 are known to cause a variety of metabolic diseases in humans, including a form of high blood pressure called Gordon’s hypertension syndrome, but the protein’s role in human obesity has not been studied. The results suggest that drugs able to regulate the production or activity of KLHL3 might offer a new approach to treating obesity.

Introduction: The binary PirA/PirB toxin expressed by Vibrio parahaemolyticus (PirABVp) is a virulent complex that causes acute hepatopancreatic necrosis disease (AHPND) in shrimps, affecting the global shrimp farming industry. AHPND is currently diagnosed by detecting pirA and pirB genes by PCR; however, several V. parahaemolyticus strains do not produce the two toxins as proteins. Thus, an immunoassay using antibodies may be the most effective tool for detecting toxin molecules. In this study, we report a sandwich ELISA-based immunoassay for the detection of PirABVp.Methods: We utilized a single-chain variable fragment (scFv) antibody library to select scFvs against the PirA or PirB subunits. Phage display panning rounds were conducted to screen and identify scFv antibodies directed against each recombinant toxin subunit. Selected scFvs were converted into IgGs to develop a sandwich immunoassay to detect recombinant and bacterial PirABVp.Results: Antibodies produced as IgG forms showed sub-nanomolar to nanomolar affinities (KD), and a pair of anti-PirA antibody as a capture and anti-PirB antibody as a detector showed a limit of detection of 201.7 ng/mL for recombinant PirABVp. The developed immunoassay detected PirABVp in the protein lysates of AHPND-causing V. parahaemolyticus (VpAHPND) and showed a significant detectability in moribund or dead shrimp infected with a VpAHPND virulent strain compared to that in non-infected shrimp.Discussion: These results indicate that the developed immunoassay is a reliable method for diagnosing AHPND by detecting PirABVp at the protein level and could be further utilized to accurately determine the virulence of extant or newly identified VpAHPND in the global shrimp culture industry.

The binary PirA/PirB toxin expressed by (PirAB ) is a virulent complex that causes acute hepatopancreatic necrosis disease (AHPND) in shrimps, affecting the global shrimp farming industry. AHPND is currently diagnosed by detecting and genes by PCR; however, several strains do not produce the two toxins as proteins. Thus, an immunoassay using antibodies may be the most effective tool for detecting toxin molecules. In this study, we report a sandwich ELISA-based immunoassay for the detection of PirAB . We utilized a single-chain variable fragment (scFv) antibody library to select scFvs against the PirA or PirB subunits. Phage display panning rounds were conducted to screen and identify scFv antibodies directed against each recombinant toxin subunit. Selected scFvs were converted into IgGs to develop a sandwich immunoassay to detect recombinant and bacterial PirAB . Antibodies produced as IgG forms showed sub-nanomolar to nanomolar affinities (K ), and a pair of anti-PirA antibody as a capture and anti-PirB antibody as a detector showed a limit of detection of 201.7 ng/mL for recombinant PirAB . The developed immunoassay detected PirAB in the protein lysates of AHPND-causing and showed a significant detectability in moribund or dead shrimp infected with a virulent strain compared to that in non-infected shrimp. These results indicate that the developed immunoassay is a reliable method for diagnosing AHPND by detecting PirAB at the protein level and could be further utilized to accurately determine the virulence of extant or newly identified in the global shrimp culture industry.

We investigated whether the standardized uptake value (SUV) of the primary tumor, the tumor length measured on a PET image, the number of (18)F-FDG PET-positive nodes, and the PET stage were independent prognostic predictors over other clinical variables in patients with esophageal squamous cell carcinoma who were undergoing curative surgery. Sixty-nine patients with newly diagnosed esophageal squamous cell carcinoma who underwent preoperative (18)F-FDG PET and curative esophagectomy were included. The events for survival analysis were defined as recurrence or metastasis and cancer-related death. The disease-free and overall survival rates of each variable were estimated by the Kaplan-Meier method. The Cox proportional hazards model was used to evaluate independent prognostic variables for multivariate survival analysis. Using univariate survival analysis, the presence of adjuvant therapy, pathologic stage, number of CT-positive nodes (0, 1, > or =2), tumor length on PET (cutoff: 3 cm, 5 cm), number of PET-positive nodes (0, 1, 2, > or =3), and PET stage (N0 M0, N1 M0, M1) were significant prognostic predictors for disease-free survival. However, only the number of PET-positive nodes was an independent significant prognostic predictor for disease-free survival in multivariate analysis (hazard ratio = 1.87, P < 0.001). In univariate survival analysis, the sex, presence of adjuvant therapy, clinical and pathologic stages, number of CT-positive nodes, maximum SUV of the primary tumor (cutoff: 6.3, 13.7), tumor length on PET, number of PET-positive nodes, and PET stage were significant prognostic predictors for overall survival. In contrast, the clinical stage (hazard ratio = 0.53, P < 0.05), pathologic stage (hazard ratio = 3.14, P < 0.005), tumor length by PET (hazard ratio = 2.74, P = 0.01), and number of PET-positive nodes (hazard ratio = 1.71, P < 0.05) were independent significant prognostic predictors for overall survival in multivariate analysis. In addition to the pathologic stage, (18)F-FDG PET provides noninvasively independent prognostic information using the number of positive lymph nodes and the tumor length on the PET image in preoperative esophageal squamous cell carcinoma. A revised TNM classification system for esophageal carcinoma may consider tumor length and the number of positive lymph nodes as important prognostic factors.

We investigated whether the standardized uptake value (SUV) of the primary tumor, the tumor length measured on a PET image, the number of (18)F-FDG PET-positive nodes, and the PET stage were independent prognostic predictors over other clinical variables in patients with esophageal squamous cell carcinoma who were undergoing curative surgery. Sixty-nine patients with newly diagnosed esophageal squamous cell carcinoma who underwent preoperative (18)F-FDG PET and curative esophagectomy were included. The events for survival analysis were defined as recurrence or metastasis and cancer-related death. The disease-free and overall survival rates of each variable were estimated by the Kaplan-Meier method. The Cox proportional hazards model was used to evaluate independent prognostic variables for multivariate survival analysis. Using univariate survival analysis, the presence of adjuvant therapy, pathologic stage, number of CT-positive nodes (0, 1, > or =2), tumor length on PET (cutoff: 3 cm, 5 cm), number of PET-positive nodes (0, 1, 2, > or =3), and PET stage (N0 M0, N1 M0, M1) were significant prognostic predictors for disease-free survival. However, only the number of PET-positive nodes was an independent significant prognostic predictor for disease-free survival in multivariate analysis (hazard ratio = 1.87, P < 0.001). In univariate survival analysis, the sex, presence of adjuvant therapy, clinical and pathologic stages, number of CT-positive nodes, maximum SUV of the primary tumor (cutoff: 6.3, 13.7), tumor length on PET, number of PET-positive nodes, and PET stage were significant prognostic predictors for overall survival. In contrast, the clinical stage (hazard ratio = 0.53, P < 0.05), pathologic stage (hazard ratio = 3.14, P < 0.005), tumor length by PET (hazard ratio = 2.74, P = 0.01), and number of PET-positive nodes (hazard ratio = 1.71, P < 0.05) were independent significant prognostic predictors for overall survival in multivariate analysis. In addition to the pathologic stage, (18)F-FDG PET provides noninvasively independent prognostic information using the number of positive lymph nodes and the tumor length on the PET image in preoperative esophageal squamous cell carcinoma. A revised TNM classification system for esophageal carcinoma may consider tumor length and the number of positive lymph nodes as important prognostic factors.

Transglutaminase2 (TG2) is a multi-functional protein involved in various cellular processes, including apoptosis, differentiation, wound healing, and angiogenesis. The malfunction of TG2 causes many human disease including inflammatory disease, celiac disease, neurodegenerative diseases, tissue fibrosis, and cancers. Protein cross-linking activity, which is representative of TG2, is activated by calcium ions and suppressed by GTP. Here, we elucidated the structure of TG2 in complex with its endogenous inhibitor, GTP. Our structure showed why GTP is the optimal nucleotide for interacting with and inhibiting TG2. In addition, sequence comparison provided information describing the evolutionary scenario of GTP usage for controlling the activity of TG2.

Studies have highlighted complex bidirectional relationships between autoimmune diseases and depressive disorders. Given that early mental health interventions have substantial public health implications, this study investigated association between optic neuritis, an autoimmune inflammatory disorder of the optic nerve, and risk of developing depressive disorders. Utilizing extensive national health insurance data encompassing almost the entire Korean population, this cohort study included 11,745 patients with optic neuritis and 58,725 age- and sex- matched controls between 2010 and 2017. The diagnosis of optic neuritis was confirmed using ICD-10 code H46 and patient medical records. The association with depression risk identified by ICD-10 codes F32 and F33 was assessed using Cox proportional hazards regression models after adjusting for demographics, lifestyle variables, and other comorbidities. Newly diagnosed optic neuritis was associated with an increased risk of depression (hazard ratio = 1.349, 95% confidence interval: 1.277–1.426), independent of potential confounding factors. Subgroup analysis revealed a stronger association for individuals under 50 years, males, current smokers, and those without hypertension. This association suggests that autoimmune neuroinflammatory responses impact mental health differently across demographics. These findings underscore the importance of implementing routine depression screening and developing targeted early intervention strategies for patients with optic neuritis, particularly for those with a high-risk of depression.

There are little direct comparative evidences of strategies between ≥50% and the absolute target goal of low-density lipoprotein cholesterol (LDL-C) level <55 mg/100 ml for the patients who underwent percutaneous coronary intervention (PCI). This study aimed to investigate the clinical impact of different strategies between 2 groups of patients who underwent PCI. A total of 3,104 patients with previous PCI were retrospectively enrolled from 2014 to 2020 at Yeungnam University Medical Center. The study population was stratified into 2 groups based on whether the LDL-C level was <55 mg/100 ml at the 1-year mark or not. Furthermore, the 50% reduction rate of LDL-C was also categorized based on whether it had decreased by ≥50% from the initial LDL-C level at the 1-year mark. The primary end point was 3-year major adverse cardiovascular events (MACEs) which were defined as a composite of cardiovascular death, nonfatal myocardial infarction, target lesion revascularization, hospitalization for heart failure, or nonfatal stroke. There was no significant difference between the LDL <55 mg/100 ml group and the LDL ≥55 mg/100 ml group in the risk of MACEs (hazard ratio 1.06, 95% confidence interval 0.81 to 1.38, p = 0.690) after propensity score matching. However, the group that achieved ≥50% reduction of LDL-C from baseline LDL-C level showed a significant reduction in the occurrence of MACEs in the subgroup of LDL-C level ≥55 mg/100 ml (hazard ratio 0.41, 95% confidence interval 0.19 to 0.89, p = 0.025) compared with the group with <50% reduction of LDL-C. In all patients, the achievement rate of target LDL-C <55 mg/100 ml and more than 50% reduction from baseline was 17.2%. In conclusion, guideline-directed management strategy of ≥50% reduction of LDL-C from the baseline will be needed to reduce the incidence of MACEs in patients with LDL-C ≥55 mg/100 ml who underwent PCI. Additional efforts to increase the target goal achievement rate of LDL-C are warranted.