ABSTRACT
A stable mitochondrial pool is crucial for healthy cell function and survival. Altered redox biology can adversely affect mitochondria through induction of a variety of cell death and survival pathways, yet the understanding of mitochondria and their dysfunction in primary human cells and in specific disease states, including asthma, is modest. Ferroptosis is traditionally considered an iron dependent, hydroperoxy-phospholipid executed process, which induces cytosolic and mitochondrial damage to drive programmed cell death. However, in this report we identify a lipoxygenase orchestrated, compartmentally-targeted ferroptosis-associated peroxidation process which occurs in a subpopulation of dysfunctional mitochondria, without promoting cell death. Rather, this mitochondrial peroxidation process tightly couples with PTEN-induced kinase (PINK)-1(PINK1)-Parkin-Optineurin mediated mitophagy in an effort to preserve the pool of functional mitochondria and prevent cell death. These combined peroxidation processes lead to altered epithelial cell phenotypes and loss of ciliated cells which associate with worsened asthma severity. Ferroptosis-targeted interventions of this process could preserve healthy mitochondria, reverse cell phenotypic changes and improve disease outcomes.
Subject(s)
Asthma , Cell Cycle Proteins , Epithelial Cells , Ferroptosis , Membrane Transport Proteins , Mitochondria , Mitophagy , Phenotype , Transcription Factor TFIIIA , Humans , Mitochondria/metabolism , Asthma/metabolism , Asthma/pathology , Epithelial Cells/metabolism , Membrane Transport Proteins/metabolism , Transcription Factor TFIIIA/metabolism , Transcription Factor TFIIIA/genetics , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/genetics , Male , Protein Kinases/metabolism , Female , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Animals , Lipid Peroxidation , Mice , Middle AgedABSTRACT
Methylenetetrahydrofolate reductase (MTHFR) is a pivotal flavoprotein connecting the folate and methionine methyl cycles, catalyzing the conversion of methylenetetrahydrofolate to methyltetrahydrofolate. Human MTHFR (hMTHFR) undergoes elaborate allosteric regulation involving protein phosphorylation and S-adenosylmethionine (AdoMet)-dependent inhibition, though other factors such as subunit orientation and FAD status remain understudied due to the lack of a functional structural model. Here, we report crystal structures of Chaetomium thermophilum MTHFR (cMTHFR) in both active (R) and inhibited (T) states. We reveal FAD occlusion by Tyr361 in the T-state, which prevents substrate interaction. Remarkably, the inhibited form of cMTHFR accommodates two AdoMet molecules per subunit. In addition, we conducted a detailed investigation of the phosphorylation sites in hMTHFR, three of which were previously unidentified. Based on the structural framework provided by our cMTHFR model, we propose a possible mechanism to explain the allosteric structural transition of MTHFR, including the impact of phosphorylation on AdoMet-dependent inhibition.
Subject(s)
Chaetomium , Methylenetetrahydrofolate Reductase (NADPH2) , S-Adenosylmethionine , Methylenetetrahydrofolate Reductase (NADPH2)/metabolism , Methylenetetrahydrofolate Reductase (NADPH2)/chemistry , Methylenetetrahydrofolate Reductase (NADPH2)/genetics , S-Adenosylmethionine/metabolism , S-Adenosylmethionine/chemistry , Allosteric Regulation , Chaetomium/enzymology , Chaetomium/metabolism , Chaetomium/genetics , Phosphorylation , Humans , Crystallography, X-Ray , Models, Molecular , Flavin-Adenine Dinucleotide/metabolism , Flavin-Adenine Dinucleotide/chemistryABSTRACT
BACKGROUND: The management of intractable secondary pneumothorax poses a considerable challenge as it is often not indicated for surgery owing to the presence of underlying disease and poor general condition. While endobronchial occlusion has been employed as a non-surgical treatment for intractable secondary pneumothorax, its effectiveness is limited by the difficulty of locating the bronchus leading to the fistula using conventional techniques. This report details a case treated with endobronchial occlusion where the combined use of transbronchoscopic oxygen insufflation and a digital chest drainage system enabled location of the bronchus responsible for a prolonged air leak, leading to the successful treatment of intractable secondary pneumothorax. CASE PRESENTATION: An 83-year-old male, previously diagnosed with chronic hypersensitivity pneumonitis and treated with long-term oxygen therapy and oral corticosteroid, was admitted due to a pneumothorax emergency. Owing to a prolonged air leak after thoracic drainage, the patient was deemed at risk of developing an intractable secondary pneumothorax. Due to his poor respiratory condition, endobronchial occlusion with silicone spigots was performed instead of surgery. The location of the bronchus leading to the fistula was unclear on CT imaging. When the bronchoscope was wedged into each subsegmental bronchus and low-flow oxygen was insufflated, a digital chest drainage system detected a significant increase of the air leak only in B5a and B5b, thus identifying the specific location of the bronchus leading to the fistula. With the occlusion of those bronchi using silicone spigots, the air leakage decreased from 200 mL/min to 20 mL/min, and the addition of an autologous blood patch enabled successful removal of the drainage tube. CONCLUSION: The combination of transbronchoscopic oxygen insufflation with a digital chest drainage system can enhance the therapeutic efficacy of endobronchial occlusion by addressing the problems encountered in conventional techniques, where the ability to identify the leaking bronchus is dependent on factors such as the amount of escaping air and the location of the fistula.
Subject(s)
Bronchoscopy , Drainage , Insufflation , Pneumothorax , Humans , Pneumothorax/therapy , Pneumothorax/surgery , Male , Aged, 80 and over , Drainage/methods , Bronchoscopy/methods , Insufflation/methods , Oxygen/administration & dosage , Bronchial Fistula/surgery , Bronchial Fistula/therapy , Tomography, X-Ray Computed , Chest Tubes , BronchiABSTRACT
BACKGROUND AND OBJECTIVE: Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a diagnostic procedure with adequate performance; however, its ability to provide specimens of sufficient quality and quantity for treatment decision-making in advanced-stage lung cancer may be limited, primarily due to blood contamination. The use of a 0.96-mm miniforceps biopsy (MFB) permits true histological sampling, but the resulting small specimens are unsuitable for the intended applications. Therefore, we introduced a 1.9-mm standard-sized forceps biopsy (SFB) and compared its utility to that of MFB. METHODS: We prospectively enrolled patients from three institutions who presented with hilar/mediastinal lymphadenopathy and suspected advanced-stage lung cancer, or those who were already diagnosed but required additional tissue specimens for biomarker analysis. Each patient underwent MFB followed by SFB three or four times through the tract created by TBNA using a 22-gauge needle on the same lymph node (LN). Two pathologists assessed the quality and size of each specimen using a virtual slide system, and diagnostic performance was compared between the MFB and SFB groups. RESULTS: Among the 60 enrolled patients, 70.0% were diagnosed with adenocarcinoma. The most frequently targeted sites were the lower paratracheal LNs, followed by the interlobar LNs. The diagnostic yields of TBNA, MFB and SFB were 91.7%, 93.3% and 96.7%, respectively. The sampling rate of high-quality specimens was significantly higher in the SFB group. Moreover, the mean specimen size for SFB was three times larger than for MFB. CONCLUSION: SFB is useful for obtaining sufficient qualitative and quantitative specimens.
Subject(s)
Lung Neoplasms , Lymphadenopathy , Humans , Lung Neoplasms/diagnostic imaging , Lung Neoplasms/pathology , Prospective Studies , Bronchoscopy/methods , Mediastinum/pathology , Image-Guided Biopsy , Lymph Nodes/diagnostic imaging , Lymph Nodes/pathology , Lymphadenopathy/pathology , Endoscopic Ultrasound-Guided Fine Needle Aspiration/methods , Surgical Instruments , Retrospective StudiesABSTRACT
Cobalamin-dependent methionine synthase (MS) is a key enzyme in methionine and folate one-carbon metabolism. MS is a large multi-domain protein capable of binding and activating three substrates: homocysteine, folate, and S-adenosylmethionine for methylation. Achieving three chemically distinct methylations necessitates significant domain rearrangements to facilitate substrate access to the cobalamin cofactor at the right time. The distinct conformations required for each reaction have eluded structural characterization as its inherently dynamic nature renders structural studies difficult. Here, we use a thermophilic MS homolog (tMS) as a functional MS model. Its exceptional stability enabled characterization of MS in the absence of cobalamin, marking the only studies of a cobalamin-binding protein in its apoenzyme state. More importantly, we report the high-resolution full-length MS structure, ending a multi-decade quest. We also capture cobalamin loading in crystallo, providing structural insights into holoenzyme formation. Our work paves the way for unraveling how MS orchestrates large-scale domain rearrangements crucial for achieving challenging chemistries.
Subject(s)
5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase , Methionine , 5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase/metabolism , Methionine/metabolism , S-Adenosylmethionine/metabolism , Folic Acid , Vitamin B 12/metabolismABSTRACT
BACKGROUND/AIM: Immune checkpoint inhibitors (ICIs) are currently a standard treatment tool for non-small cell lung cancer (NSCLC). RNA-binding motif protein 17 (RBM17), a splicing factor, is frequently over-expressed in NSCLC, but little is known about the role of RBM17 in the efficacy of ICIs for NSCLC. Thus, we investigated the correlation between RBM17 expression and ICI efficacy in NSCLC. PATIENTS AND METHODS: Biopsy or surgical specimens were collected from patients with advanced or recurrent NSCLC who received ICI monotherapy or chemo-immunotherapy in a first-line setting. RBM17 expression was examined using immunohistochemistry. The correlation between the efficacy of ICI monotherapy or chemo-immunotherapy and RBM17 expression was evaluated. RESULTS: Among the 218 cases, 115 (52.8%) cases were positive for RBM17 expression. RBM17 expression was not associated with the objective response rate (ORR) or progression-free survival (PFS) in either of the ICI monotherapy or chemo-immunotherapy groups. However, among those with a low PD-L1 expression level (PD-L1 <50%; n=86), RBM17 expression was significantly associated with a better ORR (p=0.045) and a better PFS (p<0.001) in the ICI monotherapy group, and was significantly associated with a poor ORR in the chemo-immunotherapy group (p=0.041). CONCLUSION: RBM17 might be a useful predictive marker for a higher efficacy of ICI monotherapy in NSCLC patients with a low PD-L1 expression level.
Subject(s)
Antineoplastic Agents, Immunological , Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Humans , Carcinoma, Non-Small-Cell Lung/pathology , Lung Neoplasms/pathology , B7-H1 Antigen , Antineoplastic Agents, Immunological/therapeutic use , Neoplasm Recurrence, Local , RNA Splicing FactorsABSTRACT
BACKGROUND: The relationship between the maximum lip-closing force (LCF) and malocclusion has long been studied. Recently, a method to measure the ability to control directional LCF from eight directions (upper, lower, right, left and the four directions in between) during lip pursing was established. OBJECTIVE: It is considered important to evaluate the ability to control directional LCF. The aim of this study was to investigate the ability of skeletal class III patients to control directional LCF. METHODS: Fifteen skeletal class III patients (mandibular prognathism group) and 15 people with normal occlusion (normal occlusion group) were recruited. The maximum LCF and the accuracy rate (the ratio of the matched time in which the participant was able to keep the LCF in the target range over a total time of 6 s) were measured. RESULTS: The maximum LCF was not significantly different between the mandibular prognathism group and the normal occlusion group. The accuracy rate in the mandibular prognathism group was significantly lower in all six directions than that in the individual normal occlusion group. CONCLUSION: As the accuracy rate in all six directions was significantly lower in the mandibular prognathism group than that in the normal occlusion group, occlusion and craniofacial morphology might influence lip function.
ABSTRACT
BACKGROUND: Physical activity is one of the most important prognostic factors for patients with chronic obstructive pulmonary disease (COPD). Physical activity correlates significantly with skeletal muscle mass and strength. Pulmonary rehabilitation has been conducted to improve physical activity, but its effectiveness has been inconsistent. Nutritional status is also related to physical activity in patients with COPD. The relationships between skeletal muscle mass, strength, and nutritional status evaluated using the Mini Nutritional Assessment Short Form (MNA-SF) were investigated. How nutritional status alters the relationships between physical activity, exercise capacity, skeletal muscle mass, and strength was also investigated. METHODS: This retrospective, cross-sectional study enrolled 81 outpatients with COPD. In all patients, physical activity, exercise capacity, body composition assessment, and MNA-SF were assessed. The relationships between physical activity, exercise capacity, skeletal muscle mass, and strength were examined according to the MNA-SF. RESULTS: The MNA-SF high group had significantly higher skeletal muscle mass than the MNA-SF low group when skeletal muscle strength was the covariate. In the MNA-SF low group, physical activity positively correlated with skeletal muscle mass. In the MNA-SF high group, physical activity positively correlated with skeletal muscle strength. CONCLUSIONS: This study showed that the nutritional status of patients with COPD alters the relationship between physical activity and skeletal muscle mass or strength. Optimizing rehabilitation with nutrition interventions according to nutritional status might improve physical activity in patients with COPD.
Subject(s)
Malnutrition , Pulmonary Disease, Chronic Obstructive , Humans , Nutritional Status , Cross-Sectional Studies , Retrospective Studies , Exercise , Muscle, SkeletalABSTRACT
Background: A previous report suggested that functional status does not differ between patients who received tranexamic acid and those who received placebo within the early hours of intracerebral hemorrhage (ICH). Our pilot study tested the hypothesis that 2 weeks administration of tranexamic acid would contribute to functional improvement. Methods: Consecutive patients with ICH were administered 250 mg tranexamic acid 3 times a day continuously for 2 weeks. We also enrolled historical control consecutive patients. We collected clinical data that involved hematoma size, level of consciousness, and Modified Rankin Scale (mRS) scores. Results: Univariate analysis showed that the mRS score on day 90 was better in the administration group (P = 0.0095). The mRS scores on the day of death or discharge suggested a favorable effect of the treatment (P = 0.0678). Multivariable logistic regression analysis also showed that the treatment was associated with good mRS scores on day 90 (odds ratio [OR] = 2.81, 95% confidence interval [CI]: 1.10-7.21, P = 0.0312). In contrast, ICH size was associated with poor mRS scores on day 90 (OR = 0.92, 95% CI: 0.88-0.97, P = 0.0005). After propensity score matching, there was no difference in the outcomes between the two groups. We did not detect mild and serious adverse events. Conclusion: The study could not show the significant effect of 2 weeks administration of tranexamic acid on functional outcomes of ICH patients after the matching; however, suggested that this treatment is at least safe and feasible. A larger and adequately powered trial is needed.
ABSTRACT
Three types of chromosomal translocations, t(4;14)(p16;q32), t(14;16)(q32;q23), and t(11;14)(q13;q32), are associated with prognosis and the decision making of therapeutic strategy for multiple myeloma (MM). In this study, we developed a new diagnostic modality of the multiplex FISH in immunophenotyped cells in suspension (Immunophenotyped-Suspension-Multiplex (ISM)-FISH). For the ISM-FISH, we first subject cells in suspension to the immunostaining by anti-CD138 antibody and, then, to the hybridization with four different FISH probes for genes of IGH, FGFR3, MAF, and CCND1 tagged by different fluorescence in suspension. Then, cells are analyzed by the imaging flow cytometry MI-1000 combined with the FISH spot counting tool. By this system of the ISM-FISH, we can simultaneously examine the three chromosomal translocations, i.e, t(4;14), t(14;16), and t(11;14), in CD138-positive tumor cells in more than 2.5 × 104 nucleated cells with the sensitivity at least up to 1%, possibly up to 0.1%. The experiments on bone marrow nucleated cells (BMNCs) from 70 patients with MM or monoclonal gammopathy of undetermined significance demonstrated the promising qualitative diagnostic ability in detecting t(11;14), t(4;14), and t(14;16) of our ISM-FISH, which was more sensitive compared with standard double-color (DC) FISH examining 200 interphase cells with its best sensitivity up to 1.0%. Moreover, the ISM-FISH showed a positive concordance of 96.6% and negative concordance of 98.8% with standard DC-FISH examining 1000 interphase cells. In conclusion, the ISM-FISH is a rapid and reliable diagnostic tool for the simultaneous examination of three critically important IGH translocations, which may promote risk-adapted individualized therapy in MM.
Subject(s)
Multiple Myeloma , Humans , Multiple Myeloma/diagnosis , Multiple Myeloma/genetics , Multiple Myeloma/pathology , Translocation, Genetic/genetics , Flow Cytometry , In Situ Hybridization, Fluorescence/methods , Gene Rearrangement , Chromosomes, Human, Pair 14/geneticsABSTRACT
BACKGROUND: In Japan, the fourth round of coronavirus disease (COVID-19) vaccination is ongoing and is targeted at medical staff and nursing home workers, individuals aged ≥60 years, and those with comorbidities or other high-risk factors, including body mass index (BMI) ≥30 kg/m2. The incidence of severe COVID-19 decreased markedly after widespread COVID-19 vaccination drives, and our hospital experienced a similar trend. We, therefore, examined the characteristics of our patients to clarify who benefited the most from vaccination. METHODS: We retrospectively investigated all patients hospitalized for COVID-19 in Osaka City Juso Hospital between March 1, 2021, and June 30, 2022. Using multivariable logistic analysis, we calculated the adjusted odds ratios (aORs) for severe disease after vaccination in the whole dataset and in subsets stratified by age, sex, BMI, smoking history, pre-hospitalization location, and comorbidities. RESULTS: The analysis included 1041 patients. Multivariable logistic analysis showed that vaccination was associated with a low risk of severe disease, with an aOR of 0.21 (95% confidence interval: 0.12-0.36, p < 0.001). On stratifying the analysis according to background characteristics, lower aORs for severe COVID-19 were found for patients aged ≥60 years and for those with diabetes or hypertension. Notably, patients with BMI >30 kg/m2 and those with BMI ≥18 kg/m2 and ≤30 kg/m2 benefited from vaccination. CONCLUSIONS: Individuals with diabetes or hypertension and those of age ≥60 years benefited more from vaccination than did their counterparts. We recommend extending the fourth round of vaccinations to individuals with a BMI of 18-30 kg/m2.
Subject(s)
COVID-19 , Diabetes Mellitus , Hypertension , Humans , COVID-19 Vaccines , SARS-CoV-2 , Retrospective Studies , Japan , Risk Factors , Hospitals , VaccinationABSTRACT
The flavoprotein methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of N5, N10-methylenetetrahydrofolate (CH2-H4folate) to N5-methyltetrahydrofolate (CH3-H4folate), committing a methyl group from the folate cycle to the methionine one. This committed step is the sum of multiple ping-pong electron transfers involving multiple substrates, intermediates, and products all sharing the same active site. Insight into folate substrate binding is needed to better understand this multifunctional active site. Here, we performed activity assays with Thermus thermophilus MTHFR (tMTHFR), which showed pH-dependent inhibition by the substrate analog, N5-formyltetrahydrofolate (CHO-H4folate). Our crystal structure of a tMTHFRâ¢CHO-H4folate complex revealed a unique folate-binding mode; tMTHFR subtly rearranges its active site to form a distinct folate-binding environment. Formation of a novel binding pocket for the CHO-H4folate p-aminobenzoic acid moiety directly affects how bent the folate ligand is and its accommodation in the active site. Comparative analysis of the available active (FAD- and folate-bound) MTHFR complex structures reveals that CHO-H4folate is accommodated in the active site in a conformation that would not support hydride transfer, but rather in a conformation that potentially reports on a different step in the reaction mechanism after this committed step, such as CH2-H4folate ring-opening. This active site remodeling provides insights into the functional relevance of the differential folate-binding modes and their potential roles in the catalytic cycle. The conformational flexibility displayed by tMTHFR demonstrates how a shared active site can use a few amino acid residues in lieu of extra domains to accommodate chemically distinct moieties and functionalities.
Subject(s)
Folic Acid , Methylenetetrahydrofolate Reductase (NADPH2) , Methylenetetrahydrofolate Reductase (NADPH2)/chemistry , Leucovorin/metabolism , Catalytic Domain , Folic Acid/metabolism , CatalysisABSTRACT
AIMS: Cytogenetic abnormalities involving the IGH gene are seen in up to 55% of patients with multiple myeloma. Current testing is performed manually by fluorescence in situ hybridisation (FISH) on purified plasma cells. We aimed to assess whether an automated imaging flow cytometric method that uses immunophenotypic cell identification, and does not require cell isolation, can identify IGH abnormalities. METHODS: Aspirated bone marrow from 10 patients with multiple myeloma were studied. Plasma cells were identified by CD38 and CD138 coexpression and assessed with FISH probes for numerical or structural abnormalities of IGH. Thousands of cells were acquired on an imaging flow cytometer and numerical data and digital images were analysed. RESULTS: Up to 30 000 cells were acquired and IGH chromosomal abnormalities were detected in 5 of the 10 marrow samples. FISH signal patterns seen included fused IGH signals for IGH/FGFR3 and IGH/MYEOV, indicating t(4;14) and t(11;14), respectively. In addition, three IGH signals were identified, indicating trisomy 14 or translocation with an alternate chromosome. The lowest limit of detection of an IGH abnormality was in 0.05% of all cells. CONCLUSIONS: This automated high-throughput immuno-flowFISH method was able to identify translocations and trisomy involving the IGH gene in plasma cells in multiple myeloma. Thousands of cells were analysed and without prior cell isolation. The inclusion of positive plasma cell identification based on immunophenotype led to a lowest detection level of 0.05% marrow cells. This imaging flow cytometric FISH method offers the prospect of increased precision of detection of critical genetic lesions involving IGH and other chromosomal defects in multiple myeloma.
Subject(s)
Chromosome Aberrations , Genes, Immunoglobulin Heavy Chain , Multiple Myeloma , Humans , Flow Cytometry , Multiple Myeloma/diagnosis , Multiple Myeloma/genetics , Multiple Myeloma/pathology , Translocation, Genetic , Trisomy/genetics , Genes, Immunoglobulin Heavy Chain/geneticsABSTRACT
Background: The use of endobronchial ultrasound-guided core biopsy (EBUS-CB) using forceps or cryoprobes to obtain true histological samples has improved the diagnostic yield for mediastinal and hilar lymphadenopathy. Tract creation in the bronchial wall of the central airway is primarily performed using electrocautery devices in EBUS-CB; however, their poor maneuverability and the risk of vascular injury and damage to the tip of the bronchoscope have prevented the application of EBUS-CB for diagnosing intrapulmonary lesions beyond the central locations. This report presents three cases wherein a 25-gauge transbronchial needle aspiration (TBNA) needle with high flexibility and safety was used to create a tract in the bronchial wall for EBUS-CB of the intrapulmonary lesions adjacent to the bronchi. Case Description: In all cases, EBUS-TBNA using a 25-gauge TBNA needle was performed on the intrapulmonary lesions adjacent to the bronchi, followed by EBUS-CB with 1.9-mm forceps in two cases and also with a 1.1-mm cryoprobe in one case. The EBUS-TBNA specimens revealed no abnormality or only a small number of tumor cells. However, subsequent EBUS-CB, through the tract created by EBUS-TBNA, enabled the collection of a sufficient amount of histological samples with well-preserved histoarchitecture. The histological diagnosis was made via immunostaining, and multigene mutation testing was also successfully analyzed. Conclusions: The use of a 25-gauge needle for creating a tract allows EBUS-CB for the intrapulmonary lesions and may allow for the collection of sufficient histological samples for biomarker analysis and tissue diagnosis.
ABSTRACT
Campylobacter jejuni and C. coli are one of the leading causes of gastrointestinal illnesses, and which are considered to be transmitted to humans mainly from chicken meats. Considering the less availability of quantitative contamination data in the retail chicken meats in Japan, 510 fresh chicken meats retailed at five distinct regions in Japan between June 2019 and March 2021 were examined. The quantitative testing resulted that 45.7% of the samples (254/510) were positive at mean ± standard deviation of 1.15 ± 1.03 logCFU/g, whereas 43 samples (8.4%) exceeded 3.0 logCFU/g. Seasonal comparison revealed increased bacterial counts in fall compared with spring and summer. As for the chicken slaughter age, those slaughtered at >75 days old were less contaminated than those at <75 days old. Genome sequencing analyses of 111 representative C. jejuni isolates resulted in the detection of three antimicrobial resistance genes (gyrA substitution T86I, tetO and blaOXA-61) at 25.2, 27.9 and 42.3%, respectively. In silico MLST analysis revealed the predominance of sequence types (ST)-21 clonal complex (CC), followed by ST-45CC and ST-464CC. The single nucleotide polymorphism (SNP)-based phylogenetic tree largely classified the sequenced C. jejuni isolates into two clusters (I and II), where all C. jejuni from highly contaminated samples (STs-21CC, -22CC and -45CC) belonged to cluster I, independent of both season and slaughter age. To our knowledge, this is the first example to study the current status of Campylobacter contamination levels in fresh chicken meats retailed in Japan. Our data would be contributable to future quantitative microbial risk assessment, to establish effective control measures for campylobacteriosis.
ABSTRACT
BACKGROUND: BEC-producing Clostridium perfringens is a causative agent of foodborne gastroenteritis. It was first reported in 2014, and since then, several isolates have been identified in Japan and the United Kingdom. The novel binary ADP-ribosylating toxin BEC, which consists of two components (BECa and BECb), is encoded on a plasmid that is similar to pCP13 and harbours a conjugation locus, called Pcp, encoding homologous proteins of the type 4 secretion system. Despite the high in vitro conjugation frequency of pCP13, its dissemination and that of related plasmids, including bec-harbouring plasmids, in the natural environment have not been characterised. This lack of knowledge has limited our understanding of the genomic epidemiology of bec-harbouring C. perfringens strains. RESULTS: In this study, we determined the complete genome sequences of five bec-harbouring C. perfringens strains isolated from 2009 to 2019. Each isolate contains a ~ 3.36 Mbp chromosome and 1-3 plasmids of either the pCW3-like family, pCP13-like family, or an unknown family, and the bec-encoding region in all five isolates was located on a ~ 54 kbp pCP13-like plasmid. Phylogenetic and SNP analyses of these complete genome sequences and the 211 assembled C. perfringens genomes in GenBank showed that although these bec-harbouring strains were split into two phylogenetic clades, the sequences of the bec-encoding plasmids were nearly identical (>99.81%), with a significantly smaller SNP accumulation rate than that of their chromosomes. Given that the Pcp locus is conserved in these pCP13-like plasmids, we propose a mechanism in which the plasmids were disseminated by horizontal gene transfer. Data mining showed that strains carrying pCP13-like family plasmids were unexpectedly common (58/216 strains) and widely disseminated among the various C. perfringens clades. Although these plasmids possess a conserved Pcp locus, their 'accessory regions' can accommodate a wide variety of genes, including virulence-associated genes, such as becA/becB and cbp2. These results suggest that this family of plasmids can integrate various foreign genes and is transmissible among C. perfringens strains. CONCLUSION: This study demonstrates the potential significance of pCP13-like plasmids, including bec-encoding plasmids, for the characterisation and monitoring of the dissemination of pathogenic C. perfringens strains.
Subject(s)
Clostridium perfringens , Enterotoxins , Clostridium perfringens/genetics , Enterotoxins/genetics , Genome, Bacterial , Genomics , Phylogeny , Plasmids/geneticsABSTRACT
Idiopathic obliterative bronchiolitis (OB) is a rare disease that usually requires a surgical lung biopsy. A 25-year-old woman with progressive exertional dyspnea for several months showed a severe mixed restrictive and obstructive pattern on spirometry. Chest computed tomography showed a mosaic pattern, and pulmonary ventilation-perfusion scintigraphy showed a matched defect. The bronchoscopic specimens obtained from both the alveolar and bronchiolar regions of the predicted lesion area contributed to the diagnosis of OB. She had no underlying causes of secondary OB, and she was diagnosed with idiopathic OB. Since lung transplantation was indicated, she was referred to a lung transplantation-certified hospital.
Subject(s)
Bronchiolitis Obliterans , Lung Transplantation , Adult , Biopsy/methods , Bronchiolitis Obliterans/diagnostic imaging , Bronchiolitis Obliterans/pathology , Dyspnea/pathology , Female , Humans , Lung/diagnostic imaging , Lung/pathologyABSTRACT
There are few cases describing the association of eosinophilia with hypercalcemia, and drug-induced eosinophilia with hypercalcemia has not been reported. A 74-year-old man had been diagnosed with asthma 4 months earlier. He was admitted due to eosinophilia with hypercalcemia. Chest computed tomography showed a nodule in the left lung and mediastinal lymphadenopathy. By obtaining a detailed medical history, clopidogrel was suspected as the prime cause of eosinophilia. After the discontinuation of clopidogrel, the eosinophilia with hypercalcemia, lung nodule and mediastinal lymphadenopathy improved. Clopidogrel-induced eosinophilia can potentially cause hypercalcemia. Obtaining a detailed clinical history is important in diagnosing the cause of eosinophilia.
Subject(s)
Eosinophilia , Hypercalcemia , Lymphadenopathy , Mediastinal Diseases , Aged , Clopidogrel/adverse effects , Eosinophilia/complications , Humans , Hypercalcemia/chemically induced , Hypercalcemia/complications , Lymphadenopathy/complications , MaleABSTRACT
Altered redox biology challenges all cells, with compensatory responses often determining a cell's fate. When 15 lipoxygenase 1 (15LO1), a lipid-peroxidizing enzyme abundant in asthmatic human airway epithelial cells (HAECs), binds phosphatidylethanolamine-binding protein 1 (PEBP1), hydroperoxy-phospholipids, which drive ferroptotic cell death, are generated. Peroxidases, including glutathione peroxidase 4 (GPX4), metabolize hydroperoxy-phospholipids to hydroxy derivatives to prevent ferroptotic death, but consume reduced glutathione (GSH). The cystine transporter SLC7A11 critically restores/maintains intracellular GSH. We hypothesized that high 15LO1, PEBP1, and GPX4 activity drives abnormal asthmatic redox biology, evidenced by lower bronchoalveolar lavage (BAL) fluid and intraepithelial cell GSH:oxidized GSH (GSSG) ratios, to enhance type 2 (T2) inflammatory responses. GSH, GSSG (enzymatic assays), 15LO1, GPX4, SLC7A11, and T2 biomarkers (Western blot and RNA-Seq) were measured in asthmatic and healthy control (HC) cells and fluids, with siRNA knockdown as appropriate. GSSG was higher and GSH:GSSG lower in asthmatic compared with HC BAL fluid, while intracellular GSH was lower in asthma. In vitro, a T2 cytokine (IL-13) induced 15LO1 generation of hydroperoxy-phospholipids, which lowered intracellular GSH and increased extracellular GSSG. Lowering GSH further by inhibiting SLC7A11 enhanced T2 inflammatory protein expression and ferroptosis. Ex vivo, redox imbalances corresponded to 15LO1 and SLC7A11 expression, T2 biomarkers, and worsened clinical outcomes. Thus, 15LO1 pathway-induced redox biology perturbations worsen T2 inflammation and asthma control, supporting 15LO1 as a therapeutic target.
Subject(s)
Arachidonate 15-Lipoxygenase/metabolism , Asthma/enzymology , Epithelial Cells/enzymology , Ferroptosis , Glutathione/metabolism , Respiratory Mucosa/enzymology , Signal Transduction , Cell Line , Epithelial Cells/pathology , Gene Expression Regulation , Humans , Inflammation/enzymology , Inflammation/pathology , Oxidation-Reduction , Respiratory Mucosa/pathologyABSTRACT
The fermented soy product ImmuBalance contains many active ingredients and its beneficial effects on some allergic diseases have been reported. We hypothesized that ImmuBalance could have potential effects on airway inflammation in a murine model of asthma. Mice sensitized and challenged with ovalbumin developed airway inflammation. Bronchoalveolar lavage fluid was assessed for inflammatory cell counts and levels of cytokines. Lung tissues were examined for cell infiltration and mucus hypersecretion. Oral administration of ImmuBalance significantly inhibited ovalbumin-induced eosinophilic inflammation and decreased Th2 cytokine levels in bronchoalveolar lavage fluid (p < 0.05). In addition, lung histological analysis showed that ImmuBalance inhibited inflammatory cell infiltration and airway mucus production. Our findings suggest that supplementation with ImmuBalance may provide a novel strategy for the prevention or treatment of allergic airway inflammation.