5-Formyltetrahydrofolate promotes conformational remodeling in a methylenetetrahydrofolate reductase active site and inhibits its activity.

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 tMTHFR•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.

Comparison of the specimen quality of endobronchial ultrasound-guided intranodal forceps biopsy using standard-sized forceps versus mini forceps for lung cancer: A prospective study.

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.

Combination of transbronchoscopic oxygen insufflation and a digital chest drainage system in endobronchial occlusion: a hybrid technique for localization of fistula in intractable pneumothorax.

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.

Imaging flow cytometry-based multiplex FISH for three IGH translocations in multiple myeloma.

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.

Ability to control directional lip-closing force in skeletal class III patients.

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.

Analysis of the complete genome sequences of Clostridium perfringens strains harbouring the binary enterotoxin BEC gene and comparative genomics of pCP13-like family plasmids.

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.

Invasive pulmonary aspergillosis caused by Aspergillus terreus diagnosed using virtual bronchoscopic navigation and endobronchial ultrasonography with guide sheath and successfully treated with liposomal amphotericin B.

Invasive aspergillosis is a significant cause of mortality in patients with hematological malignancy. Early diagnosis of invasive pulmonary aspergillosis (IPA) by bronchoscopy is recommended but is often difficult to perform because of small lesion size and bleeding risk due to thrombocytopenia. A 71-year-old woman had received initial induction therapy for acute myeloid leukemia. On day 22 of chemotherapy, she had a high fever, and the chest computed tomography scan revealed a 20-mm-sized nodule with a halo sign. Bronchoscopy assisted by virtual bronchoscopic navigation (VBN) and endobronchial ultrasonography with a guide sheath (EBUS-GS) was performed, and Aspergillus terreus was identified from the culture of obtained specimens. A. terreus is often resistant to amphotericin B; thus, voriconazole is usually recommended for treatment. However, the obtained A. terreus isolate showed minimal inhibitory concentrations of 2 µg/mL for voriconazole and 0.5 µg/mL for amphotericin B. Therefore, the patient was successfully treated with liposomal amphotericin B. For patients suspected of having IPA, early diagnosis and drug susceptibility testing are very important. This case suggests that bronchoscopy using VBN and EBUS-GS is helpful for accurate diagnosis and successful treatment even if the lesion is small and the patient has a bleeding risk.

Congenital bronchial atresia complicated by a lung abscess due to Aspergillus fumigatus: a case report.

BACKGROUND: Congenital bronchial atresia is a rare pulmonary abnormality characterized by the disrupted communication between the central and the peripheral bronchus and is typically asymptomatic. Although it can be symptomatic especially when infections occur in the involved areas, fungal infections are rare complications in patients with bronchial atresia. We report a case of congenital bronchial atresia complicated by a fungal infection. CASE PRESENTATION: A 30-year-old man with no previous history of immune dysfunction was brought to a nearby hospital and diagnosed with a left lung abscess. Although antimicrobial treatment was administered, it was ineffective, and he was transferred to our hospital. Since diagnostic imaging findings and bronchoscopy suggested congenital bronchial atresia and a fungal infection, he was treated with voriconazole and surgical resection was subsequently performed. A tissue culture detected Aspergillus fumigatus and histopathological findings were compatible with bronchial atresia. After discharge, he remained well and voriconazole was discontinued 5 months after the initiation of therapy. CONCLUSION: Bronchial atresia is a rare disease that is seldom complicated by a fungal infection, which is also a rare complication; however, physicians should consider fungal infections in patients with bronchial atresia who present with infections resistant to antimicrobial treatment.

Pre-pollination barriers between two sympatric Arisaema species in northern Shikoku Island, Japan.

PREMISE: The genus Arisaema (Araceae) has rapidly diversified in Japan, and multiple species often coexist in the field. Although Japanese Arisaema species hybridize from artificial crossing, hybrid individuals are rare in mixed populations; suggesting the presence of effective pre-pollination barriers. We examined the following reproductive barriers between A. sikokianum and A. tosaense: habitat, phenology, and pollinator isolations. METHODS: Habitat isolation was examined by interspecific comparisons of microhabitat conditions at a mixed site and of altitude at the sampling site of herbarium specimens. Phenological isolation was evaluated by comparing seasonal transition in apparent spathe condition and frequency of insect visitation. Pollinator isolation was examined by comparing floral visitor assemblages between the two Arisaema species. To avoid overestimation of pollinator isolation due to seasonal changes in insect assemblages, we also compared visitor assemblages between natural and late-flowering A. sikokianum, where the latter was experimentally introduced and blooming with a natural A. tosaense population. RESULTS: Microhabitat conditions and sampling elevations of herbarium specimens overlapped between the two Arisaema species. At the population level, A. sikokianum and A. tosaense flowered for 39 and 52 days, respectively, with 13 days overlap. Insect visitation in A. sikokianum decreased before the seasonal overlap. Floral visitor assemblages differed between the two Arisaema species, while the difference between natural and late-flowering A. sikokianum was less distinct. CONCLUSIONS: Phenological and pollinator isolation contribute to reproductive isolation between the two Arisaema species and should enable the two species to coexist in this area.

Astaxanthin Suppresses Cigarette Smoke-Induced Emphysema through Nrf2 Activation in Mice.

Oxidative stress plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). The activation of nuclear factor erythroid 2-related factor 2 (Nrf2) is a key cellular defense mechanism against oxidative stress. Recent studies have shown that astaxanthin protects against oxidative stress via Nrf2. In this study, we investigated the emphysema suppression effect of astaxanthin via Nrf2 in mice. Mice were divided into four groups: control, smoking, astaxanthin, and astaxanthin + smoking. The mice in the smoking and astaxanthin + smoking groups were exposed to cigarette smoke for 12 weeks, and the mice in the astaxanthin and astaxanthin + smoking groups were fed a diet containing astaxanthin. Significantly increased expression levels of Nrf2 and its target gene, heme oxygenase-1 (HO-1), were found in the lung homogenates of astaxanthin-fed mice. The number of inflammatory cells in the bronchoalveolar lavage fluid (BALF) was significantly decreased, and emphysema was significantly suppressed. In conclusion, astaxanthin protects against oxidative stress via Nrf2 and ameliorates cigarette smoke-induced emphysema. Therapy with astaxanthin directed toward activating the Nrf2 pathway has the potential to be a novel preventive and therapeutic strategy for COPD.

Ability to control directional lip-closing force during voluntary lip pursing in healthy young adults.

BACKGROUND: It is considered important to evaluate the ability to control lip-closing force (LCF). OBJECTIVE: This study aimed to investigate the ability to control directional LCF. METHODS: The experimental system included an apparatus developed to measure LCF during lip pursing in eight directions (upper, lower, right, left and the four directions in between) and a display showing the exerted LCF and a target value in each direction in real time. Twenty subjects (10 men and 10 women) were instructed to maintain the LCF at a specific target value using visual feedback. Based on our preliminary experiments, the target value was set as 50% of the maximum LCF, and the range was set at the target value ±8%. The accuracy rate was defined as the ratio of the matched time, in which the subject was able to keep the LCF in the target range, to the total 3 seconds. RESULTS: The accuracy rate of men was higher than in women in the lower, lower left and lower right directions. The accuracy rate of the directional LCF differed significantly depending on the direction. In assessing the accuracy rate for each directional LCF, the rates of upper and lower directional LCF were significantly higher than those of oblique directional LCF. No significant relationship was observed between the accuracy rate and the maximum LCF except for one direction in men subjects. CONCLUSIONS: Our findings suggest that the ability to control directional LCF is affected by sex and the force direction.

Coordination chemistry controls the thiol oxidase activity of the B12-trafficking protein CblC.

The cobalamin or B12 cofactor supports sulfur and one-carbon metabolism and the catabolism of odd-chain fatty acids, branched-chain amino acids, and cholesterol. CblC is a B12-processing enzyme involved in an early cytoplasmic step in the cofactor-trafficking pathway. It catalyzes the glutathione (GSH)-dependent dealkylation of alkylcobalamins and the reductive decyanation of cyanocobalamin. CblC from Caenorhabditis elegans (ceCblC) also exhibits a robust thiol oxidase activity, converting reduced GSH to oxidized GSSG with concomitant scrubbing of ambient dissolved O2 The mechanism of thiol oxidation catalyzed by ceCblC is not known. In this study, we demonstrate that novel coordination chemistry accessible to ceCblC-bound cobalamin supports its thiol oxidase activity via a glutathionyl-cobalamin intermediate. Deglutathionylation of glutathionyl-cobalamin by a second molecule of GSH yields GSSG. The crystal structure of ceCblC provides insights into how architectural differences at the α- and ß-faces of cobalamin promote the thiol oxidase activity of ceCblC but mute it in wild-type human CblC. The R161G and R161Q mutations in human CblC unmask its latent thiol oxidase activity and are correlated with increased cellular oxidative stress disease. In summary, we have uncovered key architectural features in the cobalamin-binding pocket that support unusual cob(II)alamin coordination chemistry and enable the thiol oxidase activity of ceCblC.

Structure and mechanism of the chromatin remodelling factor ISW1a.

Site-specific recognition of DNA in eukaryotic organisms depends on the arrangement of nucleosomes in chromatin. In the yeast Saccharomyces cerevisiae, ISW1a and related chromatin remodelling factors are implicated in establishing the nucleosome repeat during replication and altering nucleosome position to affect gene activity. Here we have solved the crystal structures of S. cerevisiae ISW1a lacking its ATPase domain both alone and with DNA bound at resolutions of 3.25 Å and 3.60 Å, respectively, and we have visualized two different nucleosome-containing remodelling complexes using cryo-electron microscopy. The composite X-ray and electron microscopy structures combined with site-directed photocrosslinking analyses of these complexes suggest that ISW1a uses a dinucleosome substrate for chromatin remodelling. Results from a remodelling assay corroborate the dinucleosome model. We show how a chromatin remodelling factor could set the spacing between two adjacent nucleosomes acting as a 'protein ruler'.

Structure of Human B12 Trafficking Protein CblD Reveals Molecular Mimicry and Identifies a New Subfamily of Nitro-FMN Reductases.

In mammals, B12 (or cobalamin) is an essential cofactor required by methionine synthase and methylmalonyl-CoA mutase. A complex intracellular pathway supports the assimilation of cobalamin into its active cofactor forms and delivery to its target enzymes. MMADHC (the methylmalonic aciduria and homocystinuria type D protein), commonly referred to as CblD, is a key chaperone involved in intracellular cobalamin trafficking, and mutations in CblD cause methylmalonic aciduria and/or homocystinuria. Herein, we report the first crystal structure of the globular C-terminal domain of human CblD, which is sufficient for its interaction with MMADHC (the methylmalonic aciduria and homocystinuria type C protein), or CblC, and for supporting the cytoplasmic cobalamin trafficking pathway. CblD contains an α+ß fold that is structurally reminiscent of the nitro-FMN reductase superfamily. Two of the closest structural relatives of CblD are CblC, a multifunctional enzyme important for cobalamin trafficking, and the activation domain of methionine synthase. CblD, CblC, and the activation domain of methionine synthase share several distinguishing features and, together with two recently described corrinoid-dependent reductive dehalogenases, constitute a new subclass within the nitro-FMN reductase superfamily. We demonstrate that CblD enhances oxidation of cob(II)alamin bound to CblC and that disease-causing mutations in CblD impair the kinetics of this reaction. The striking structural similarity of CblD to CblC, believed to be contiguous in the cobalamin trafficking pathway, suggests the co-option of molecular mimicry as a strategy for achieving its function.

Risk of Cerebral Infarction in Japanese Hemodialysis Patients: Miyazaki Dialysis Cohort Study (MID study).

BACKGROUND/AIMS: Predictors including the preventive effects of antiplatelet and anticoagulant drugs on cerebral infarction (CI) events have not yet been clarified in dialysis patients. The aim of the present study was to examine the risk of CI and preventive effects of these drugs in Japanese hemodialysis patients. METHODS: Patients receiving maintenance hemodialysis (n=1,551, median age (interquartile range), 69.0 (59.0-78.0) years; 41.5% female) were enrolled in the Miyazaki Dialysis Cohort Study and prospectively followed-up for 3 years. Kaplan-Meier and Cox's regression analyses were used to clarify the risk of CI. RESULTS: Eighty-four patients developed CI at an incidence of 21.5/1000 patients per year. The presence of a previous history of CI, atrial fibrillation (AF), and diabetes mellitus in addition to age were also identified as predictive factors for new CI, whereas no relationship was observed between antiplatelet and/or anticoagulant usage and CI. Furthermore, no significant difference was noted in the frequency of CI events between patients with AF who received warfarin and those who did not. CONCLUSIONS: The incidence of CI was higher in dialysis patients with a previous history of CI and AF; however, the preventive effects of antiplatelet/anticoagulant drugs on the development of CI were not evident.

Glycaemic control is a predictor of infection-related hospitalization on haemodialysis patients: Miyazaki Dialysis Cohort study (MID study).

AIM: Although infection is the second leading cause of death in maintenance haemodialysis patients, the effects of glycaemic control on infection in diabetic haemodialysis patients have not yet been examined in detail. We examined the relationship between diabetes or glycemic control and infection-related hospitalization (IRH) in haemodialysis patients. METHODS: Patients receiving maintenance haemodialysis (n = 1551, 493 diabetic patients) were enrolled in this prospective cohort study in December 2009 and followed-up for 3 years. IRH during the follow-up period was abstracted from medical records. Kaplan-Meier and Cox regression analyses were used to investigate the relationship between diabetes or glycaemic control and IRH. RESULTS: The Kaplan-Meier analysis revealed that the risk of IRH was significantly higher in haemodialysis patients with diabetes, particularly in those with poorly controlled HbA1c levels (HbA1c ≥ 7.0%), than in haemodialysis patients without diabetes. When patients with ≥HbA1c 7.0% were divided into two groups using a median value of HbA1c, the risk of IRH was significantly higher in those with the poorest glycaemic control (HbA1c ≥ 7.4%), an older age, or lower albumin levels. The multivariable-adjusted hazard ratio for the risk of IRH was not higher in the second criteria of HbA1c (HbA1c 7.0-7.3%), but was significantly higher in the group with the poorest glycaemic control (HbA1c ≥ 7.4%) than in those in the good control criterion (HbA1c < 7.0%). CONCLUSIONS: Although diabetes is a risk factor for IRH among maintenance haemodialysis patients, the relationship between glycaemic control and the risk of infection is not linear. Therefore, the risk of infection may increase in a manner that is dependent on the glycaemic control threshold.

Impaired nuclear factor erythroid 2-related factor 2 expression increases apoptosis of airway epithelial cells in patients with chronic obstructive pulmonary disease due to cigarette smoking.

BACKGROUND: Cigarette smoking-induced oxidative stress is known to be a key mechanism in COPD pathogenesis. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a central transcription factor that regulates the antioxidant defense system. The aim of this study was to compare Nrf2 expression in COPD subjects and control subjects, and to determine the role of Nrf2 in protecting against oxidative stress-induced apoptosis. METHODS: We enrolled 8 COPD subjects and 7 control subjects in this study. We performed bronchial brushing by bronchoscopy and obtained bronchial epithelial cells from the airways. Nrf2 expression in bronchial epithelial cells was evaluated by real-time PCR and Western blotting. We examined the effect of 10 or 15 % cigarette smoke extract (CSE) induced A549 cells apoptosis using a time-lapse cell imaging assay with caspase-3/7 activation detecting reagent and performed Terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling assay for confirming A549 cells apoptosis. We also examined the effects of Nrf2 knockdown and, 0.1, 0.5, and 1.0 mM N-acetyl cysteine on CSE-induced apoptosis. Statistical analyses were performed using t-test, paired t-test or an analysis of variance followed by the Tukey-Kramer method. RESULTS: Nrf2 mRNA expression in COPD subjects was significantly lower than that in control subjects and Nrf2 mRNA were negatively correlated with pack year. Nrf2 protein in COPD subjects was significantly lower than that in control subjects. CSE-induced A549 cells apoptosis was increased in a time-, concentration-dependent manner, and was significantly increased by Nrf2 knockdown. N-acetyl cysteine significantly ameliorated CSE-induced apoptosis. CONCLUSIONS: Nrf2 expression was lower in COPD patients than in control subjects. Nrf2 might have a protective role against apoptosis caused by CSE-induced oxidative stress. These results suggest an involvement of Nrf2 in COPD and administration of antioxidants to patients with COPD might be a basic therapeutic option.

Synthesis of the Tetrasaccharide Repeating Unit from Acinetobacter baumannii Serogroup O18 Capitalizing on Phosphorus-Containing Leaving Groups.

The first convergent synthesis of the tetrasaccharide repeating unit of the polymeric O antigen isolated from Acinetobacter baumannii serogroup O18 has been achieved. The ManNAcß1→4Gal and GalNAcß1→3Gal units were successfully obtained through ß-selective glycosylation with 2-azido-4,6-O-benzylidene-2-deoxymannosyl diphenyl phosphate and Tf2NH-promoted glycosylation with 2-acetamido-2-deoxygalactosyl diethyl phosphite, respectively. The disaccharide units could be coupled with the aid of TMSClO4 as an activator of the diphenyl phosphate leaving group, and global deprotection completed the synthesis of the tetrasaccharide.

Designing multiplex PCR system of Campylobacter jejuni for efficient typing by improving monoplex PCR binary typing method.

Campylobacter jejuni is responsible for the majority of Campylobacter infections. As the molecular epidemiological study of outbreaks, pulsed-field gel electrophoresis (PFGE) is performed in general. But PFGE has several problems. PCR binary typing (P-BIT) method is a typing method for Campylobacter spp. that was recently developed, and was reported to have a similar discriminatory power and stability to those of PFGE. We modified the P-BIT method from 18 monoplex PCRs to two multiplex PCR systems (mP-BIT). The same results were obtained from monoplex PCRs using original primers and multiplex PCR in the representative isolates. The mP-BIT can analyze 48 strains at a time by using 96-well PCR systems and can identify C. jejuni because mP-BIT includes C. jejuni marker. The typing of the isolates by the mP-BIT and PFGE demonstrated generally concordant results and the mP-BIT method (D = 0.980) has a similar discriminatory power to that of PFGE with SmaI digest (D = 0.975) or KpnI digest (D = 0.987) as with original article. The mP-BIT method is quick, simple and easy, and comes to be able to perform it at low cost by having become a multiplex PCR system. Therefore, the mP-BIT method with two multiplex PCR systems has high potential for a rapid first-line surveillance typing assay of C. jejuni and can be used for routine surveillance and outbreak investigations of C. jejuni in the future.

Sonographic appearances of cervical lymph nodes in healthy young Japanese adults: Association with age, sex, and body mass index.

PURPOSE: We investigated whether there was any association between age, sex, and body mass index (BMI) and nodal morphology and vascular pattern in healthy young Japanese adults. METHODS: Three neck regions in 100 healthy subjects, 19-40 years old, were examined by gray-scale and color Doppler sonography. Vascular pattern was classified into three groups: avascular, hilar, or scattered. A linear mixed-effect model was used to identify associations of age, sex, or BMI with the short-axis diameter and the short-to-long axis diameter ratio (S/L). A cumulative link mixed model was used to identify any association between age, sex, BMI, and vascular pattern. RESULTS: In the upper cervical region, a decrease in the short-axis diameter was statistically significantly associated with aging (p = 0.04), and an increase in the short-axis diameter was significantly associated with greater BMI (p < 0.001). An increase in short-axis diameter was significantly associated with female sex (p = 0.02) and higher BMI (p = 0.002) in the submandibular region, whereas it was associated with higher BMI (p = 0.001) in the submental region. A greater S/L was significantly associated with higher BMI and female sex in all regions. The scattered vascular pattern tended to be associated with lower BMI (p = 0.051) in the upper cervical region, but it was significantly associated with higher BMI (p = 0.01) in the submental region. CONCLUSIONS: Nodal morphology and vascular pattern may be associated with age, sex, and BMI.