High-level phenol bioproduction by engineered Pichia pastoris in glycerol fed-batch fermentation using an efficient pertraction system.

This study aimed to establish a high-level phenol bioproduction system from glycerol through metabolic engineering of the yeast Pichia pastoris (Komagataella phaffii). Introducing tyrosine phenol-lyase to P. pastoris led to a production of 59 mg/L of phenol in flask culture. By employing a strain of P. pastoris that overproduces tyrosine-a precursor to phenol-we achieved a phenol production of 1052 mg/L in glycerol fed-batch fermentation. However, phenol concentrations exceeding 1000 mg/L inhibited P. pastoris growth. A phenol pertraction system utilizing a hollow fiber membrane contactor and tributyrin as the organic solvent was developed to reduce phenol concentration in the culture medium. Integrating this system with glycerol fed-batch fermentation resulted in a 214 % increase in phenol titer (3304 mg/L) compared to glycerol fed-batch fermentation alone. These approaches offer a significant framework for the microbial production of chemicals and materials that are highly toxic to microorganisms.

Current status of stroke in hemodialysis patients on a remote island.

OBJECTIVES: Hemodialysis patients have a higher incidence of stroke than healthy individuals. Hemodialysis patients living on remote islands are subject to additional distance and transportation difficulties. Therefore, we aimed to study the association between stroke and hemodialysis in patients living on remote islands. MATERIALS AND METHODS: We conducted a retrospective cohort study based on the medical records of maintenance hemodialysis patients in Shinkamigoto-Cho, Nagasaki, Japan, between June 1, 2005, and June 31, 2022. The clinical characteristics, probability of hemorrhagic stroke, acute ischemic stroke-free rate, and survival probability with or without a history of anticoagulant/antiplatelet use were evaluated. The survival probability among the hemorrhagic stroke, acute ischemic stroke, and non-stroke groups was also evaluated. RESULTS: This study involved 142 patients. Nine patients (6.3%) had intracerebral hemorrhage, one (0.7%) had subarachnoid hemorrhage, eight (5.6%) had acute ischemic stroke, and 124 (87.3%) had no stroke. The number of patients with severe disabilities (modified Rankin Scale 5/6) was significantly higher in the hemorrhagic stroke group. The probability of hemorrhagic stroke and acute ischemic stroke-free rate, or survival probability with or without a history of anticoagulant/antiplatelet use, were not significantly different. The acute ischemic stroke group was not associated with a lower survival probability than the other groups. The hemorrhagic stroke group had a significantly lower survival probability than the acute ischemic stroke group. CONCLUSIONS: This is the first study to report the status of stroke in hemodialysis patients living on remote islands, thus providing valuable information for improved stroke management in such patients.

A lesson from polybutylene succinate plastisphere to the discovery of novel plastic degrading enzyme genes in marine vibrios.

Polybutylene succinate (PBS) is an eco-friendly green plastic. However, PBS was shown as being non-biodegradable in marine environments, and up until now, only a limited number of PBS-degrading marine microbes have been discovered. We first set up in vitro PBS- and PBSA (polybutylene succinate adipate)-plastispheres to characterize novel PBS-degrading marine microbes. Microbial growth and oxygen consumption were observed in both PBS- and PBSA-plastispheres enriched with natural seawater collected from Usujiri, Hokkaido, Japan, and Vibrionaceae and Pseudoalteromonadaceae were significantly enriched on these films. Further gene identification indicated that vibrios belonging to the Gazogenes clade possess genes related to a PBS degrading enzyme (PBSase). The PBS degradation assay for six Gazogenes clade vibrios identified Vibrio ruber, Vibrio rhizosphaerae, and Vibrio spartinae as being capable of degrading PBS. We further identified the gene responsible for PBSase from the type strain of V. ruber, and the purified recombinant vibrio PBSase was found to have low-temperature adaptation and was active under high NaCl concentrations. We also provided docking models between the vibrio PBSase and PBS and PBSA units to show how vibrio PBSase interacts with each substrate compared to the Acidovorax PBSase. These results could contribute to a more sustainable society through further utilization of PBS in marine environments and plastic recycling.

Complete His-ventricular block, atrial flutter and ventricular tachycardia as arrhythmogenic activities in a patient with takotsubo cardiomyopathy.

An association of atrial arrhythmias with takotsubo cardiomyopathy (TTC) has not been described previously. Here we report a 65-year-old male patient with TTC. The sudden appearance of atrioventricular block and subsequent bradycardia are believed to be key contributing factors for the development of TTC. Both ventricular tachyarrhythmia and various atrial arrhythmias, such as atrial flutter and atrial fibrillation, were observed during the initial management of the patient's TTC. We speculate that both the left ventricular contractile dysfunction and the arrhythmogenic activities may share a common underlying etiology in advanced heart failure patients with TTC. .

Darkfield adapter for whole slide imaging: adapting a darkfield internal reflection illumination system to extend WSI applications.

We present a new method for whole slide darkfield imaging. Whole Slide Imaging (WSI), also sometimes called virtual slide or virtual microscopy technology, produces images that simultaneously provide high resolution and a wide field of observation that can encompass the entire section, extending far beyond any single field of view. For example, a brain slice can be imaged so that both overall morphology and individual neuronal detail can be seen. We extended the capabilities of traditional whole slide systems and developed a prototype system for darkfield internal reflection illumination (DIRI). Our darkfield system uses an ultra-thin light-emitting diode (LED) light source to illuminate slide specimens from the edge of the slide. We used a new type of side illumination, a variation on the internal reflection method, to illuminate the specimen and create a darkfield image. This system has four main advantages over traditional darkfield: (1) no oil condenser is required for high resolution imaging (2) there is less scatter from dust and dirt on the slide specimen (3) there is less halo, providing a more natural darkfield contrast image, and (4) the motorized system produces darkfield, brightfield and fluorescence images. The WSI method sometimes allows us to image using fewer stains. For instance, diaminobenzidine (DAB) and fluorescent staining are helpful tools for observing protein localization and volume in tissues. However, these methods usually require counter-staining in order to visualize tissue structure, limiting the accuracy of localization of labeled cells within the complex multiple regions of typical neurohistological preparations. Darkfield imaging works on the basis of light scattering from refractive index mismatches in the sample. It is a label-free method of producing contrast in a sample. We propose that adapting darkfield imaging to WSI is very useful, particularly when researchers require additional structural information without the use of further staining.

[Necessity of clinical decision support system created by medical staff].

To support patient safety, we have established a new system that collates medical facility clinical records, examination results and orders, and implementation information comprehensively in real time, checks for consistency and validity, and sends warnings to the appropriate people at the appropriate time. Because our system actually corrects inaccurate operation information, it is different from most existing facilities for patient safety in that it reconstructs information independently from the HIS (Hospital Information System). We were permitted to send warning messages not only to the doctor who entered the orders, but also to the chief of medical staff and team members. For the warning method, we tried screen flashes and chimes, mobile phone messages, and high quality interactive voice responses. We also investigated the degree of message usefulness. Therein, by not relying on "authenticity" and "readability," but by exhaustively collecting and appropriately revising in alignment with the use of information, we have created an original system that collects accurate information. This original system was established by medical staff members. The appropriate revisions mentioned herein are items which meticulously reflect the medical professional's comments and selected operation and signify why a "Clinical Decision Support System created by medical staff" is necessary.

Cerebrospinal fluid and serum levels of vitamin B6 in status epilepticus children.

Vitamin B6 (B6) is an essential cofactor of glutamate decarboxylase and catalyzes the decarboxylation of the excitatory neurotransmitter glutamate to the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). Although immediate administration of B6 to patients with acute encephalopathy with febrile convulsive status epilepticus (AEFCSE) is effective, it is not known whether prolonged seizures in status epilepticus (SE) children prevent the transport of B6 to the central nervous system (CNS) and/or induce the consumption of B6 inside the CNS. We evaluated the B6 concentration in cerebrospinal fluid (CSF) and serum in SE children. Further, we performed a sequential serum B6 analysis on days 1 and 2 after admission and on the day before discharge. Among the several vitamers of B6, we used pyridoxal (PL) as a representative of B6 in this study. We enrolled 15 SE children (8 boys and 7 girls; age range,1-11years; average age, 3.3years) and 21 control children (3 boys and 18 girls; age range, 7months-14years; average age, 3.0years) and each group was divided into 2 subgroups according to age (4months-1year and 2-14years). We found no significant differences in the CSF PL levels, CSF/serum PL ratios, and serum PL levels in the SE and control subgroups. Our results suggest that prolonged seizures do not result in B6 deficiency in CSF and serum in SE children. Whenever necessary, B6 should be administered to SE children with caution to prevent possible adverse effects.

An inter-laboratory collaborative study by the Non-Genotoxic Carcinogen Study Group in Japan, on a cell transformation assay for tumour promoters using Bhas 42 cells.

The Bhas promotion assay is a cell culture transformation assay designed as a sensitive and economical method for detecting the tumour-promoting activities of chemicals. In order to validate the transferability and applicability of this assay, an inter-laboratory collaborative study was conducted with the participation of 14 laboratories. After confirmation that these laboratories could obtain positive results with two tumour promoters, 12-O-tetradecanoylphorbol-13-acetate (TPA) and lithocholic acid (LCA), 12 coded chemicals were assayed. Each chemical was tested in four laboratories. For eight chemicals, all four laboratories obtained consistent results, and for two of the other four chemicals, only one of the four laboratories showed inconsistent results. Thus, the rate of consistency was high. During the study, several issues were raised, each of which were analysed step-by-step, leading to revision of the protocol of the original assay. Among these issues were the importance of careful maintenance of mother cultures and the adoption of test concentrations for toxic chemicals. In addition, it is suggested that three different types of chemicals show positive promoting activity in the assay. Those designated as T-type induced extreme growth enhancement, and included TPA, mezerein, PDD and insulin. LCA and okadaic acid belonged to the L-type category, in which transformed foci were induced at concentrations showing growth-inhibition. In contrast, M-type chemicals, progesterone, catechol and sodium saccharin, induced foci at concentrations with little or slight growth inhibition. The fact that different types of chemicals similarly induce transformed foci in the Bhas promotion assay may provide clues for elucidating mechanisms of tumour promotion.

Toxicity studies of tetradecanoic acid, 2-sulfo-, 1-methylester, sodium salt (C14-MES).

We evaluated the toxicity of tetradecanoic acid methyl ester sodium salt (C14-MES), a major component of fabric detergents, following the test guidelines of the Organization for Economic Cooperation and Development. The rat acute oral LD(50) was 1,000 mg/kg in males and 500 mg/kg in females. Applying the combined repeated dose and reproductive/developmental toxicity screening test (ReproTox), we exposed groups of Crj:CD (SD) IGS rats to C14-MES in the diet at concentrations of 0, 0.3, 0.6, or 1.2%. We observed decreases in fibrinogen levels and longer prothrombin time at the 1.2% treated level in females and decreases in serum triglyceride levels in both sexes at the 0.6% and 1.2% treatment levels, but the effects were not clinically significant. The no-observed-effect-level (NOEL) for repeated dose toxicity was 0.3% (175 mg/kg body weight/day for males, 249 for females). The NOEL for reproduction/developmental toxicity was 1.2% (740 mg/kg for males, 1039 for females). C14-MES was negative in the reverse gene mutation assay and the chromosomal aberration test and did not induce skin sensitization in the guinea pig maximization test. These data confirm that C14-MES is of low hazard potential.

Biosynthesis of 2-deoxystreptamine by three crucial enzymes in Streptomyces fradiae NBRC 12773.

NeoA, B, and C encoded in the neomycin biosynthetic gene cluster have been enzymatically confirmed to be responsible to the formation of 2-deoxystreptamine (DOS) in Streptomyces fradiae. NeoC was functionally characterized as 2-deoxy-scyllo-inosose synthase, which catalyzes the carbocycle formation from D-glucose-6-phosphate to 2-deoxy-scyllo-inosose. Further, NeoA appeared to catalyze the oxidation of 2-deoxy-scyllo-inosamine (DOIA) with NAD(P)+ forming 3-amino-2,3-dideoxy-scyllo-inosose (amino-DOI). Consequently, NeoA was characterized as 2-deoxy-scyllo-inosamine dehydrogenase. Finally, amino-DOI produced by NeoA from DOIA was transformed into DOS by NeoB. Since NeoB (Neo6) was also reported to be L-glutamine:2-deoxy-scyllo-inosose aminotransferase, all the enzymes in the DOS biosynthesis were characterized for the first time.

Application of in vitro mutagenesis to identify the gene responsible for cold agglutination phenotype of Streptococcus mutans.

A previously unidentified protein with an apparent molecular mass of 120 kDa was detected in some Streptococcus mutans strains including the natural isolate strain Z1. This protein was likely involved in the cold-agglutination of the strain, since a correlation between this phenotype and expression of the 120 kDa protein was found. We have applied random mutagenesis by in vitro transposition with the Himar1 minitransposon and isolated three cold-agglutination-negative mutants of this strain from approximately 2,000 mutants screened. A 2.5 kb chromosomal fragment flanking the minitransposon in one of the three mutants was amplified by PCR-based chromosome walking and the minitransposon insertion in the other two mutants occurred also within the same region. Nucleotide sequencing of the region revealed a 1617 nt open reading frame specifying a putative protein of 538 amino acid residues with a calculated molecular weight of 57,192. The deduced eight amino acid sequence following a putative signal sequence completely coincided with the N-terminal octapeptide sequence of the 120 kDa protein determined by the Edman degradation. Therefore, the 1617 nt gene unexpectedly encoded the 120 kDa protein from S. mutans. Interestingly, this gene encoded a collagen adhesin homologue. In vitro mutagenesis using the Himar1 minitransposon was successfully applied to S. mutans.