NAD+ enhances the activity and thermostability of S-adenosyl-L-homocysteine hydrolase from Pyrococcus horikoshii OT3.

S-Adenosyl-L-methionine (SAM) and S-adenosyl-L-homocysteine (SAH) are important biochemical intermediates. SAM is the major methyl donor for diverse methylation reactions in vivo. The SAM to SAH ratio serves as a marker of methylation capacity. Stable isotope-labeled SAM and SAH are used to measure this ratio with high sensitivity. SAH hydrolase (EC 3.13.2.1; SAHH), which reversibly catalyzes the conversion of adenosine and L-homocysteine to SAH, is used to produce labeled SAH. To produce labeled SAH with high efficiency, we focused on the SAHH of Pyrococcus horikoshii OT3, a thermophilic archaeon. We prepared recombinant P. horikoshii SAHH using Escherichia coli and investigated its enzymatic properties. Unexpectedly, the optimum temperature and thermostability of P. horikoshii SAHH were much lower than its optimum growth temperature. However, addition of NAD+ to the reaction mixture shifted the optimum temperature of P. horikoshii SAHH to a higher temperature, suggesting that NAD+ stabilizes the structure of the enzyme.

Ultrasound-guided femoral vascular access in pediatric cardiac catheterization.

BACKGROUND: In pediatric catheterization, the palpation and landmark (PL) technique is widely used for femoral arterial and venous (FAV) cannulation. Over the past decade, the ultrasound-guided (US) technique has replaced the PL technique. This study aimed to assess the clinical impact of application of the US technique on the success rate and completion time of FAV cannulation during cardiac catheterization in children. METHODS: This is a retrospective observational study of consecutive pediatric patients who underwent cardiac catheterization in a tertiary care children's hospital from April 2016 to March 2022. The association between FAV cannulation success rate and the US technique was analyzed using multiple logistic regression analysis by adjusting for potential confounders, including patient and operator characteristics and procedural details. RESULTS: A total of 749 patients (PL, 378; US, 371) were analyzed. The odds ratio (OR) of the US technique success rate for FAV cannulation was 2.03, 95% confidence interval (CI), 1.10-3.73; p = 0.02. The OR of the cannulation success rate of children aged <1 year was 0.16 (95% CI, 0.03-0.97; p = 0.046). CONCLUSIONS: The US-guided technique was associated with an increased success rate of FAV cannulation, compared with the PL technique. Moreover, age < 1 year was an independent factor associated with a lower success rate of FAV cannulation. The US-guided technique might be an effective procedure in FAV cannulation during cardiac catheterization in children.

Production and characterization of cyanocobalamin-enriched tomato (Solanum lycopersicum) fruits grown using hydroponics.

BACKGROUND: Vitamin B12 is an essential vitamin that is absent in plant-derived foods such as fruits and vegetables. This can result in an increased risk of developing vitamin B12 deficiency in strict vegetarians (vegans). There are several studies that have aimed to enhance nutrients in food crops. The purpose of the present study was to fortify tomato fruits with vitamin B12 (or cyanocobalamin). RESULTS: Tomato plants were grown for 70 days in hydroponic culture pots and treated with 5 µm of cyanocobalamin on days 1-24 after the fruiting, and then harvested for tomato fruits. The ripened tomato fruits contained 4.0 × 10-7  g of cyanocobalamin per 100 g of dry weight and showed a significant increase in glucose and lycopene levels. CONCLUSION: The present study highlights the use of a cyanocobalamin-supplementation system for the production of B12 fortified tomato fruits that can help prevent B12 deficiency in vegetarians. © 2022 Society of Chemical Industry.

Lemon myrtle extract inhibits lactate production by Streptococcus mutans.

Backhousia citriodora (lemon myrtle) extract has been found to inhibit glucansucrase activity, which plays an important role in biofilm formation by Streptococcus mutans. In addition to glucansucrase, various virulence factors in S. mutans are involved in the initiation of caries. Lactate produced by S. mutans demineralizes the tooth enamel. This study investigated whether lemon myrtle extract can inhibit S. mutans lactate production. Lemon myrtle extract reduced the glycolytic pH drop in S. mutans culture and inhibited lactate production by at least 46%. Ellagic acid, quercetin, hesperetin, and myricetin, major polyphenols in lemon myrtle, reduced the glycolytic pH drop and lactate production, but not lactate dehydrogenase activity. Furthermore, these polyphenols reduced the viable S. mutans cell count. Thus, lemon myrtle extracts may inhibit S. mutans-mediated acidification of the oral cavity, thereby preventing dental caries and tooth decay.

Valvular changes after aortic valve neo-cuspidization in children: A case series.

BACKGROUND: Aortic valve neo-cuspidization (AVNeo), a procedure wherein the aortic valve is reconstructed utilizing an autologous pericardium, has recently been more commonly performed in children. However, the postoperative morphological changes in the aortic valve of pediatric patients remain unknown. The current study aimed to describe the intraoperative and postoperative findings of aortic regurgitation (AR) and stenosis (AS) after AVNeo in children. METHODS: This case series describes the morphological changes in AR and AS, and their severity, between the perioperative period and 3 months postoperative period after AVNeo in children (<18 years) who underwent AVNeo between April 2016 and March 2020. Data were collected at two measurement points: (i) intraoperative transesophageal echocardiography after weaning from cardiopulmonary bypass (io-TEE); (ii) postoperative transthoracic echocardiography 3 months after the procedure (po-TTE). RESULTS: Seven patients were included in this case series. The number of postoperative AR sites and the ratio of AR jet area to the left ventricular outflow tract area showed a tendency to decrease between io-TEE and po-TTE. All AR sites were integrated during the postoperative period. One patient identified developed intraoperative AS, which maintained its severity after AVNeo. CONCLUSIONS: Most cases exhibited spontaneous improvement in AR, while one developed postoperative AS. Further prospective investigation is, therefore, needed to explore surgical outcomes following AVNeo among children.

Dityrosine Crosslinking of Collagen and Amyloid-ß Peptides Is Formed by Vitamin B12 Deficiency-Generated Oxidative Stress in Caenorhabditis elegans.

(1) Background: Vitamin B12 deficiency in Caenorhabditis elegans results in severe oxidative stress and induces morphological abnormality in mutants due to disordered cuticle collagen biosynthesis. We clarified the underlying mechanism leading to such mutant worms due to vitamin B12 deficiency. (2) Results: The deficient worms exhibited decreased collagen levels of up to approximately 59% compared with the control. Although vitamin B12 deficiency did not affect the mRNA expression of prolyl 4-hydroxylase, which catalyzes the formation of 4-hydroxyproline involved in intercellular collagen biosynthesis, the level of ascorbic acid, a prolyl 4-hydroxylase coenzyme, was markedly decreased. Dityrosine crosslinking is involved in the extracellular maturation of worm collagen. The dityrosine level of collagen significantly increased in the deficient worms compared with the control. However, vitamin B12 deficiency hardly affected the mRNA expression levels of bli-3 and mlt-7, which are encoding crosslinking-related enzymes, suggesting that deficiency-induced oxidative stress leads to dityrosine crosslinking. Moreover, using GMC101 mutant worms that express the full-length human amyloid ß, we found that vitamin B12 deficiency did not affect the gene and protein expressions of amyloid ß but increased the formation of dityrosine crosslinking in the amyloid ß protein. (3) Conclusions: Vitamin B12-deficient wild-type worms showed motility dysfunction due to decreased collagen levels and the formation of highly tyrosine-crosslinked collagen, potentially reducing their flexibility. In GMC101 mutant worms, vitamin B12 deficiency-induced oxidative stress triggers dityrosine-crosslinked amyloid ß formation, which might promote its stabilization and toxic oligomerization.

5-hydroxymethyl-2-furaldehyde purified from Japanese pear (Pyrus pyrifolia Nakai cv. Nijisseiki) juice concentrate inhibits melanogenesis in B16 mouse melanoma cells.

Pear juice concentrate prepared by boiling Japanese pear (Pyrus pyrifolia Nakai cv. Nijisseiki) juice can significantly inhibit the activity of tyrosinase, a key enzyme in melanin synthesis in human skin. Using the ethanol extract of pear juice concentrate, we homogeneously purified an active compound that was identified as 5-hydroxymethyl-2-furaldehyde (5-HMF) through 1H- and 13C-NMR and mass spectroscopy. We observed that 5-HMF inhibited the monophenolase and diphenolase activities of mushroom tyrosinase as a mixed-type inhibitor (K i values of 3.81 and 3.70 mmol/L, respectively). In B16 mouse melanoma cells, treatment with 170 µmol/L of 5-HMF significantly reduced α-melanocyte-stimulated melanin synthesis by suppressing the cyclic adenosine monophosphate-dependent signaling pathway involved in melanogenesis. The results of our study indicated that 5-HMF can be potentially used as a skin-lightening agent in the cosmetic industry. Abbreviations: AC: adenylate cyclase; CREB: cAMP response element-binding protein; dhFAME: S-(-)-10,11-Dihydroxyfarnesoic acid methyl ester; DMEM: dulbecco's modified eagle medium; l-DOPA: 3-(3,4-Dihydroxyphenyl)- l-alanine; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HEPES: 4-(2-Hydroxyethyl)-1-piperazine ethane sulfonic acid; 5-HMF: 5-Hydroxymethyl-2-furaldehyde; MITF: microphthalmia-associated transcription factor; α-MSH: α-Melanocyte-stimulating hormone; PKA: protein kinase A; PVDF: polyvinylidene difluoride; SDS: sodium dodecyl sulfate; TRP1: tyrosinase-related protein 1; TRP2: tyrosinase-related protein 2.

Biological Activity of Pseudovitamin B12 on Cobalamin-Dependent Methylmalonyl-CoA Mutase and Methionine Synthase in Mammalian Cultured COS-7 Cells.

Adenyl cobamide (commonly known as pseudovitamin B12) is synthesized by intestinal bacteria or ingested from edible cyanobacteria. The effect of pseudovitamin B12 on the activities of cobalamin-dependent enzymes in mammalian cells has not been studied well. This study was conducted to investigate the effects of pseudovitamin B12 on the activities of the mammalian vitamin B12-dependent enzymes methionine synthase and methylmalonyl-CoA mutase in cultured mammalian COS-7 cells to determine whether pseudovitamin B12 functions as an inhibitor or a cofactor of these enzymes. Although the hydoroxo form of pseudovitamin B12 functions as a coenzyme for methionine synthase in cultured cells, pseudovitamin B12 does not activate the translation of methionine synthase, unlike the hydroxo form of vitamin B12 does. In the second enzymatic reaction, the adenosyl form of pseudovitamin B12 did not function as a coenzyme or an inhibitor of methylmalonyl-CoA mutase. Experiments on the cellular uptake were conducted with human transcobalamin II and suggested that treatment with a substantial amount of pseudovitamin B12 might inhibit transcobalamin II-mediated absorption of a physiological trace concentration of vitamin B12 present in the medium.

A lemon myrtle extract inhibits glucosyltransferases activity of Streptococcus mutans.

Streptococcus mutans is a bacterium found in human oral biofilms (dental plaques) that is associated with the development of dental caries. Glucosyltransferases (GTFs) are key enzymes involved in dental plaque formation, and compounds that inhibit their activities may prevent dental caries. We developed a screening system for GTF-inhibitory activities, and used it to profile 44 types of herbal tea extracts. Lemon myrtle (Backhousia citriodora) extract exhibited the highest GTF-inhibitory activity, with an IC50 for GTF in solution of 0.14 mg mL-1. Furthermore, lemon myrtle extracts had the third-highest polyphenol content of all tested extracts, and strongly inhibited S. mutans biofilm. Interestingly, lemon myrtle extracts did not inhibit cell growth.

Biochemistry and Physiology of Vitamins in Euglena.

Euglena gracilis Z requires vitamins B1 and B12 for growth. It takes up and accumulates large amounts of these exogenous vitamins through energy-dependent active transport systems. Except for these essential vitamins, E. gracilis Z has the ability to synthesize all human vitamins. Euglena synthesizes high levels of antioxidant vitamins such as vitamins C and E, and, thus, are used as nutritional supplements for humans and domestic animals. Methods to effectively produce vitamins in Euglena have been investigated.Previous biochemical studies indicated that E. gracilis Z contains several vitamin-related novel synthetic enzymes and metabolic pathways which suggests that it is a highly suitable organism for elucidating the physiological functions of vitamins in comparative biochemistry and biological evolution. E. gracilis Z has an unusual biosynthetic pathway for vitamin C, a hybrid of the pathways found in animals and plants. This chapter presents up-to-date information on the biochemistry and physiological functions of vitamins in this organism.

Functional and structural characteristics of methylmalonyl-CoA mutase from Pyrococcus horikoshii.

Methylmalonyl-CoA mutase (MCM) requires 5'-deoxyadenosylcobalamin (AdoCbl) as a cofactor and is widely distributed in organisms from bacteria and animals. Although genes encoding putative MCMs are present in many archaea, they are separately encoded in large and small subunits. The large and small subunits of archaeal MCM are similar to the catalytic and AdoCbl-binding domains of human MCM, respectively. In Pyrococcus horikoshii OT3, putative genes PH1306 and PH0275 encode the large and small subunits, respectively. Because information on archaeal MCM is extremely restricted, we examined the functional and structural characteristics of P. horikoshii MCM. Reconstitution experiments using recombinant PH0275 and PH1306 showed that these proteins assemble in equimolar ratios and form of heterotetrameric complexes in the presence of AdoCbl. Subsequent immunoprecipitation experiments using anti-PH0275 and anti-PH1306 antibodies suggested that PH0275 and PH1306 form a complex in P. horikoshii cells in the presence of AdoCbl.

The influence of a whole food vegan diet with Nori algae and wild mushrooms on selected blood parameters.

BACKGROUND: Vegan and vegetarian diets could overcome many diseases of civilization. This study examines whether a whole food vegan diet with Nori algae and wild mushrooms can provide a sufficient quantity of critical nutrients. METHODS: Five blood samples (Baseline to Time 5) were taken over eight months from 75 subjects (10 vegans without B12 supplementation who consumed Nori algae and wild mushrooms, 20 vegans with supplementation, 40 vegetarians, 5 meat-eaters). Blood was analyzed for blood cell counts, total vitamin B12, holotranscobalamin, homocysteine, methylmalonic acid, vitamin B6, folic acid, ferritin, TSH, zinc, creatinine, vitamin D2 and D3. RESULTS: In the vegan group without supplementation, all means were within the tolerance (holotranscobalamin, homocystein) or normal, except for elevated methylmalonic acid and diminished vitamin D. This group developed significantly higher vitamin D2 levels. The vegan group with B12 supplementation and the vegetarian group showed normal values for all parameters. CONCLUSIONS: Vegans following a whole food diet had a borderline supply of vitamin B12. Folic acid, vitamin B6, TSH, iron metabolism, and the blood count were in the normal range. Vegans taking dietary supplements demonstrated satisfactory overall results. An ingestion of sundried mushrooms can contribute to the supply of vitamin D.

The sulfamide moiety affords higher inhibitory activity and oral bioavailability to a series of coumarin dual selective RAF/MEK inhibitors.

Introducing a sulfamide moiety to our coumarin derivatives afforded enhanced Raf/MEK inhibitory activity concomitantly with an acceptable PK profile. Novel sulfamide 17 showed potent HCT116 cell growth inhibition (IC50=8 nM) and good PK profile (bioavailability of 51% in mouse), resulting in high in vivo antitumor efficacy in the HCT116 xenograft (ED50=4.8 mg/kg). We confirmed the sulfamide moiety showed no negative impact on tests run on the compound to evaluate DMPK (PK profiles in three animal species, CYP inhibition and CYP induction) and the safety profile (hERG and AMES tests). Sulfamide 17 had favorable properties that warranted further preclinical assessment.

Enantioselective synthesis of derivatives and structure-activity relationship study in the development of NA255 as a novel host-targeting anti-HCV agent.

Hepatitis C virus (HCV) infection represents a serious health-care problem. Previously we reported the identification of NA255 from our natural products library using a HCV sub-genomic replicon cell culture system. Herein, we report how the absolute stereochemistry of NA255 was determined and an enantioselective synthetic method for NA255 derivatives was developed. The structure-activity relationship of the NA255 derivatives and rat pharmacokinetic profiles of the representative compounds are disclosed.

Efficacy of in-situ simulation training using evaluation checklists for sudden oxygen supply failure during general anesthesia: A preliminary report.

Introduction: Sudden oxygen supply failure (OSF) is a life-threatening consequence that may be triggered by natural disasters. Anesthesiologists are required to manage OSF promptly in such catastrophic situations. However, the current evidence regarding the efficacy of anesthesia training for sudden OSF is insufficient. This preliminary study aimed to introduce our in-situ simulation training utilizing evaluation checklists for a sudden OSF situation during general anesthesia and to evaluate the efficacy of the training program for anesthesia providers. Methods: This is a preliminary single-center, prospective study. We developed an OSF simulation scenario utilizing evaluation checklists with key actions to manage OSF. The training session comprised four components: orientation, benchmark evaluation (pre-test) according to the checklists, a short didactic lecture, and post-lecture evaluation (post-test). The scenario comprised two steps wherein the participants were supposed to utilize different oxygen supply sources immediately after OSF (Step 1) and minimize the amount of oxygen consumption (Step 2). Results: Fifteen anesthesia providers were enrolled. The score for all anesthesia providers in the post-test was significantly higher than that in the pre-test (median 8 [IQR: 8, 8], 3 [IQR: 3, 4], P < 0.001, respectively). The successful performance rates of all anesthesia providers in one key action of all the four in Step 1 and four of all the six in Step 2 were significantly higher in the post-test than in the pre-test. Conclusions: Our in-situ training method utilizing evaluation checklists for a sudden OSF situation improved overall performance of anesthesia providers.

Association Between Plasma Fibrinogen Concentration After Cardiopulmonary Bypass and Postoperative Blood Loss in Children Undergoing Cardiac Surgery: A Retrospective Cohort Study.

Background Intraoperative hypofibrinogenemia is a major factor associated with increased postoperative blood loss in adult cardiac surgery. However, previous pediatric studies on this topic did not sufficiently adjust for potential confounders and variations in surgeons' techniques. Therefore, evidence for the association between hypofibrinogenemia and postoperative blood loss after cardiac surgery in children remains insufficient. In this study, we aimed to evaluate the association between postoperative blood loss and hypofibrinogenemia by adjusting for potential confounders and the effects of differences in surgeons' techniques. Methodology This single-center, retrospective, cohort study included children who underwent cardiac surgery with cardiopulmonary bypass from April 2019 to March 2022. Multilevel logistic regression models with mixed effects were used to evaluate the association of major blood loss in the first six hours postoperatively with fibrinogen concentration at the end of cardiopulmonary bypass. The difference in the surgeon's techniques was adjusted as a random effect for the model. The model included potential confounders identified as risk factors in previous studies. Results A total of 401 patients were included. A fibrinogen concentration ≤150 mg/dL (adjusted odds ratio (aOR) = 2.08; 95% confidence interval (CI) = 1.18-3.67; p = 0.011) and the presence of cyanotic disease (aOR = 2.34; 95% CI = 1.10-4.97; p = 0.027) were associated with major blood loss in the first six postoperative hours. Conclusions A fibrinogen concentration ≤150 mg/dL and the presence of cyanotic disease were associated with postoperative blood loss in pediatric cardiac surgery. Maintaining a fibrinogen concentration >150 mg/dL is recommended, especially for patients with cyanotic diseases.

Determination of Vitamin B12 and Folate Compounds in Commercially Available Edible Seaweed Products.

BACKGROUND: Information on the contents of both vitamin B12 and folate in edible seaweeds is limited, of which deficiencies disrupt methionine biosynthesis to accumulate homocysteine as a risk factor of cardiovascular diseases. METHODS: Both vitamins were determined in commercially available edible seaweed products using high-performance liquid chromatography. RESULTS: Dried purple laver (Neopyropia yezoensis) products contain higher levels of vitamin B12 (approximately 30-60 µg/100 g dry weight) and folate compounds (approximately 880-1300 µg/100 g dry weight) than other seaweed products, such as kombu (Saccharina japonica), hijiki (Sargassum fusiformis), and wakame (Undaria pinnatifida). 5-methyltetrahydrofolate was the major folate compound in purple laver products. 5-formyltetrahydrofolate was found at a moderate level, whereas tetrahydrofolate, 5,10-metenyltetrahydrofolate, 10-formyltetrahydrofolate, and folic acid were found to be minor folate compounds. CONCLUSIONS: These findings suggest that dried purple laver (nori) products are suitable sources of vitamin B12 and folate compounds for humans, especially vegetarians.

Epidemiology of adverse events attributed to airway management in paediatric anaesthesia: protocol for the prospective, multicentre, registry-based, cross-sectional Japan Pediatric Difficult Airway in Anesthesia study (J-PEDIA).

INTRODUCTION: Failure to secure an airway during general anaesthesia is a major cause of adverse events (AEs) in children. The safety of paediatric anaesthesia may be improved by identifying the incidence of AEs and their attributed risk factors. The aim of the current study is to obtain real-world data on the incidence of adverse peri-intubation events and assess their association with patient characteristics (including the prevalence of difficult airway features) and choice of anaesthesia management. These data can be used to develop a targeted education programme for anaesthesia providers towards quality improvement activities. METHODS AND ANALYSIS: This prospective, multicentre, registry-based, cross-sectional study will be conducted in four tertiary care hospitals in Japan from June 2022 to May 2025. Children <18 years of age undergoing surgical and/or diagnostic test procedures under general anaesthesia or sedation by anaesthesiologists will be enrolled in this study. Data on patient characteristics, discipline of anaesthesia providers and methodology of airway management will be collected through a standardised verification system. The exposure of interest is the presence of difficult airway features defined based on the craniofacial appearance. The primary and secondary endpoints are all AEs associated with airway management and reduced peripheral capillary oxygen saturation values. Potential confounders are related to the failure to secure the airway and variations in the anaesthesia providers' levels, adjusted using hierarchical multivariable regression models with mixed effects. The sample size was calculated to be approximately 16 000 assuming a 99% probability of obtaining a 95% Wilson CI with±0.3% of the half-width for the 2.0% of the incidence of critical AEs. ETHICS AND DISSEMINATION: The study protocol was approved by the Institutional Review Board at Aichi Children's Health and Medical Center (2021051). The results will be reported in a peer-reviewed journal and a relevant academic conference. TRIAL REGISTRATION NUMBER: UMIN000047351.

Sedation with dexmedetomidine and propofol in children with Fontan circulation undergoing cardiac catheterization: A descriptive study.

Background: A combination of dexmedetomidine and propofol is considered advantageous for maintaining spontaneous breathing with a satisfactory depth of anesthesia. However, the incidence of upper airway obstruction under sedation with dexmedetomidine and propofol in patients with Fontan circulation remains unanswered. This study aimed to evaluate upper airway patency and oxygen desaturation during sedation with dexmedetomidine and propofol for cardiac catheterization in pediatric patients with Fontan circulation. Methods: In this descriptive study, we reviewed medical records of patients with Fontan circulation who underwent cardiac catheterization between December 2018 and August 2020 at a single-center 200-bed academic children's hospital in Japan. Results: A total of 35 patients with Fontan circulation sedated with a departmental protocol of dexmedetomidine and propofol infusion for cardiac catheterization were reviewed. Overall, the incidence of airway interventions and oxygen desaturation were 31.4% and 28.6%, respectively. In children with a history of snoring and additional use of intravenous midazolam, the rates of airway interventions were 50% and 100%, respectively. In patients ≤2 years old with recent upper respiratory infection (URI) symptoms, oxygen desaturation rate was 75%. Conclusions: In children with Fontan circulation, the incidence rate of upper airway obstruction was high under sedation with dexmedetomidine and propofol during cardiac catheterization, which is commonly considered safe in children without Fontan circulation. A history of snoring, an additional bolus of IV midazolam, and the presence of recent URI symptoms in patients ≤2 years old are potential risks for upper airway obstruction.