No Detectable Coagulation Activation After Vitamin K (MK-7) Supplementation in Patients on Dialysis With Functional Vitamin K Deficiency: A One-Year Randomized, Placebo-Controlled Study.

OBJECTIVE: Patients on dialysis treatment have poor functional vitamin K status, and this may increase the risk of vascular calcification. Vitamin K supplementation may therefore be relevant in patients on dialysis, but the procoagulant effects have not been studied. We evaluated effects of menaquinone-7 (MK-7) supplementation on biomarkers of coagulation in patients on dialysis. METHODS: Double-blinded, placebo-controlled study in 123 patients on dialysis randomized to 52 weeks of vitamin K (MK-7, 360 µg/daily, n = 61) or placebo (n = 62). Measurements at baseline and after 52 weeks of intervention included thrombin generation (endogenous thrombin potential, peak thrombin concentration, time to peak, and lag time); clot activities of vitamin K-dependent coagulation factors (F) II, VII, IX, and X; prothrombin fragment 1 + 2 (F1+2); and proteins induced by vitamin K absence II (PIVKA-II). Between-group differences (vitamin K vs. placebo) at 52 weeks were determined with an analysis of covariance. Within-group changes in vitamin K and placebo groups were analyzed with a paired t-test. Vascular adverse events and serious adverse events were registered based on hospital records, laboratory data, and participant interviews and compared between groups using Fisher's exact test or Pearson's Chi-Squared test. RESULTS: A between-group difference at 52 weeks was observed for PIVKA-II (P < .001). PIVKA-II decreased significantly from baseline to 52 weeks in the vitamin K group, but not in the placebo group. We observed no between-group differences or within-group changes for biomarkers of coagulation, except for FVII clot activity which was reduced in the placebo group (P = .04), and no between-group differences in adverse events and serious adverse events. CONCLUSION: One year of vitamin K supplementation in patients on dialysis has no detectable effects on biomarkers of coagulation activation, clot activities of vitamin K-dependent coagulation factors, and vascular events or death, indicating no procoagulant effects of this treatment.

Menaquinone-7 ameliorates cerebrovascular calcification-associated memory decline in aged mice.

Menaquinone (MK)-7 is a vitamin K2 analog that functions as a cofactor of γ-glutamyl carboxylase involved in the activation of vitamin K (VK)-dependent proteins. The present study aimed to evaluate the effect of MK-7 on memory and cognitive function in aged C57BL/6 mice. Eighteen-month-old mice were raised for a further 4 months, fed on a standard or calcium-rich diet (3 % [w/w]), and were orally given MK-7 (40 and 400 µg/day/mouse) five times per week during the same period. The Morris water maze (MWM) test was performed at 19 and 22 months. The aged mice showed noticeable memory declines in the MWM test at all time points compared with 6-week-old mice, and this memory loss was significantly restored by the daily administration of high-dose MK-7 for 4 months. MK-7 administration also improved micro-computed tomography-based cerebrovascular calcification and aging-associated declines in growth arrest-specific 6, total and carboxylated matrix Gla proteins, and ganglioside levels in the brain of aged mice. It serologically reduced phosphorous levels in the blood, but not the urea, cholesterol, and calcium. Taken together, the long-term administration of MK-7 significantly improved age-related memory and cognitive impairments, possibly through inhibition of cerebrovascular calcification in aged mice, indicating that it can be used to develop new drugs for improving memory and cognitive function in older adults.

The effect of menaquinone-7 supplementation on dp-ucMGP, PIVKAII, inflammatory markers, and body composition in type 2 diabetes patients: a randomized clinical trial.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is a common disorder that is characterized by chronic hyperglycemia and chronic inflammation, which also have a reinforcing effect on each other. The present research studied the effects of menaquinone (MK-7) supplementation on serum dp-ucMGP (dephospho uncarboxylated Matrix Gla Protein), PIVKAII (Prothrombin Induced by Vitamin K Absence), inflammatory markers and body composition indices in type 2 diabetes mellitus (T2DM) patients. METHODS: This 12-week double-blind placebo-controlled randomized clinical trial allocated 60 T2DM patients equally into a MK-7 (200 mcg/day) group or a placebo group. All patients also received dietary advice at the beginning of study and their dietary intakes were checked using a 3-day food record. The body composition of each patient was also measured and their vitamin K status was assessed using the ELISA method to measure serum dp-ucMGP and PIVKAII. In addition, inflammatory status indices were also measured, including hsCRP (high-sensitivity C-reactive protein), IL-6 (interleukin-6) and TNF-α (tumor necrosis factor alpha). All measurements were made both before and after the intervention period. RESULTS: In total 45 patients completed the trial (MK-7 group = 23 and placebo group = 22). The calorie and macronutrient intake of the two groups were similar pre and post intervention. There were statistically significant increases in dietary vitamin K intake for both groups over the course of the study (p < 0.05), but the intergroup differences were not significant. The body composition indices (i.e., body fat percentage, fat mass, fat free mass, muscle mass, bone mass and total body water) were not significantly different between groups or across the trial. The serum levels of the vitamin K markers, PIVKAII and dp-ucMGP, decreased significantly in the MK-7 group over the course of the study (p < 0.05), but there was no decrease in the placebo group. However, after adjusting for the baseline levels and changes in vitamin K intake, the between group differences were only significant for PIVKAII (p < 0.05). Following the intervention, the serum levels of the inflammatory markers (hsCRP, IL-6, and TNF-α) were significantly lower in the MK-7 group (p < 0.05), but not in the placebo group. However, the between group differences in the inflammatory markers were not statistically significant. CONCLUSIONS: Although further studies are needed, it appears that MK-7 supplementation can be effective in improving PIVKAII levels, but not for improving dp-ucMGP, inflammatory status or the body composition indices of T2DM patients. TRIAL REGISTRATION NUMBER: This study was prospectively registered at the Iranian Registry of Clinical Trials on the 20th of May 2019 (ID: IRCT20100123003140N22).

Development of a liquid chromatography mass spectrometry method for the determination of vitamin K1, menaquinone-4, menaquinone-7 and vitamin K1-2,3 epoxide in serum of individuals without vitamin K supplements.

OBJECTIVES: Vitamin K and metabolites have a beneficial role in blood coagulation, bone metabolism and growth. However, the determination of vitamin K concentrations in the blood in patients consuming a diet with naturally occurring vitamin K is currently challenging. We aim to develop a cost-effective and rapid method to measure vitamin K metabolites with potential application for clinics and research. METHODS: We developed a simple liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the determination of vitamin K1, menaquinone-4 (MK-4), menaquinone-7 (MK-7) and vitamin K1-2,3 epoxide in human serum and validated the method in a study cohort of 162 patients tested for carbohydrate malabsorption and in 20 patients with oral phenprocoumon intake. RESULTS: The overall precision (CVs) ranged between 4.8 and 17.7% in the specified working range (0.06-9.0 nmol/L for all analytes except for MK-7 with 0.04-6.16 nmol/L). In the malabsorption cohort samples, measured values were obtained for all different vitamin K metabolites except for vitamin K1-2,3 epoxide. This metabolite could be detected only in patients with phenprocoumon intake. The good performance of the method is especially achieved by the interaction of three factors: the use of lipase in the sample preparation, the use of an atypical fluorinated reversed phase column, and a logarithmic methanol gradient. CONCLUSIONS: The described method is able to determine the concentration of four vitamin K metabolites in a time-efficient, simple and cost-effective manner. It can be suitable for both routine clinics and research.

Multiple Dietary Vitamin K Forms Are Converted to Tissue Menaquinone-4 in Mice.

BACKGROUND: Vitamin K is a term that comprises a family of structurally related quinones, phylloquinone (PK) and the menaquinones (MKn), that share a common naphthoquinone ring but vary in sidechain length (n) and saturation. Dietary PK is a biosynthetic precursor to tissue menaquinone-4 (MK4), but little is known about the absorption and metabolism of dietary MKn. OBJECTIVE: To characterize the absorption and metabolism of dietary MKn relative to PK. METHODS: In the 4-week diet study, 10-week-old male and female C57BL/6 mice were pair-fed a vitamin K deficient diet (control) or a diet supplemented with 5.0 µmol/kg total PK, MK4, and/or MK9 (separately and in combination). In the 1-week stable isotope study, 12-week-old mice were pair-fed diets containing 2.2 µmol/kg PK (unlabeled control), 2H7PK, 13C11MK4, 2H7MK7, or 2H7MK9. Vitamin K tissue content was quantified by HPLC and/or LC-MS, and concentrations were compared by sex and diet group using 2-factor ANOVA. RESULTS: Regardless of the form(s) of vitamin K provided in the diet, tissue MK4 concentrations did not differ across equimolar supplemented groups in the kidney, adipose, reproductive organ, bone, or pancreas in either males or females in the diet study (all P values > 0.05). Isotopic labeling confirmed the naphthoquinone ring of MK4 in tissues originated from the administered dietary PK or MKn. Despite equimolar supplementation, accumulation of the administered dietary form differed across diet groups in small intestinal segments (all P values < 0.002) and the liver (P < 0.001). Female mice had greater total vitamin K than males in every tissue examined (P < 0.05). CONCLUSIONS: Dietary PK, MK4, MK7, and MK9 all served as precursors to tissue MK4 in mice. This study expands our understanding of vitamin K metabolism and supports a common conversion mechanism of all dietary vitamin K forms to MK4. Further investigation of the metabolism and physiological roles of MK4 that may be independent of classical vitamin K function is warranted.

Separation of menaquinone-7 geometric isomers by semipreparative high-performance liquid chromatography with silver complexation and identification by nuclear magnetic resonance.

Dietary supplements containing vitamin K2 are often used to prevent osteoporosis, vascular calcification and coronary heart disease. It has been shown that some of these products contain a mixture of menaquinone-7 geometric isomers. Since the geometric shape may influence biological activity, there was a need for a semipreparative method to isolate single compounds for further studies. Here, we present an argentation chromatographic method for the separation of menaquinone-7 isomers and an nuclear magnetic resonance (NMR) methodology for the configuration assignment of isoprenoid side chain. The DFT calculations were performed to determine more energetically favorable complexes between the cis or trans menaquinone-7 isomers and the silver cation. Seventeen components were resolved, and fractions were collected and subjected to NMR study. Structures and chemical shifts for thirteen new compounds were assigned, and the identity of three known compounds was confirmed.

Fat-Soluble Vitamin Supplementation Using Liposomes, Cyclodextrins, or Medium-Chain Triglycerides in Cystic Fibrosis: A Randomized Controlled Trial.

Fat-soluble vitamin deficiency remains a challenge in cystic fibrosis (CF), chronic pancreatitis, and biliary atresia. Liposomes and cyclodextrins can enhance their bioavailability, thus this multi-center randomized placebo-controlled trial compared three-month supplementation of fat-soluble vitamins in the form of liposomes or cyclodextrins to medium-chain triglycerides (MCT) in pancreatic-insufficient CF patients. The daily doses were as follows: 2000 IU of retinyl palmitate, 4000 IU of vitamin D3, 200 IU of RRR-α-tocopherol, and 200 µg of vitamin K2 as menaquinone-7, with vitamin E given in soybean oil instead of liposomes. All participants received 4 mg of ß-carotene and 1.07 mg of vitamin K1 to ensure compliance with the guidelines. The primary outcome was the change from the baseline of all-trans-retinol and 25-hydroxyvitamin D3 concentrations and the percentage of undercarboxylated osteocalcin. Out of 75 randomized patients (n = 28 liposomes, n = 22 cyclodextrins, and n = 25 MCT), 67 completed the trial (89%; n = 26 liposomes, n = 18 cyclodextrins, and n = 23 MCT) and had a median age of 22 years (IQR 19-28), body mass index of 20.6 kg/m2 [18.4-22.0], and forced expiratory volume in 1 s of 65% (44-84%). The liposomal formulation of vitamin A was associated with the improved evolution of serum all-trans-retinol compared to the control (median +1.7 ng/mL (IQR -44.3-86.1) vs. -38.8 ng/mL (-71.2-6.8), p = 0.028). Cyclodextrins enhanced the bioavailability of vitamin D3 (+9.0 ng/mL (1.0-17.0) vs. +3.0 ng/mL (-4.0-7.0), p = 0.012) and vitamin E (+4.34 µg/mL (0.33-6.52) vs. -0.34 µg/mL (-1.71-2.15), p = 0.010). Liposomes may augment the bioavailability of vitamin A and cyclodextrins may strengthen the supplementation of vitamins D3 and E relative to MCT in pancreatic-insufficient CF but further studies are required to assess liposomal vitamin E (German Clinical Trial Register number DRKS00014295, funded from EU and Norsa Pharma).

Simultaneous Determination of Vitamins D3 (Calcitriol, Cholecalciferol) and K2 (Menaquinone-4 and Menaquinone-7) in Dietary Supplements by UHPLC.

The content and composition of dietary supplements is of great interest due to their increasing consumption and variety of available brand offered in the market. Accurate determination of vitamins is important for the improvement of dietary supplement quality and nutrition assessments. In this regard, the simultaneous determination of vitamin D3 (calcitriol-CT and cholecalciferol-CHL) and K2 (menaquinone-4-MK-4 and menaquinone-7-MK-7) in dietary supplements was developed by using ultra-high-pressure liquid chromatography (UHPLC). The overall runtime per sample was above 35 min, with the retention times of 2.40, 6.59, 7.06, and 32.6 min for vitamin D3 (CT and CHL) and vitamin K2 (MK-4 and MK-7), respectively. The limits of detection and limits of quantification for the target nutritional compounds ranged between 0.04-0.05 µg/mL, respectively. The validation results indicated that the method had reasonable linearity (R2 ≥ 0.9990), good recovery (>82%), satisfactory intra-day precision (≤1.9%) and inter-day precision (≤3.5%), and high selectivity and specificity. The validated UHPLC method was demonstrated to be precise, accurate, and robust for the simultaneous determination of vitamins D3 (CT and CHL) and K2 (MK-4 and MK-7) in dietary supplements.

Inhibition of the futalosine pathway for menaquinone biosynthesis suppresses Chlamydia trachomatis infection.

Chlamydia trachomatis, an obligate intracellular bacterium with limited metabolic capabilities, possesses the futalosine pathway for menaquinone biosynthesis. Futalosine pathway enzymes have promise as narrow-spectrum antibiotic targets, but the activity and essentiality of chlamydial menaquinone biosynthesis have yet to be established. In this work, menaquinone-7 (MK-7) was identified as a C. trachomatis-produced quinone through liquid chromatography-tandem mass spectrometry. An immunofluorescence-based assay revealed that treatment of C. trachomatis-infected HeLa cells with the futalosine pathway inhibitor docosahexaenoic acid (DHA) reduced inclusion number, inclusion size, and infectious progeny. Supplementation with MK-7 nanoparticles rescued the effect of DHA on inclusion number, indicating that the futalosine pathway is a target of DHA in this system. These results open the door for menaquinone biosynthesis inhibitors to be pursued in antichlamydial development.

Chryseobacterium panacisoli sp. nov., isolated from ginseng-cultivation soil with ginsenoside-converting activity.

A Gram-stain-negative, non-motile, non-spore-forming, aerobic, rod-shaped and yellow-pigmented bacterium, designated strain Gsoil 183T, was isolated from ginseng-cultivation soil sampled in Pocheon Province, Republic of Korea. This bacterium was characterized to determine its taxonomic position by using a polyphasic approach. Strain Gsoil 183T grew at 10-37 °C and at pH 5.0-9.0 on tryptic soy agar. Strain Gsoil 183T had ß-glucosidase activity, which was responsible for its ability to convert ginsenoside Rb1 (one of the dominant active components of ginseng) to F2. Based on 16S rRNA gene sequencing, strain Gsoil 183T clustered with species of the genus Chryseobacterium and appeared to be closely related to Chryseobacterium sediminis LMG 28695T (99.1 % sequence similarity), Chryseobacterium lactis NCTC 11390T (98.6%), Chryseobacterium rhizoplanae LMG 28481T (98.6%), Chryseobacterium oncorhynchi CCUG 60105T (98.5%), Chryseobacterium viscerum CCUG 60103T (98.4%) and Chryseobacterium joostei DSM 16927T (98.3%). Menaquinone MK-6 was the predominant respiratory quinone and the major fatty acids were iso-C15 : 0, iso-C17 : 0-3OH and summed feature 3 (C16 : 1 ω6c and/or C16 : 1 ω7c). The polar lipids were phosphatidylethanolamine, six unidentified glycolipids, five unidentified aminolipids and three unidentified lipids. The G+C content of the genomic DNA was 36.6 mol%. Digital DNA-DNA hybridization between strain Gsoil 183T and the type strains of C. sediminis, C. lactis, C. rhizoplanae, C. oncorhynchi, C. viscerum and C. joostei resulted in values below 70 %. Strain Gsoil 183T could be differentiated genotypically and phenotypically from the recognized species of the genus Chryseobacterium. The isolate therefore represents a novel species, for which the name Chryseobacterium panacisoli sp. nov. is proposed, with the type strain Gsoil 183T (=KACC 15033T=LMG 23397T).

Rossellomorea arthrocnemi sp. nov., a novel plant growth-promoting bacterium used in heavy metal polluted soils as a phytoremediation tool.

Strain EAR8T is a root endophyte isolated from Arthrocnemum macrostachyum plants collected from the Odiel marshes, Huelva (Spain). It presented in vitro plant growth-promoting properties and improved the plant growth and heavy metal accumulation in polluted soils playing an important role in phytoremediation strategies. Phenotypically, strain EAR8T cells were Gram-positive, aerobic and non-motile rods with terminal oval endospores and non-swollen sporangia which form beige, opaque, butyrous, raised and irregular colonies with undulate margins. The strain was able to grow between 15-45 °C, at pH 6.0-9.0 and tolerated 0-25 % NaCl (w/v) showing optimal growth conditions on trypticase soy agar plates supplemented with 2.5 % NaCl (w/v) at pH 7.0 and 37 °C for 24 h. Chemotaxonomic analyses showed that the isolate has meso-diaminopimelic acid as the peptidoglycan in the cell wall and MK-7 as the major respiratory quinone. The predominant fatty acids were anteiso-C15 : 0 and iso-C15 : 0 and the polar lipid profile was composed of diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Phylogenetic analyses based on the whole proteomes of closest sequenced relatives confirmed that strain EAR8T is affiliated to the genus Rossellomorea and forms a clade with Rossellomorea vietnamensis 15-1T with maximum support. Genome analyses showed that EAR8T has indole-3-acetic acid and siderophore biosynthesis and transporters genes and genes related to resistance against heavy metals. Phenotypic and phylogenomic comparative studies suggested that strain EAR8T is a new representative of the genus Rossellomorea and the name Rossellomorea arthrocnemi sp. nov. is proposed. Type strain is EAR8T (=CECT 9072T=DSM 103900T).

Menaquinone 4 Reduces Bone Loss in Ovariectomized Mice through Dual Regulation of Bone Remodeling.

Epidemiologic studies showed that higher vitamin K (VK) consumption correlates with a reduced risk of osteoporosis, yet the dispute remains about whether VK is effective in improving bone mineral density (BMD). We sought to discover the anti-osteoporotic effect of menaquinone-4 (MK-4) and evaluate the expression of critical genes related to bone formation and bone resorption pathways in the body. Fifty female C57BL/6 mice (aged 13 weeks) were randomly arranged to a sham-operated group (SHAM, treated with corn oil) and four ovariectomized groups that were administered corn oil (OVX group), estradiol valerate (EV, 2 mg/kg body weight as the positive control), low or high doses of VK (LVK and HVK; 20 and 40 mg MK-4/kg body weight, respectively) by gavage every other day for 12 weeks. Body and uterine weight, serum biochemical indicators, bone microarchitecture, hematoxylin-eosin (HE) staining, and the mRNA expression of critical genes related to bone formation and bone resorption pathways were assessed. Either dose of MK-4 supplementation increased the alkaline phosphatase (ALP), decreased the undercarboxylated osteocalcin (ucOC) and tartrate-resistant acid phosphatase (TRACP, p < 0.05) levels, and presented higher BMD, percent bone volume (BV/TV), trabecular thickness (Tb.Th), and lower trabecular separation (Tb.Sp) and structure model index (SMI, p < 0.05) compared with the OVX group. Additionally, both doses of MK4 increased the mRNA expression of Runx2 and Bmp2 (p < 0.05), whereas the doses down-regulated Pu.1 and Nfatc1 (p < 0.05) mRNA expression, the high dose decreased Osx and Tgfb (p < 0.05) mRNA expression, and the low dose decreased Mitd and Akt1 (p < 0.05) mRNA expression. These data show the dual regulatory effects of MK-4 on bone remodeling in ovariectomized mice: the promotion of bone anabolic activity and inhibition of osteoclast differentiation, which provides a novel idea for treating osteoporosis.

First Clinical Trial of a Newly Developed, Low Menaquinone-7, Fermented Soybean Natto in Warfarin-Dependent Patients.

Bacillus subtilis fermented soybeans (natto) contain high vitamin K2 levels, mostly as menaquinone-7 (MK-7), and must be avoided by warfarin-dependent patients. This is the first report which demonstrates the characteristics and clinical relevance of a low MK-7 natto for such patients. We generated a novel, mutant B. subtilis strain TTCC2051 with short-term fermentation and reduced MK-7 production, yielding 19-24% of the normal MK-7 content. After functional assessments and a preclinical trial, 10 warfarin-dependent patients underwent a clinical trial with a 7-day ingestion test of the low MK-7 natto. Functional assessments were satisfactory, and the preclinical trial showed no increases in plasma MK-7 levels after 7 days of ingestion. In the clinical trial, 20 g/day of the low MK-7 natto significantly increased plasma MK-7 levels while 10 g/day did not. However, neither dose of low MK-7 natto changed international normalized ratio of prothrombin time (PT-INR) values in either group. The low MK-7 natto neither changed PT-INR values nor precipitated adverse events if ingested with a once-daily maximum of 20 g (46 µg of MK-7). Thus, this novel food product has potential for consumption by warfarin-dependent patients.

Effect of Vitamin K-Mediated PXR Activation on Drug-Metabolizing Gene Expression in Human Intestinal Carcinoma LS180 Cell Line.

The pregnane X receptor (PXR) is the key regulator of our defense mechanism against foreign substances such as drugs, dietary nutrients, or environmental pollutants. Because of increased health consciousness, the use of dietary supplements has gradually increased, and most of them can activate PXR. Therefore, an analysis of the interaction between drugs and nutrients is important because altered levels of drug-metabolizing enzymes or transporters can remarkably affect the efficiency of a co-administered drug. In the present study, we analyzed the effect of vitamin K-mediated PXR activation on drug metabolism-related gene expression in intestine-derived LS180 cells via gene expression studies and western blotting analyses. We demonstrated that menaquinone 4 (MK-4), along with other vitamin Ks, including vitamin K1, has the potential to induce MDR1 and CYP3A4 gene expression. We showed that PXR knockdown reversed MK-4-mediated stimulation of these genes, indicating the involvement of PXR in this effect. In addition, we showed that the expression of MDR1 and CYP3A4 genes increased synergistically after 24 h of rifampicin and MK-4 co-treatment. Our study thus elucidates the importance of drug-nutrient interaction mediated via PXR.

Aminofutalosine Deaminase in the Menaquinone Pathway of Helicobacter pylori.

Helicobacter pylori is a Gram-negative bacterium that is responsible for gastric and duodenal ulcers. H. pylori uses the unusual mqn pathway with aminofutalosine (AFL) as an intermediate for menaquinone biosynthesis. Previous reports indicate that hydrolysis of AFL by 5'-methylthioadenosine nucleosidase (HpMTAN) is the direct path for producing downstream metabolites in the mqn pathway. However, genomic analysis indicates jhp0252 is a candidate for encoding AFL deaminase (AFLDA), an activity for deaminating aminofutolasine. The product, futalosine, is not a known substrate for bacterial MTANs. Recombinant jhp0252 was expressed and characterized as an AFL deaminase (HpAFLDA). Its catalytic specificity includes AFL, 5'-methylthioadenosine, 5'-deoxyadenosine, adenosine, and S-adenosylhomocysteine. The kcat/Km value for AFL is 6.8 × 104 M-1 s-1, 26-fold greater than that for adenosine. 5'-Methylthiocoformycin (MTCF) is a slow-onset inhibitor for HpAFLDA and demonstrated inhibitory effects on H. pylori growth. Supplementation with futalosine partially restored H. pylori growth under MTCF treatment, suggesting AFL deamination is significant for cell growth. The crystal structures of apo-HpAFLDA and with MTCF at the catalytic sites show a catalytic site Zn2+ or Fe2+ as the water-activating group. With bound MTCF, the metal ion is 2.0 Å from the sp3 hydroxyl group of the transition state analogue. Metabolomics analysis revealed that HpAFLDA has intracellular activity and is inhibited by MTCF. The mqn pathway in H. pylori bifurcates at aminofutalosine with HpMTAN producing adenine and depurinated futalosine and HpAFLDA producing futalosine. Inhibition of cellular HpMTAN or HpAFLDA decreased the cellular content of menaquinone-6, supporting roles for both enzymes in the pathway.

Safety and Efficacy of Vitamin K Antagonists versus Rivaroxaban in Hemodialysis Patients with Atrial Fibrillation: A Multicenter Randomized Controlled Trial.

BACKGROUND: In patients with normal renal function or early stage CKD, the risk-benefit profile of direct oral anticoagulants (DOACs) is superior to that of vitamin K antagonists (VKAs). In patients on hemodialysis, the comparative efficacy and safety of DOACs versus VKAs are unknown. METHODS: In the Valkyrie study, 132 patients on hemodialysis with atrial fibrillation were randomized to a VKA with a target INR of 2-3, 10 mg rivaroxaban daily, or rivaroxaban and vitamin K2 for 18 months. Patients continued the originally assigned treatment and follow-up was extended for at least an additional 18 months. The primary efficacy end point was a composite of fatal and nonfatal cardiovascular events. Secondary efficacy end points were individual components of the composite outcome and all-cause death. Safety end points were life-threatening, major, and minor bleeding. RESULTS: Median (IQR) follow-up was 1.88 (1.01-3.38) years. Premature, permanent discontinuation of anticoagulation occurred in 25% of patients. The primary end point occurred at a rate of 63.8 per 100 person-years in the VKA group, 26.2 per 100 person-years in the rivaroxaban group, and 21.4 per 100 person-years in the rivaroxaban and vitamin K2 group. The estimated competing risk-adjusted hazard ratio for the primary end point was 0.41 (95% CI, 0.25 to 0.68; P=0.0006) in the rivaroxaban group and 0.34 (95% CI, 0.19 to 0.61; P=0.0003) in the rivaroxaban and vitamin K2 group, compared with the VKA group. Death from any cause, cardiac death, and risk of stroke were not different between the treatment arms, but symptomatic limb ischemia occurred significantly less frequently with rivaroxaban than with VKA. After adjustment for competing risk of death, the hazard ratio for life-threatening and major bleeding compared with the VKA group was 0.39 (95% CI, 0.17 to 0.90; P=0.03) in the rivaroxaban group, 0.48 (95% CI, 0.22 to 1.08; P=0.08) in the rivaroxaban and vitamin K2 group and 0.44 (95% CI, 0.23 to 0.85; P=0.02) in the pooled rivaroxaban groups. CONCLUSIONS: In patients on hemodialysis with atrial fibrillation, a reduced dose of rivaroxaban significantly decreased the composite outcome of fatal and nonfatal cardiovascular events and major bleeding complications compared with VKA. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: Oral Anticoagulation in Hemodialysis, NCT03799822.

The effects of eggshell calcium (Biomin H® ) and its combinations with alfacalcidol (1α-hydroxyvitamin D3) and menaquinone-7 (vitamin K2) on ovariectomy-induced bone loss in a rat model of osteoporosis.

The purpose of this study was to investigate the impact of eggshell calcium (Biomin H® dietary supplement) and its combinations with alfacalcidol (1α-hydroxyvitamin D3 ) and menaquinone-7 (vitamin K2 ) on ovariectomy-induced bone loss in rats. Adult female rats (n = 48) were divided into 6 groups of 8 individuals each: sham-operated rats (SHAM); ovariectomized (OVX) rats untreated; OVX rats treated with Biomin H® (BIO); OVX rats simultaneously receiving Biomin H® , vitamin D3 (BIO + D3 ); OVX rats simultaneously treated with Biomin H® , vitamin K2 (BIO + K2 ) and OVX rats treated with Biomin H® , vitamin D3 , vitamin K2 (BIO + D3  + K2 ) during 8 weeks. Biochemical parameters, bone mineral density (BMD), bone mineral content (BMC) and femoral bone microstructure were determined. Plasma calcium and phosphate were increased in BIO + D3 and BIO + D3  + K2 groups as compared to OVX. Alkaline phosphatase was elevated in OVX, BIO versus SHAM, BIO + D3  + K2 groups. When compared to OVX group, decreased urine deoxypyridinoline was observed in all treated groups and femoral BMD, BMC were higher in BIO, BIO + D3 , BIO + D3  + K2 groups. The BIO + K2 rats had similar densitometrical values than OVX individuals. Microcomputed tomography revealed increased trabecular relative bone volume (due to an increase in trabecular number) in BIO + D3 , BIO + D3  + K2 as compared to OVX. The higher relative bone volume in BIO + D3 , BIO + D3  + K2 groups was also accompanied by an increase in bone surface. In the cortical bone, an enhanced periosteal bone apposition was identified in BIO, BIO + D3 , BIO + K2 , BIO + D3  + K2 groups. The rats from BIO + D3  + K2 group had a higher area of primary osteon's vascular canals. In BIO + D3 , BIO + K2 , BIO + D3  + K2 groups, an increased area of secondary osteons was determined in comparison with OVX. Our results indicate the beneficial effect of triple application of Biomin H® , vitamin D3 , vitamin K2 , as well as simultaneous administration of Biomin H® , vitamin D3 on the inhibition of ovariectomy-induced bone loss in a rat model of osteoporosis.

Steptomyces fagopyri sp. nov., a novel actinomycete isolated from rhizospheric soil of Fagopyrum dibotrys.

A novel actinomycete, designated strain QMT-28T, was isolated from rhizosphere soil of Fagopyrum dibotrys collected from Shuangfeng, Hunan Province, PR China. Strain QMT-28T grew well on International Streptomyces Project series media and formed well-developed, branched substrate hyphae and aerial mycelium that differentiated into loose spiral spore chains consisting of cylindrical spores with smooth surfaces. The diagnostic diamino acid was ll-diaminopimelic acid and the whole-cell sugars were galactose and glucose. The predominant fatty acids were C18 : 1 cis9, summed feature 6 (C18 : 2 cis 9,12/C18 : 0 a) and C16 : 0. The polar lipids included diphosphatidylglycerol, hydroxy phospatidylethanolamine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannosides, phospholipids of unknown structure containing glucosamine and several unidentified phospholipids. The major menaquinones were MK-9, MK-9(H2), MK-9(H4), MK-9(H6) and MK-9(H8). The genome size of strain QMT-28T was about 8.7 Mbp with a G+C content of 71.2 mol%. Phylogenetic analysis showed that the novel strain was closely related to Streptomyces olivochromogenes DSM 40451T (99.5 % similarity), Streptomyces mirabilis NBRC 13450T (98.9 %), Streptomyces kanamyceticus NBRC 13414T (98.9 %), Streptomyces kaempferi I37T (98.9 %) and Streptomyces arcticus ZLN234T (98.8 %). However, the average nucleotide identity values, the digital DNA-DNA hybridization values and the multilocus sequence analysis evolutionary distances between this strain and closely related strains showed that it belonged to a distinct species. In addition, these results were also supported by differences in the phenotypic characteristics between QMT-28T and five closely related type strains. Consequently, strain QMT-28T should represent a novel species of the genus Streptomyces, with the suggested name Streptomyces fagopyri sp. nov. The type strain is QMT-28T (=CICC 24808T=JCM 33796T).

Microbispora clausenae sp. nov., an endophytic actinobacterium isolated from the surface-sterilized stem of a Thai medicinal plant, Clausena excavala Burm. f.

An endophytic actinobacterium, strain CLES2T, was discovered from the surface-sterilized stem of a Thai medicinal plant, Clausena excavala Burm. f., collected from the Phujong-Nayoa National Park, Ubon Ratchathani Province, Thailand. The results of a polyphasic taxonomic study identified this strain as a member of the genus Microbispora and a Gram-stain-positive, aerobic actinobacterium. It had well-developed substrate mycelia, which were non-motile and possessed paired spores. A phylogenetic evaluation based on 16S rRNA gene sequence analysis placed this strain in the family Streptosporangiaceae, being most closely related to Microbispora bryophytorum NEAU-TX2-2T (99.4 %), Microbispora camponoti 2C-HV3T (99.2 %), Microbispora catharanthi CR1-09T (99.2 %) and Microbispora amethystogenes JCM 3021T and Microbispora fusca NEAU-HEGS1-5T (both at 99.1 %). The major cellular fatty acid of this strain was iso-C16 : 0 and major menaquinone was MK-9(H4). The polar lipid profile of strain CLES2T contained diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylinositol and phosphatidylinositol dimannosides. These chemotaxonomic data confirmed the affiliation of strain CLES2T to the genus Microbispora. The DNA G+C content of this strain was 70 mol%. Digital DNA-DNA hybridization and average nucleotide identity blast values between strain CLES2T and M. catharanthi CR1-09T were 62.4 and 94.0 %, respectively. The results of the polyphasic study allowed the genotypic and phenotypic differentiation of strain CLES2T from its closest species with valid names. The name proposed for the new species is Microbispora clausenae sp. nov. The type strain is CLES2T (=DSM 101759T=NRRL B-65340T).