Chryseobacterium metallicongregator, sp. nov., a bacterium possessing metallophore activity towards rare earth elements.

A polyphasic taxonomic study was carried out on strain ES2T, isolated from sediment of a wetland created to remediate acid drainage from a coal mine. The rod-shaped bacterium formed yellow/orange pigmented colonies and produced the pigment flexirubin. The 16S rRNA gene sequence results assigned the strain to Chryseobacterium, with 98.9 and 98.3 % similarity to Chryseobacterium vietnamense and Chryseobacterium cucumeris, respectively. Computation of the average nucleotide identity and digital DNA-DNA hybridization values with the closest phylogenetic neighbours of ES2T revealed genetic differences at the species level, which were further substantiated by differences in several physiological characteristics. The dominant fatty acids of strain ES2T were iso-C15 : 0, iso-C17 : 1 ω9c, iso C17 : 0 3-OH, and iso-C15 : 0 2-OH. The DNA G+C content was 35.5 mol%. The major polar lipid was phosphatidylethanolamine while menaquinone-6 was the only menaquinone found. This bacterium has been previously shown to possess metallophore activity towards rare earth elements, and based on genome sequencing, possesses all required genes for siderophore production/activity, possibly identifying the source of this unique ability. On the basis of the results obtained here, this bacterium is assigned to the genus Chryseobacterium as representing a new species with the name Chryseobacterium metallicongregator sp. nov., type strain ES2T (=NRRL B-65679T=KCTC 102120T).

Maribacter halichondriae sp. nov., isolated from the marine sponge Halichondria panicea, displays features of a sponge-associated life style.

A new member of the family Flavobacteriaceae (termed Hal144T) was isolated from the marine breadcrumb sponge Halichondria panicea. Sponge material was collected in 2018 at Schilksee which is located in the Kiel Fjord (Baltic Sea, Germany). Phylogenetic analysis of the full-length Hal144T 16S rRNA gene sequence revealed similarities from 94.3 to 96.6% to the nearest type strains of the genus Maribacter. The phylogenetic tree of the 16S rRNA gene sequences depicted a cluster of strain Hal144T with its closest relatives Maribacter aestuarii GY20T (96.6%) and Maribacter thermophilus HT7-2T (96.3%). Genome phylogeny showed that Maribacter halichondriae Hal144T branched from a cluster consisting of Maribacter arenosus, Maribacter luteus, and Maribacter polysiphoniae. Genome comparisons of strain Maribacter halichondriae Hal144T with Maribacter sp. type strains exhibited average nucleotide identities in the range of 75-76% and digital DNA-DNA hybridisation values in the range of 13.1-13.4%. Compared to the next related type strains, strain Hal144T revealed unique genomic features such as phosphoenolpyruvate-dependent phosphotransferase system pathway, serine-glyoxylate cycle, lipid A 3-O-deacylase, 3-hexulose-6-phosphate synthase, enrichment of pseudogenes and of genes involved in cell wall and envelope biogenesis, indicating an adaptation to the host. Strain Hal144T was determined to be Gram-negative, mesophilic, strictly aerobic, flexirubin positive, resistant to aminoglycoside antibiotics, and able to utilize N-acetyl-ß-D-glucosamine. Optimal growth occurred at 25-30 °C, within a salinity range of 2-6% sea salt, and a pH range between 5 and 8. The major fatty acids identified were C17:0 3-OH, iso-C15:0, and iso-C15:1 G. The DNA G + C content of strain Hal144T was 41.4 mol%. Based on the polyphasic approach, strain Hal144T represents a novel species of the genus Maribacter, and we propose the name Maribacter halichondriae sp. nov. The type strain is Hal144T (= DSM 114563T = LMG 32744T).

Ruicaihuangia caeni gen. nov., sp. nov., a novel taxon within the family Microbacteriaceae isolated from sludge.

A Gram-stain-positive bacterium, designated YN-L-19T, was isolated from a sludge sample collected from a pesticide-manufacturing plant. Cells of YN-L-19T were strictly aerobic, non-spore-forming, non-motile and ovoid-shaped. Colonies were small, smooth and yellow. Growth occurred at 10-37 °C (optimum, 30 °C), pH 5.0-9.0 (optimum, 7.0) and 0-3.0 % (w/v) NaCl (optimum 0.5 %). Phylogenetic analysis based on genome and 16S rRNA gene sequences indicated that YN-L-19T was affiliated to the family Microbacteriaceae and most closely related to Diaminobutyricimonas aenilata, Terrimesophilobacter mesophilus, Planctomonas deserti and Curtobacterium luteum. The major cellular fatty acids of YN-L-19T were anteiso-C15 : 0, anteiso-C17 : 0, iso-C16 : 0 and C16 : 0. The predominant menaquinone was MK-7. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, glycolipid and one unidentified lipid. The average amino acid identity values between strain YN-L-19T and the related strains were 57.9-61.9 %, which were below the genus boundary (70 %). On the basis of the evidence presented in this study, strain YN-L-19T represents a novel species of a new genus in the family Microbacteriaceae, for which the name Ruicaihuangia caeni gen. nov., sp. nov. (type strain YN-L-19T=CCTCC AB 2022401T= KCTC 49935T) is proposed.

Safety evaluation of vitamin K2 (menaquinone-7) via toxicological tests.

This study aims to evaluate the safety of MK-7 produced by fermentation process using a Bacillus subtilis var. natto strain for human ingestion via acute oral toxicity, repeated dose 90-day oral toxicity, 28-day recovery test, and genotoxicity tests. The acute oral toxicity test results indicated that all subjects survived at the dose of 5000 mg/kg with no toxic effects. For the repeated dose 90-day oral toxicity test, MK-7 was administered to rats at 500, 1500, and 4500 mg/kg for 90 d. No abnormal findings were detected in clinical observations or in clinical pathological and histopathological examinations. The no-observed-adverse-effect level(NOAEL) was determined to be 4500 mg/kg/d, the maximum dose tested. For the evaluation of genotoxicity, reverse mutation, chromosomal aberration, and micronucleus tests were performed. In the reversion mutation test, vitamin K2 did not induce reversion in bacterial strains, and no chromosomal abnormality was observed in the chromosomal abnormality test using Chinese hamster lung cells. In the micronucleus test, micronuclei were not induced using ICR mouse bone marrow cells. All the toxicity test results suggest that vitamin K2 produced by fermentation processes using Bacillus subtilis var. natto induced no toxicological changes under the experimental conditions.

Rathayibacter tanaceti sp. nov., a Novel Actinobacterium from Tanacetum vulgare Infested by Foliar Nematode Aphelenchoides sp.

Two novel yellow-pigmented, rod-shaped and non-motile coryneform actinobacteria, strains VKM Ac-2596T and VKM Ac-2761, were isolated from a plant Tanacetum vulgare (Asteraceae) infested by foliar nematode Aphelenchoides sp. The strains exhibited the highest 16S rRNA gene sequence similarities to Rathayibacter agropyri CA4T (99.71%), Rathayibacter rathayi DSM 7485T (99.65%) and Rathayibacter iranicus VKM Ac-1602T (99.65%). The pairwise average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between VKM Ac-2596T and VKM Ac-2671 towards the type strains of Rathayibacter species did not exceed 85.24% and 29.40%, respectively, that are well below the thresholds for species delineation. The target strains had key chemotaxonomic properties typical of the genus Rathayibacter, namely, the DAB-based peptidoglycan, rhamnose and mannose as the predominant sugars and a rhamnomannan in the cell, the major menaquinone MK-10 and fatty acids of iso-anteiso type, with a large proportion of anteiso-15:0. The strains showed clear differences from the recognized Rathayibacter species in several phenotypic characteristics, including the difference in the composition of cell wall glycopolymers. Based on the results obtained in this study and the data published previously, we provide a description of a new species, Rathayibacter tanaceti sp. nov., with DL-642T (= VKM Ac-2596T = LMG 33114T) as the type strain.

Sciscionella sediminilitoris sp. nov., a Marine Actinomycete Isolated from Cape Rochado, Malaysia, and the Emendations to the Description of the Genus Sciscionella.

In this study, we employed a polyphasic approach to determine the taxonomic position of a newly isolated actinomycete, designated SE31T, obtained from a sediment sample collected at Cape Rochado, Malaysia. Phylogenetic analysis of the 16S rRNA gene sequence revealed that strain SE31T belonged to the family Pseudonocardiaceae and exhibited the highest sequence similarity (98.9%) to Sciscionella marina. Further genomic analysis demonstrated a 93.4% average nucleotide identity and 54.4% digital DNA-DNA hybridization relatedness between strain SE31T and S. marina. The chemotaxonomic characteristics of strain SE31T were typical of the genus Sciscionella, including cell-wall chemotype IV (with meso-diaminopimelic acid as the diagnostic diamino acid, and arabinose and galactose as whole-cell sugars). The identified polar lipids of strain SE31T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylmethylethanolamine, and hydroxyphosphatidymethylethanolamine. The primary menaquinone observed was MK-9(H4), and the major cellular fatty acid was iso-C16:0. The genomic DNA size of strain SE31T was determined to be 7.4 Mbp with a G+C content of 68.7%. Based on these comprehensive findings, strain SE31T represents a novel species within the genus Sciscionella, in which the name Sciscionella sediminilitoris sp. nov. is proposed. The type strain of Sciscionella sediminilitoris is SE31T (= DSM 46824T = TBRC 5134T).

Vitamin K2 protects mice against non-alcoholic fatty liver disease induced by high-fat diet.

Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases worldwide and there is a huge unmet need to find safer and more effective drugs. Vitamin K has been found to regulate lipid metabolism in the liver. However, the effects of vitamin K2 on NAFLD is unclear. This study aims to evaluate the preventive and therapeutic effects of vitamin K2 in the process of fatty liver formation and to explore molecular mechanisms the associated with lipid metabolism. A non-alcoholic fatty liver model was established by high-fat diet administration for three months. Vitamin K2 significantly reduced the body weight, abdominal circumference and body fat percentage of NAFLD mice. Vitamin K2 also showed histological benefits in reducing hepatic steatosis. NAFLD mice induced by high-fat diet showed increased HMGR while vitamin K2 intervention could reverse the pathological lterations. Adiponectin (APN) is an endogenous bioactive polypeptide or protein secreted by adipocytes. We detected APN, SOD, AlaDH and other indicators that may affect the state of high-fat diet mice, but the experimental results showed that the above indicators did not change significantly. It is worth noting that the effect of vitamin K2 supplementation on the lipid-lowering effect of uc OC in vivo needs to be further explored. This study first reported the protective effect of vitamin K2 on high-fat diet-induced NAFLD in mice. The protective effect of vitamin K2 may be related to the improvement of lipid metabolism disorder in NAFLD.

Comparative genomics analysis and transposon mutagenesis provides new insights into high menaquinone-7 biosynthetic potential of Bacillus subtilis natto.

This research combined Whole-Genome sequencing, intraspecific comparative genomics and transposon mutagenesis to investigate the menaquinone-7 (MK-7) synthesis potential in Bacillus subtilis natto. First, Whole-Genome sequencing showed that Bacillus subtilis natto BN-P15-11-1 contains one single circular chromosome in size of 3,982,436 bp with a GC content of 43.85 %, harboring 4,053 predicted coding genes. Next, the comparative genomics analysis among strain BN-P15-11-1 with model Bacillus subtilis 168 and four typical Bacillus subtilis natto strains proves that the closer evolutionary relationship Bacillus subtilis natto BN-P15-11-1 and Bacillus subtilis 168 both exhibit strong biosynthetic potential. To further dig for MK-7 biosynthesis latent capacity of BN-P15-11-1, we constructed a mutant library using transposons and a high throughput screening method using microplates. We obtained a YqgQ deficient high MK-7 yield strain F4 with a yield 3.02 times that of the parent strain. Experiments also showed that the high yield mutants had defects in different transcription and translation regulatory factor genes, indicating that regulatory factor defects may affect the biosynthesis and accumulation of MK-7 by altering the overall metabolic level. The findings of this study will provide more novel insights on the precise identification and rational utilization of the Bacillus subtilis subspecies for biosynthesis latent capacity.

Myceligenerans pegani sp. nov., an endophytic actinomycete isolated from Peganum harmala L. in Xinjiang, PR China.

An endophytic actinomycete designated TRM65318T, was isolated from the root of Peganum harmala L. Its taxonomic status was determined using a polyphasic approach. Comparative 16S rRNA gene sequence analysis indicated that strain TRM65318T is phylogenetically most closely related to Myceligenerans salitolerans XHU 5031T (98.15 %) and Myceligenerans xiligouense DSM 15700T (97.78 %). The peptidoglycan belonged to type A4α. The polar lipids were phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol, two unknown lipids and three glycolipids. The predominant menaquinones were MK-9(H4) and MK-9(H6) and the whole-cell sugars contained glucose, mannose and galactose. Major fatty acids were anteiso-C15 : 0, iso-C15 : 0 and C16 : 0. Strain TRM65318T had a genome size of 5881012 bp with a genome G+C content of 71.79 mol%. The average nucleotide identity and DNA-DNA hybridization values between strain TRM65318T and the most closely related species were much lower than the thresholds commonly used to define species. At the same time, differences in phenotypic and genotypic data showed that strain TRM65318T could be clearly distinguished from M. salitolerans XHU 5031T. Therefore, it is concluded that strain TRM65318T represents a novel species of the genus of Myceligenerans. The proposed name for this organism is Myceligenerans pegani sp. nov., with type strain TRM65318T (=CCTCC AA 2019057T=LMG 31679T).

Description of Chryseobacterium fluminis sp. nov., a keratinolytic bacterium isolated from a freshwater river.

A Gram-stain-negative, aerobic, rod-shaped bacterial strain, designated MMS21-Ot14T, was isolated from a freshwater river, and shown to represent a novel species of the genus Chryseobacterium on the basis of the results from a polyphasic approach. The 16S rRNA gene sequence analysis revealed that MMS21-Ot14T represented a member of the genus Chryseobacterium of the family Weeksellaceae and was closely related to Chryseobacterium hagamense RHA2-9T (97.52 % sequence similarity), Chryseobacterium gwangjuense THG A18T (97.46 %) and Chryseobacterium gregarium P 461/12T (97.27 %). The optimal growth of MMS21-Ot14T occurred at 25-30 °C, pH 6.0-7.0 and in the absence of NaCl. MMS21-Ot14T was capable of hydrolysing casein, starch, DNA, Tween 20 and tyrosine. The strain also showed keratinolytic activity with keratin azure and decolourising activity with remazol brilliant blue R (RBBR), which indicated potential ability to degrade keratin and lignin. The main polar lipids of MMS21-Ot14T were phosphatidylethanolamine, unidentified aminophospholipids, unidentified aminolipids, an unidentified phospholipid and several unidentified lipids. The predominant fatty acids of MMS21-Ot14T were iso-C15 : 0 and iso-C17 : 0 3-OH, and the major isoprenoid quinone was menaquinone 6 (MK-6). The whole genome of MMS21-Ot14T was 5 062 016 bp in length with a DNA G+C content of 37.7 %. The average nucleotide identity and digital DNA-DNA hybridisation values between MMS21-Ot14T and phylogenetically related members of the genus Chryseobacterium were well below the threshold values for species delineation. It is evident from the results of this study that MMS21-Ot14T should be classified as representing a novel species of the genus Chryseobacterium, for which the name Chryseobacterium fluminis sp. nov. (type strain, MMS21-Ot14T = KCTC 92255T = LMG 32529T) is proposed.

Three novel species of the genus Flavobacterium, Flavobacterium odoriferum sp. nov., Flavobacterium fragile sp. nov. and Flavobacterium luminosum sp. nov., isolated from activated sludge.

Three Gram-stain-negative, rod-shaped, non-spore-forming bacteria were isolated from activated sludge samples. The results of phylogenetic analysis based on the 16S rRNA gene sequences indicated that the three strains, designated HXWNR29T, HXWNR69T and HXWNR70T, had the highest sequence similarity to the type strains Flavobacterium cheniae NJ-26T, Flavobacterium channae KSM-R2A30T and Flavobacterium amniphilum KYPY10T with similarities of 97.66 %, 98.66 and 98.14 %, respectively. The draft genomes of these three strains were 2.93 Mbp (HXWNR29T), 2.69 Mbp (HXWNR69T) and 2.65 Mbp (HXWNR70T) long with DNA G+C contents of 31.84 %, 32.83 % and 34.66 %, respectively. These genomes contained many genes responsible for carbohydrate degradation and antibiotic resistance. The major fatty acids (>5 %) included iso-C15 : 0, iso-C15 : 0 3-OH and iso-C17 : 0 3-OH. The major menaquinone was MK-6 for all the three strains. The average nucleotide identity (ANI; 72.7-88.5 %) and digital DNA-DNA hybridization (dDDH; 19.6-35.3 %) results further indicated that these three strains represented three novel species within the genus Flavobacterium, for which the names Flavobacterium odoriferum sp. nov. (type strain HXWNR29T = KCTC 92446T = CGMCC 1.61821T), Flavobacterium fragile sp. nov. (type strain HXWNR69T = KCTC 92468T = CGMCC 1.61442T) and Flavobacterium luminosum sp. nov. (type strain HXWNR70T = KCTC 92447T = CGMCC 1.61443T) are proposed.

Reduction of neutral lipid reservoirs, bioconversion and untargeted metabolomics reveal distinct roles for vitamin K isoforms on lipid metabolism.

Vitamin K isoforms are known as co-factors for the synthesis of blood-clotting proteins, but several other bioactivities were reported. In this work, we isolated a vitamin K1-analogue (OH-PhQ) from the cyanobacterium Tychonema sp. LEGE 07196 with lipid reducing activity. OH-PhQ reduced neutral lipid reservoirs with an EC50 value of 31 µM after 48 h exposure in zebrafish larvae, while other vitamin K isoforms had EC50 values of 21.1 µM (K2) and 1.2 µM (K3). No lipid reducing activity was observed for K1 up to 50 µM. The presence of vitamin K isoforms was studied in zebrafish after exposure (OH-PhQ, K1, K2 and K3), and a clear preference for bioconversion was observed to retain K1 and OH-PhQ. Untargeted metabolomics revealed different biological effects for vitamin K isoforms on the subclass and metabolite level, but similarities were present on the compound class level, particularly on the regulation of glycerophospholipids. Our data showed for the first time a lipid reducing activity of OH-PhQ and performed a comparative analysis of vitamin K isoforms, which could be important for the development of future nutraceuticals or food supplements.

Flavivirga spongiicola sp. nov. and Flavivirga abyssicola sp. nov., Isolated from Marine Environments.

Two novel Gram-stain-negative, strictly-aerobic, rod-shaped (1.2 ± 3.4 µm × 0.3 ± 0.7 µm), and non-motile marine bacterial species, designated MEBiC05379T and MEBiC07777T, were isolated from a marine sponge Pseudaxinella sp. in Gangneung City and deep-sea sediments of the Ulleung basin in the East Sea of Korea, respectively. The 16S rRNA gene sequence analysis revealed high levels of similarities between these strains and members of the genus Flavivirga (97.0-98.4% sequence identities). Both novel strains revealed as mesophilic, neutrophilic in pH and slightly halophilic. Similar to those of other Flavivirga members, the primary cellular fatty acids of both strains were iso-C15:0, iso-C15:1 G, iso-C15:03-OH, and iso-C17:0 3-OH, with MEBiC05379T and MEBiC07777T containing relatively higher proportions of C12:0 and summed feature 3 (C16:1ω7c and/or C16:1ω6c). In both taxa, the major isoprenoid quinone was MK-6. The DNA G + C contents of MEBiC05379T and MEBiC07777T genomes were 32.62 and 32.46 mol%, respectively. Compared to other members of Flavivirga, both strains exhibited similar DNA G + C ratio and fatty acids pattern, yet enzyme expression and carbon sources utilization pattern were different. Genomes of the genus Flavivirga showed enzyme preferences to fucoidan and sulfated galactans. Considering the monophyly rule, AAI values delineate the genus Flavivirga from adjacent genera calculated to be 76.0-78.7%. Based on the phenotypic, genomic and biochemical data, strains for MEBiC05379T and MEBiC07777T thus represent two novel species in the genus Flavivirga, for which the names Flavivirga spongiicola sp. nov. (MEBiC05379T [= KCTC 92527 T = JCM 16662 T]), and Flavivirga abyssicola sp. nov. (MEBiC07777T [= KCTC 92563 T = JCM 36477 T]) are proposed.

Speluncibacter jeojiensis gen. nov. sp. nov., a novel bacterium of the order Mycobacteriales isolated from a cave and a proposal of Speluncibacteraceae fam. nov.

Two Gram-stain-positive, aerobic, non-spore-forming, non-motile, irregular rod-shaped actinobacteria, designated as D2-41T and D3-21, were isolated from soil samples collected in a natural cave in Jeju, Republic of Korea. Both of the isolates were shown to share 100 % 16S rRNA sequence identity. The cell wall contained meso-diaminopimelic acid, arabinose and galactose. The predominant menaquinone was MK-8(H2). The polar lipids contained phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, an unidentified aminolipid, an unidentified aminoglycolipid, an unidentified phospholipid and two unidentified lipids. The predominant fatty acids were C16 : 0 and summed feature 3 (C16 : 1 ω7c and/or iso-C15 : 0 2-OH). Mycolic acids of C30-C38 were present. The 16S rRNA gene trees showed that the organisms occupied a distinct position remotely located from recognized genera within the order Mycobacteriales, albeit with the 16S rRNA gene similarities of 97.0-97.1 % with Rhodococcus olei, Rhodococcus rhodnii and Rhodococcus triatomae. The genome sizes and DNA G+C contents of strains D2-41T and D3-21 were 4.77-4.88 Mbp and 69.8 mol%, respectively. Both of the isolates shared an average nucleotide identity of 99.4 % and digital DNA-DNA hybridization of 95.2 % to each other, revealing that strains D2-41T and D3-21 belonged to the same species. In the core genome-based phylogenomic tree, both of the isolates were found to be closely associated with members of the genus Tomitella. However, strains D2-41T and D3-21 revealed the highest amino acid identity values (mean 66.5 %, range 66.2-67.0 % with the genus Prescottella of the family Nocardiaceae, followed by the genus Tomitella (mean 64.1 %, range 63.6-64.7 %) of the family Tomitellaceae. Based on the combined data obtained here, the novel isolates belong to a new genus of the new family for which the name Speluncibacter jeojiensis gen. nov. sp. nov. is proposed, with Speluncibacteraceae fam. nov. The type strain is strain D2-41T (=KACC 17930T=DSM 101875T).

Gut microbiome for predicting immune checkpoint blockade-associated adverse events.

BACKGROUND: The impact of the gut microbiome on the initiation and intensity of immune-related adverse events (irAEs) prompted by immune checkpoint inhibitors (ICIs) is widely acknowledged. Nevertheless, there is inconsistency in the gut microbial associations with irAEs reported across various studies. METHODS: We performed a comprehensive analysis leveraging a dataset that included published microbiome data (n = 317) and in-house generated data from 16S rRNA and shotgun metagenome samples of irAEs (n = 115). We utilized a machine learning-based approach, specifically the Random Forest (RF) algorithm, to construct a microbiome-based classifier capable of distinguishing between non-irAEs and irAEs. Additionally, we conducted a comprehensive analysis, integrating transcriptome and metagenome profiling, to explore potential underlying mechanisms. RESULTS: We identified specific microbial species capable of distinguishing between patients experiencing irAEs and non-irAEs. The RF classifier, developed using 14 microbial features, demonstrated robust discriminatory power between non-irAEs and irAEs (AUC = 0.88). Moreover, the predictive score from our classifier exhibited significant discriminative capability for identifying non-irAEs in two independent cohorts. Our functional analysis revealed that the altered microbiome in non-irAEs was characterized by an increased menaquinone biosynthesis, accompanied by elevated expression of rate-limiting enzymes menH and menC. Targeted metabolomics analysis further highlighted a notably higher abundance of menaquinone in the serum of patients who did not develop irAEs compared to the irAEs group. CONCLUSIONS: Our study underscores the potential of microbial biomarkers for predicting the onset of irAEs and highlights menaquinone, a metabolite derived from the microbiome community, as a possible selective therapeutic agent for modulating the occurrence of irAEs.

Role of connexin 32 in the directional differentiation of induced pluripotent stem cells into hepatocytes.

This study aimed to explore the role of connexin 32 (Cx32) in the directional differentiation of induced pluripotent stem cells (iPSCs) into hepatocytes. Urine-derived epithelial cells were collected from the fresh urine of a healthy donor and transducted with reprogramming plasmid mixture to generate iPSCs. The iPSCs were then directionally differentiated into hepatocytes. During the differentiation, the upregulated and downregulated groups were treated with vitamin K2 (VK2) and 2-aminoethoxyboronate diphenylester (2-APB) to increase and inhibit Cx32 expression, respectively. The control group was not treated with the regulatory factor. Expression of Cx32 and hepatocyte-specific markers, including AFP, hepatocyte nuclear factor 4α (HNF-4α), albumin (ALB) and cytokeratin 18 (CK18) were detected. It indicated that Cx32 expression was not observed in iPSCs, but gradually increased during the process of hepatic differentiation from iPSCs. Upregulation of Cx32 expression by VK2 treatment promoted hepatocyte maturation and enhanced the expression of the aforementioned hepatic specific markers, whereas downregulation of Cx32 expression by 2-APB treatment had the opposite effects. In conclusion, urine-derived iPSCs could be directionally differentiated into hepatocytes. Up-regulation of Cx32 improves the efficiency and maturity of differentiation of iPSCs into hepatocytes, and Cx32 may be a promoting factor during the process of hepatic differentiation from iPSCs.

Adaptive laboratory evolution for improved tolerance of vitamin K in Bacillus subtilis.

Menaquinone-7 (MK-7), a subtype of vitamin K2 (VK2), assumes crucial roles in coagulation function, calcium homeostasis, and respiratory chain transmission. The production of MK-7 via microbial fermentation boasts mild technological conditions and high biocompatibility. Nevertheless, the redox activity of MK-7 imposes constraints on its excessive accumulation in microorganisms. To address this predicament, an adaptive laboratory evolution (ALE) protocol was implemented in Bacillus subtilis BS011, utilizing vitamin K3 (VK3) as a structural analog of MK-7. The resulting strain, BS012, exhibited heightened tolerance to high VK3 concentrations and demonstrated substantial enhancements in biofilm formation and total antioxidant capacity (T-AOC) when compared to BS011. Furthermore, MK-7 production in BS012 exceeded that of BS011 by 76% and 22% under static and shaking cultivation conditions, respectively. The molecular basis underlying the superior performance of BS012 was elucidated through genome and transcriptome analyses, encompassing observations of alterations in cell morphology, variations in central carbon and nitrogen metabolism, spore formation, and antioxidant systems. In summation, ALE technology can notably enhance the tolerance of B. subtilis to VK and increase MK-7 production, thus offering a theoretical framework for the microbial fermentation production of other VK2 subtypes. Additionally, the evolved strain BS012 can be developed for integration into probiotic formulations within the food industry to maintain intestinal flora homeostasis, mitigate osteoporosis risk, and reduce the incidence of cardiovascular disease. KEY POINTS: • Bacillus subtilis was evolved for improved vitamin K tolerance and menaquinone-7 (MK-7) production • Evolved strains formed wrinkled biofilms and elongated almost twofold in length • Evolved strains induced sporulation to improve tolerance when carbon was limited.

Aquimarina aquimarini sp. nov. and Aquimarina spinulae sp. nov., novel bacterial species with versatile natural product biosynthesis potential isolated from marine sponges.

This study describes two Gram-negative, flexirubin-producing, biofilm-forming, motile-by-gliding and rod-shaped bacteria, isolated from the marine sponges Ircinia variabilis and Sarcotragus spinosulus collected off the coast of Algarve, Portugal. Both strains, designated Aq135T and Aq349T, were classified into the genus Aquimarina by means of 16S rRNA gene sequencing. We then performed phylogenetic, phylogenomic and biochemical analyses to determine whether these strains represent novel Aquimarina species. Whereas the closest 16S rRNA gene relatives to strain Aq135T were Aquimarina macrocephali JAMB N27T (97.8 %) and Aquimarina sediminis w01T (97.1 %), strain Aq349T was more closely related to Aquimarina megaterium XH134T (99.2 %) and Aquimarina atlantica 22II-S11-z7T (98.1 %). Both strains showed genome-wide average nucleotide identity scores below the species level cut-off (95 %) with all Aquimarina type strains with publicly available genomes, including their closest relatives. Digital DNA-DNA hybridization further suggested a novel species status for both strains since values lower than 70 % hybridization level with other Aquimarina type strains were obtained. Strains Aq135T and Aq349T grew from 4 to 30°C and with between 1-5 % (w/v) NaCl in marine broth. The most abundant fatty acids were iso-C17 : 03-OH and iso-C15 : 0 and the only respiratory quinone was MK-6. Strain Aq135T was catalase-positive and ß-galactosidase-negative, while Aq349T was catalase-negative and ß-galactosidase-positive. These strains hold unique sets of secondary metabolite biosynthetic gene clusters and are known to produce the peptide antibiotics aquimarins (Aq135T) and the trans-AT polyketide cuniculene (Aq349T), respectively. Based on the polyphasic approach employed in this study, we propose the novel species names Aquimarina aquimarini sp. nov. (type strain Aq135T=DSM 115833T=UCCCB 169T=ATCC TSD-360T) and Aquimarina spinulae sp. nov. (type strain Aq349T=DSM 115834T=UCCCB 170T=ATCC TSD-361T).

Menaquinone-4 attenuates ferroptosis by upregulating DHODH through activation of SIRT1 after subarachnoid hemorrhage.

BACKGROUND: Menaquinone-4(MK-4), the isoform of vitamin K2 in the brain, exerts neuroprotective effects against a variety of central nervous system disorders. This study aimed to demonstrate the anti-ferroptosis effects of MK-4 in neurons after SAH. METHODS: A subarachnoid hemorrhage (SAH) model was prepared by endovascular perforation in mice. In vitro hemoglobin stimulation of primary cortical neurons mimicked SAH. MK-4, Brequinar (BQR, DHODH inhibitor), and Selisistat (SEL, SIRT1 inhibitor) were administered, respectively. Subsequently, WB, immunofluorescence was used to determine protein expression and localization, and transmission electron microscopy was used to observe neuronal mitochondrial structure while other indicators of ferroptosis were measured. RESULTS: MK-4 treatment significantly upregulated the protein levels of DHODH; decreased GSH, PTGS2, NOX1, ROS, and restored mitochondrial membrane potential. Meanwhile, MK-4 upregulated the expression of SIRT1 and promoted its entry into the nucleus. BQR or SEL partially abolished the protective effect of MK-4 on, neurologic function, and ferroptosis. CONCLUSIONS: Taken together, our results suggest that MK-4 attenuates ferroptosis after SAH by upregulating DHODH through the activation of SIRT1.

Observation of the neuroprotective efficacy of vitamin K in a streptozocin-induced diabetes model in chick embryos.

Diabetes mellitus (DM) is a metabolic disease characterized by hyperglycemia due to insulin deficiency and/or resistance. Vitamin K (VK) is a group of fat-soluble molecules, including naturally occurring vitamin K1 (phylloquinone). vitamin K2 (menaquinone), and synthetic vitamin K3 (menadione). Beyond coagulation, the health benefits of VK have been described to play different roles in both physiological and pathological processes such as inflammation, energy metabolism, neuroprotection, cellular growth, and survival. It was aimed to observe the antioxidant and/or neuroprotective activity of vitamin K1 in our model of chick embryo diabetic neuropathy (DN) induced by streptozotocin (STZ). Ninety White Leghorn, fertile and 0-day-old SPF (specific pathogen-free) eggs (57 ± 4 gr) were used in the study. Chick embryo blood brain tissues were taken for biochemical evaluation. Plasma insulin and glucose levels were measured. In addition, brain tissue total antioxidant level (TAS), total oxidant level (TOS), malondialdehyde (MDA), and vascular endothelial growth factor (VEGF) levels were measured. Plasma glucose levels were higher in the STZ-treated groups and lower in the treatment groups. Plasma insulin levels were observed to be higher in STZ groups in groups treated with high VK. Low TAS, high MDA, TOS, and VEGF levels were recorded in brain tissue STZ groups. Low VEGF, TOS, and MDA levels were recorded in the group treated with the highest VK, while high TAS levels were observed. In our STZ-induced chick embryo diabetic neuropathy model, we observed that VK1 reduced oxidant damage by showing antioxidant properties or by modulating antioxidant enzymes.