Conductive single enzyme nanocomposites prepared by in-situ growth of nanoscale polyaniline for high performance enzymatic bioelectrode.

Enzyme-based electrochemical biosensors hold great promise for applications in health/disease monitoring, drug discovery, and environmental monitoring. However, inherently non-conductive nature of proteinaceous enzymes often hampers effective electron transfer at enzyme-electrode interface, limiting biosensor performance of enzyme bioelectrodes. To address this problem, we present an approach to synthesize polyaniline (PAN)-based conductive single enzyme nanocomposites of glucose oxidase (GOx) (denoted as PAN-GOx). To prevent multimerization of enzymes during nanocomposite synthesis and enable single enzyme wrapping, we activate GOx surface with phenylamine groups based on the programmed diffusion of reactants in the reaction solution. Subsequent in-situ polymerization enables the synthesis of nanoscale conductive PAN layer (∼2.7 nm thickness) grafted from individual GOx molecule. PAN-GOx retains 83% and 74% of its specific activity and catalytic efficiency, respectively, compared to free GOx, while demonstrating a ∼500% improved conductivity. Furthermore, PAN-GOx-based glucose biosensors show an approximately 16- and 3-fold higher sensitivity compared to biosensors prepared by using free GOx and a mixture of PAN and GOx, respectively. This study provides a facile method to fabricate conductive single enzyme nanocomposites with enhanced electron transfer, which can potentially be further modified and/or compounded with conductive materials for demonstrating high performance enzymatic bioelectrodes.

Does the femoral component design modified in consideration of Asian anatomical characteristics fit better than the conventional design for Korean femora in reality?

INTRODUCTION: We aimed to investigate the anatomical and clinical advantages of an Asian-specific femoral component design with a high femoral aspect ratio, compared with the conventional femoral component design. MATERIALS AND METHODS: A retrospective analysis of the operation and outpatient clinic records of 239 knees operated on using an anatomically modified femoral component design (MFCD, Group A) and 153 knees operated on using a conventional femoral component design (CFCD, Group B) in Korean patients was performed. Three subgroups were created based on the mediolateral size of the two different femoral component designs. The geometric accommodation of each femoral component was assessed using intraoperatively measured femoral posterior condylar resection and posterior condylar trimming amounts. Clinical outcomes were assessed using a range of motion (ROM) and patient-reported outcome measurements. RESULTS: In the comparison between Groups A and B, the mean combined bilateral posterior condylar trimming (XPCT) was 2.91 [2SD: - 4.12-9.94] and 1.45 [2SD: - 5.89-8.80], and the median XPCT was 3 and 1.5. In the largest subgroup (subgroup 2), Groups A and B included 100 and 112 patients, all six posterior condylar resection and trimming parameters were significantly larger in Group A. Preoperative and postoperative ROM and Hospital for Special Surgery scores were similar between the two groups. Preoperative Western Ontario and McMaster Universities Arthritis Index (WOMAC) was higher in Group A. However, postoperative WOMAC was similar between the groups. Perioperative improvement in WOMAC index was significantly greater in Group B. CONCLUSIONS: The Asian-specific femoral component design resulted in more resection and trimming of the femoral posterior condyle than the conventional design despite it was not associated with different clinical outcomes. Surgeons should be aware of unexpected excessive posterior condylar resection and formation of large flexion gap when using femoral component design with high femoral aspect ratio.

Sub-lethal effects of metals and pesticides on the freshwater dinoflagellate Palatinus apiculatus and environmental implications.

Microalgae are unicellular, photosynthetic organisms in aquatic environments and are sensitive to water quality and contaminants. While green algae and diatoms are widely used for toxicity assessments, there is a relatively limited amount of toxicity data available for freshwater dinoflagellates. Here, we evaluated the sub-lethal effects of the metals Cu, Cr, Ni, and Zn and the herbicides atrazine and S-metolachlor on the freshwater dinoflagellate Palatinus apiculatus. Based on the 72-h median effective concentration (EC50), P. apiculatus showed sensitive responses to metals in the order of Cu (0.052 mg L-1), Cr (0.085 mg L-1), Zn (0.098 mg L-1), and Ni (0.13 mg L-1). Among the tested herbicides, P. apiculatus was more sensitive to atrazine (0.0048 mg L-1) than S-metolachlor (0.062 mg L-1). In addition, we observed morphological alterations and significant increases in reactive oxygen species (ROS) production in cells exposed to 0.05 mg L-1 of Cu and 0.005 mg L-1 of atrazine. These indicated that metals and pesticides induced oxidative stress in cellular metabolic processes and consequently caused severe physiological damage to the cells. Our results provide baseline data on the toxic effects of typical environmental contaminants on freshwater dinoflagellate, suggesting that P. apiculatus could be used as a bioindicator in freshwater toxicity assessments. PRACTITIONER POINTS: The sub-lethal effects of metals and pesticides on the freshwater dinoflagellate Palatinus apiculatus were evaluated. Palatinus sensitively responded to metals and pesticides; of test chemicals, atrazine (0.0048 mg L-1 of EC50) was the most sensitive. Metals and pesticides induced oxidative stress and consequently caused severe physiological damage to the Palatinus cells. The freshwater dinoflagellate Palatinus can be used as a bioindicator in freshwater toxicity assessments.

Antimicrobial adhesive self-healing hydrogels for efficient dental biofilm removal from periodontal tissue.

OBJECTIVES: Oral biofilms, including pathogens such as Porphyromonas gingivalis, are involved in the initiation and progression of various periodontal diseases. However, the treatment of these diseases is hindered by the limited efficacy of many antimicrobial materials in removing biofilms under the harsh conditions of the oral cavity. Our objective is to develop a gel-type antimicrobial agent with optimal physicochemical properties, strong tissue adhesion, prolonged antimicrobial activity, and biocompatibility to serve as an adjunctive treatment for periodontal diseases. METHODS: Phenylboronic acid-conjugated alginate (Alg-PBA) was synthesized using a carbodiimide coupling agent. Alg-PBA was then combined with tannic acid (TA) to create an Alg-PBA/TA hydrogel. The composition of the hydrogel was optimized to enhance its mechanical strength and tissue adhesiveness. Additionally, the hydrogel's self-healing ability, erosion and release profile, biocompatibility, and antimicrobial activity against P. gingivalis were thoroughly characterized. RESULTS: The Alg-PBA/TA hydrogels, with a final concentration of 5 wt% TA, exhibited both mechanical properties comparable to conventional Minocycline gel and strong tissue adhesiveness. In contrast, the Minocycline gel demonstrated negligible tissue adhesion. The Alg-PBA/TA hydrogel also retained its rheological properties under repeated 5 kPa stress owing to its self-healing capability, whereas the Minocycline gel showed irreversible changes in rheology after just one stress cycle. Additionally, Alg-PBA/TA hydrogels displayed a sustained erosion and TA release profile with minimal impact on the surrounding pH. Additionally, the hydrogels exhibited potent antimicrobial activity against P. gingivalis, effectively eliminating its biofilm without compromising the viability of MG-63 cells. SIGNIFICANCE: The Alg-PBA/TA hydrogel demonstrates an optimal combination of mechanical strength, self-healing ability, tissue adhesiveness, excellent biocompatibility, and sustained antimicrobial activity against P. gingivalis. These attributes make it superior to conventional Minocycline gel. Thus, the Alg-PBA/TA hydrogel is a promising antiseptic candidate for adjunctive treatment of various periodontal diseases.

Environmental Factors Modulate Saxitoxins (STXs) Production in Toxic Dinoflagellate Alexandrium: An Updated Review of STXs and Synthesis Gene Aspects.

The marine dinoflagellate Alexandrium is known to form harmful algal blooms (HABs) and produces saxitoxin (STX) and its derivatives (STXs) that cause paralytic shellfish poisoning (PSP) in humans. Cell growth and cellular metabolism are affected by environmental conditions, including nutrients, temperature, light, and the salinity of aquatic systems. Abiotic factors not only engage in photosynthesis, but also modulate the production of toxic secondary metabolites, such as STXs, in dinoflagellates. STXs production is influenced by a variety of abiotic factors; however, the relationship between the regulation of these abiotic variables and STXs accumulation seems not to be consistent, and sometimes it is controversial. Few studies have suggested that abiotic factors may influence toxicity and STXs-biosynthesis gene (sxt) regulation in toxic Alexandrium, particularly in A. catenella, A. minutum, and A. pacificum. Hence, in this review, we focused on STXs production in toxic Alexandrium with respect to the major abiotic factors, such as temperature, salinity, nutrients, and light intensity. This review informs future research on more sxt genes involved in STXs production in relation to the abiotic factors in toxic dinoflagellates.

Adlercreutzia faecimuris sp. nov., producing propionate and acetate isolated from mouse feces.

A novel strictly anaerobic bacterium, strain JBNU-10 T, was isolated from BALB/c mouse feces. Cells of the strain JBNU-10 T were Gram-stain positive, non-motile and rod-shaped. Optimum growth occurred at 37℃, with 1% (w/v) NaCl and at pH 7. Phylogenetic analysis based on 16S rRNA gene sequence showed that strain JBNU-10 T belonged to the genus Adlercreutzia and were closely related to Adlercreutzia muris WCA-131-CoC-2 T (95.90%). The genome sequencing of strain JBNU-10 T revealed a genome size of 2,790,983 bp, a DNA G + C content of 69.4 mol%. It contains a total of 2,266 CDSs, 5 rRNA genes and 49 tRNA genes. According to the data obtained strain JBNU-10 T shared ANI value below 77.6- 67.7%, dDDH value below 23.8% with the closely type species. Strain JBNU-10 T possessed iso-C16:0 DMA, C18:1 CIS 9 FAME, and C18:0 DMA as the major fatty acids and had DMMK-6. The major end products of fermentation is propionate and acetate. Based on phylogenetic, physiological and chemotaxonomic characteristics, strain JBNU-10 T represent a novel species of the genus Adlercreutzia. The type strain is JBNU-10 T (= KCTC 25028 T = CCUG 75610 T).

Development of saxitoxin biosynthesis gene sxtB-targeted qPCR assay for the quantification of toxic dinoflagellates Alexandrium catenella (group I) and A. pacificum (group IV) occurring in the Korean coast.

Toxic dinoflagellate Alexandrium can produce saxitoxins (STXs) and cause paralytic shellfish poisoning (PSP), and thus they are monitored for environmental safety management. Microscopic discrimination of dinoflagellates is difficult to distinguish between toxic and non-toxic species due to their similar morphology. Meanwhile, an alternative quantitative PCR (qPCR) assay is sensitive, rapid, and cost-effective for harmful species monitoring. Herein, we developed a novel qPCR assay to detect the STXs biosynthesis gene sxtB of Alexandrium catenella and A. pacificum, the leading cause of PSP outbreaks in Asian coasts and worldwide. The newly designed sxtB TaqMan probes target the species without any positive signal in other relative dinoflagellates. Deming regression analysis revealed that the sxtB copy number of A. catenella and A. pacificum was 3.6 and 4.1 copies per cell, respectively. During the blooming periods (April 13th-14th, 2020), only A. catenella cells were detected through the qPCR assay, ranging from 5.0 × 10 to 2.5 × 104 eq cells L-1. In addition, sxtB qPCR quantified more accurately compared to large subunit (LSU) rRNA targeting qPCR assay that overestimate cell density. Besides, the sensitivity of sxtB was higher compared to the microscope when the species were rarely present (5.0 × 102 cells L-1). These suggest that the sxtB qPCR assay can be applied to toxic Alexandrium monitoring in the Korean coast, even in the early stage of bloomings.

Polyphyletic origin of saxitoxin biosynthesis genes in the marine dinoflagellate Alexandrium revealed by comparative transcriptomics.

The marine dinoflagellate Alexandrium is known to form harmful algal blooms, and at least 14 species within the genus can produce saxitoxins (STXs). STX biosynthesis genes (sxt) are individually revealed in toxic dinoflagellates; however, the evolutionary history remains controversial. Herein, we determined the transcriptome sequences of toxic Alexandrium (A. catenella and A. pacificum) and non-toxic Alexandrium (A. fraterculus and A. fragae) and characterized their sxt by focusing on evolutionary events and STX production. Comparative transcriptome analysis revealed higher homology of the sxt in toxic Alexandrium than in non-toxic species. Notably, non-toxic Alexandrium spp. were found to have lost two sxt core genes, namely sxtA4 and sxtG. Expression levels of 28 transcripts related to eight sxt core genes showed that sxtA, sxtG, and sxtI were relatively high (>1.5) in the toxic group compared to the non-toxic group. In contrast, the non-toxic group showed high expression levels in sxtU (1.9) and sxtD (1.7). Phylogenetic tree comparisons revealed distinct evolutionary patterns between 28S rDNA and sxtA, sxtB, sxtI, sxtD, and sxtU. However, similar topology was observed between 28S rDNA, sxtS, and sxtH/T. In the sxtB and sxtI phylogeny trees, toxic Alexandrium and cyanobacteria were clustered together, separating from non-toxic species. These suggest that Alexandrium may acquire sxt genes independently via horizontal gene transfer from toxic cyanobacteria and other multiple sources, demonstrating monocistronic transcripts of sxt in dinoflagellates.

Biocomposite thermoplastic polyurethanes containing evolved bacterial spores as living fillers to facilitate polymer disintegration.

The field of hybrid engineered living materials seeks to pair living organisms with synthetic materials to generate biocomposite materials with augmented function since living systems can provide highly-programmable and complex behavior. Engineered living materials have typically been fabricated using techniques in benign aqueous environments, limiting their application. In this work, biocomposite fabrication is demonstrated in which spores from polymer-degrading bacteria are incorporated into a thermoplastic polyurethane using high-temperature melt extrusion. Bacteria are engineered using adaptive laboratory evolution to improve their heat tolerance to ensure nearly complete cell survivability during manufacturing at 135 °C. Furthermore, the overall tensile properties of spore-filled thermoplastic polyurethanes are substantially improved, resulting in a significant improvement in toughness. The biocomposites facilitate disintegration in compost in the absence of a microbe-rich environment. Finally, embedded spores demonstrate a rationally programmed function, expressing green fluorescent protein. This research provides a scalable method to fabricate advanced biocomposite materials in industrially-compatible processes.

Phocaeicola acetigenes sp. nov., producing acetic acid and iso-butyric acid, isolated faeces from a healthy human.

An obligately anaerobic, non-motile, Gram-stain-negative, and rod-shaped strain KGMB11183T was isolated from the feces of healthy Koreans. The growth of strain KGMB11183T occurred at 30-45 °C (optimum 37 °C), at pH 6-9 (optimum pH 7), and in the presence of 0-0.5% NaCl (optimum 0%). Strain KGMB11183T showed 16S rRNA gene sequence similarities of 95.4% and 94.2% to the closest recognized species, Phocaeicola plebeius M12T, and Phocaeicola faecicola AGMB03916T. Phylogenetic analysis showed that strain KGMB11183T is a member of the genus Phocaeiocla. The major end products of fermentation are acetic acid and isobutyric acid. The major cellular fatty acids (> 10%) of this isolate were C18:1 cis 9, anteiso-C15:0, and summed feature 11 (iso-C17:0 3-OH and/or C18:2 DMA). The assembled draft genome sequences of strain KGMB11183T consisted of 3,215,271 bp with a DNA G + C content of 41.4%. According to genomic analysis, strain KGMB11183T has a number of genes that produce acetic acid. The genome of strain KGMB11183T encoded the starch utilization system (Sus) operon, SusCDEF suggesting that strain uses many complex polysaccharides that cannot be digested by humans. Based on the physiological, chemotaxonomic, phenotypic, and phylogenetic data, strain KGMB11183T is regarded a novel species of the genus Phocaeicola. The type strain is KGMB11183T (= KCTC 25284T = JCM 35696T).

Gordonibacter faecis sp. nov., producing urolithin C from ellagic acid, isolated from feces of healthy Korean subjects.

A Gram-stain-positive, anaerobic, motile, and short rod-shaped bacterium, designated KGMB12511T, was isolated from the feces of healthy Koreansubjects. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain KGMB12511T was closely related to Gordonibacter pamelaeae 7-10-1-bT (95.2%). The draft genome of KGMB12511T comprised 33 contigs and 2,744 protein-coding genes. The DNA G + C content was 59.9% based on whole-genome sequences. The major cellular fatty acids (>10%) of strain KGMB12511T were C18:1 cis9, C18:1 cis9 DMA (dimethylacetal), and C16:0 DMA. The predominant polar lipids included a diphosphatydilglycerol, four glycolipids, and an unidentified phospholipid. The major respiratory quinones were menaquinone 6 (MK-6) and monomethylmenaquinone 6 (MMK-6). Furthermore, HPLC analysis demonstrated the ability of strain KGMB12511T to convert ellagic acid into urolithin. Based on a comprehensive analysis of phenotypic, chemotaxonomic, and phylogenetic data, strain KGMB12511T represents a novel species in the genus Gordonibacter. The type strain is KGMB12511T (= KCTC 25343T = NBRC 116190T).

Nuclear genome of dinoflagellates: Size variation and insights into evolutionary mechanisms.

Recent progress in high-throughput sequencing technologies has dramatically increased availability of genome data for prokaryotes and eukaryotes. Dinoflagellates have distinct chromosomes and a huge genome size, which make their genomic analysis complicated. Here, we reviewed the nuclear genomes of core dinoflagellates, focusing on the genome and cell size. Till now, the genome sizes of several dinoflagellates (more than 25) have been measured by certain methods (e.g., flow cytometry), showing a range of 3-250 pg of genomic DNA per cell. In contrast to their relatively small cell size, their genomes are huge (about 1-80 times the human haploid genome). In the present study, we collected the genome and cell size data of dinoflagellates and compared their relationships. We found that dinoflagellate genome size exhibits a positive correlation with cell size. On the other hand, we recognized that the genome size is not correlated with phylogenetic relatedness. These may be caused by genome duplication, increased gene copy number, repetitive non-coding DNA, transposon expansion, horizontal gene transfer, organelle-to-nucleus gene transfer, and/or mRNA reintegration into the genome. Ultimate verification of these factors as potential causative mechanisms would require sequencing of more dinoflagellate genomes in the future.

Jatrophihabitans cynanchi sp. nov., isolated from rhizosphere soil of Cynanchum wilfordii.

A novel actinobacterial strain, SB3-54T was isolated from rhizosphere soil of Cynanchum wilfodill, Jaecheon, Chungcheongbuk-do, Republic of Korea. Cells of strain SB3-54T were Gram-stain-positive, aerobic, rod-shaped, and flagellated which formed pale yellow colonies on Reasoner's 2A (R2A) agar. Growth occurred at 15-30 °C (optimum 25 °C), pH 5-8 (optimum pH 7), and 0-2.5% NaCl (optimum 0%). Phylogenetic and phylogenomic analyses showed that strain SB3-54T formed a separate lineage in the genus Jatrophihabitans with Jatrophihabitans telluris N237T. Strain SB3-54T was positive for catalase activity. Genomic analysis showed that SB3-54T has plant-beneficial function contributing (referred to as PBFC) genes such as root colonization and plant protection from oxidative stress. Furthermore, genome of SB3-54T contained gene clusters related to cytokinin biosynthesis, auxin response, tryptophan biosynthesis, siderophore biosynthesis and bacterial toxin-antitoxin systems. Strain SB3-54T contained iso-C16:0 as the major fatty acid and MK-9(H4) and MK-9(H6) as the predominant quinones. The organism had meso-diaminopimelic acid as the diagnostic diamino acid in the peptidoglycan. The major polar lipids were diphosphatidylglycerol, phosphatidylinositol polymannosides, two unidentified aminoglycophospholipids and three unidentified phospholipids. Based on phylogenetic, physiological and chemotaxonomic characteristics, strain SB3-54T represents a novel species of the genus Jatrophihabitans. The type strain is SB3-54T (= KCTC 49134T = NBRC 114108T).

A novel plant growth-promoting rhizobacterium, Rhizosphaericola mali gen. nov., sp. nov., isolated from healthy apple tree soil.

The rhizosphere microbial community is closely associated with plant disease by regulating plant growth, agricultural production, nutrient availability, plant hormone and adaptation to environmental changes. Therefore, it is very important to identify the rhizosphere microbes around plant roots and understand their functions. While studying the differences between the rhizosphere microbiota of healthy and diseased apple trees to find the cause of apple tree disease, we isolated a novel strain, designated as B3-10T, from the rhizosphere soil of a healthy apple tree. The genome relatedness indices between strain B3-10T and other type species of family Chitinophagaceae were in the ranges of 62.4-67.0% for ANI, 18.6-32.1% for dDDH, and 39.0-56.6% for AAI, which were significantly below the cut­off values for the species delineation, indicating that strain B3-10T could be considered to represent a novel genus in family Chitinophagaceae. Interestingly, the complete genome of strain B3-10T contained a number of genes encoding ACC-deaminase, siderophore production, and acetoin production contributing to plant-beneficial functions. Furthermore, strain B3-10T was found to significantly promote the growth of shoots and roots of the Nicotiana benthamiana, which is widely used as a good model for plant biology, demonstrating that strain B3-10T, a rhizosphere microbe of healthy apple trees, has the potential to promote growth and reduce disease. The phenotypic, chemotaxonomic, phylogenetic, genomic, and physiological properties of this plant growth-promoting (rhizo)bacterium, strain B3-10T supported the proposal of a novel genus in the family Chitinophagaceae, for which the name Rhizosphaericola mali gen. nov., sp. nov. (= KCTC 72123T = NBRC 114178T).

Two novel glutathione S-transferase (GST) genes in the toxic marine dinoflagellate Alexandrium pacificum and their transcriptional responses to environmental contaminants.

The present study identified two novel glutathione S-transferase (GST) genes from the toxic dinoflagellate Alexandrium pacificum and examined their molecular characteristics and transcriptional responses to algicides and environmental contaminants. Bioinformatic analysis revealed that both ApGSTs are cytosolic, belonging to the chi-like class (ApGST1) and an undefined class (ApGST2). The overall expression of ApGSTs showed similar patterns depending on the exposed contaminants, while they were differently regulated by polychlorinated biphenyl (PCB). Copper treatments (CuCl2 and CuSO4) did not significantly induce the expression of ApGSTs. The highest up-regulations of ApGST1 and ApGST2 were under 6-h treatments of 0.10 and 0.50 mg L-1 NaOCl. Interestingly, only ApGST1 increased significantly after 0.10, 0.50, and 1.00 mg L-1 of PCB exposure (6 h). Intracellular reactive oxygen species (ROS) increased considerably under NaOCl; however, it was not significantly higher in the PCB-treated cells. GST activity was increased by NaOCl and PCB treatments, but only PCB caused apoptosis. These results suggest that GSTs are involved in the first line of phase II detoxification, protecting dinoflagellate cells against oxidative damage.

Pseudomonas aestuarii sp. nov., isolated from tidal flat sediment.

Two novel Pseudomonas strains, SA3-5T and SA3-6, were isolated from a tidal flat (getbol) in the Republic of Korea. Strains SA3-5T and SA3-6 were subjected to polyphasic characterization to determine their taxonomic affiliations. Cells were Gram-stain-negative, aerobic, rod-shaped and motile by using peritrichous flagella. Based on their 16S rRNA gene sequences, strains SA3-5T and SA3-6 exhibited a high degree of similarity (100 %) and were classified within the genus Pseudomonas. Furthermore, the closest related species to SA3-5T and SA3-6 were Pseudomonas taeanensis MS-3T (98.3 %). The ranges of average nucleotide identity and digital DNA-DNA hybridization values between SA3-5T and closely related species were 75.9-89.1% and 21.3-38.7%, respectively, both of which being below the thresholds for delineating novel strains. Strain SA3-5T and SA3-6 contained C16 : 1 ω6с and/or C16 : 1 ω7с (summed feature 3), C16 : 0 and C18 : 1 ω6с and/or C18 : 1 ω7с (summed feature 8) as the major fatty acids. The predominant respiratory quinone was Q-9. The DNA G+C content of strain SA3-5T was 62.5 mol%. Based on their combined phenotypic, chemotaxonomic and phylogenetic characterisitics, strains SA3-5T and SA3-6 represent a novel species of the genus Pseudomonas for which the name Pseudomonas aestuarii sp. nov. is proposed. The type strain is SA3-5T (=KCTC 92395T=JCM 35697T).

Alkalimarinus alittae sp. nov., isolated from gut of marine sandworm (Alitta virens) and emended description of the genus Alkalimarinus.

A novel, Gram-stain-negative, aerobic, motile, catalase- and oxidase-negative bacterial strain, designated A2M4T, was isolated from the gut contents of a marine sandworm Alitta virens, collected from the eastern coast of the Republic of Korea. Strain A2M4T formed translucent circular colonies and showed rod-shaped cells with peritrichous flagella. Optimal growth of strain A2M4T occurred at 25 °C, pH 7.0 and in the presence of 2 % (w/v) NaCl. Phylogenetic analysis based on the 16S rRNA gene sequences showed that strain A2M4T was closely related to Alkalimarinus sediminis FA028T, with the highest sequence similarity of 98.9 %. The complete genome sequence of strain A2M4T was 4.25 Mbp in size and the genomic G+C content, calculated from the genome sequence, was 43.2 mol%. A comparison between the genome sequence of strain A2M4T and that of its closest relative, A. sediminis FA028T, showed an average nucleotide identity value of 76.63 % and a digital DNA-DNA hybridization value of 22.2 %. Strain A2M4T contained Q-9 as the sole respiratory isoprenoid quinone and the major polar lipids were phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine. The major cellular fatty acids of strain A2M4T were C14 : 0, C16 : 0 and summed feature 3 (comprising C16 : 1 ω7c and/or C16 : 1 ω6c). Based on its phenotypic, chemotaxonomic and genomic characteristics, strain A2M4T represents a novel species of the genus Alkalimarinus, for which the name Alkalimarinus alittae sp. nov. is proposed. The type is strain A2M4T (=KCTC 92030T=JCM 35924T). The description of the genus Alkalimarinus has also been emended.

Effects of nitrate on the saxitoxins biosynthesis revealed by sxt genes in the toxic dinoflagellate Alexandrium pacificum (group IV).

The dinoflagellate Alexandrium pacificum (group IV) is of particular interest because of its involvement in harmful algal blooms and production of saxitoxin (STX), which causes paralytic shellfish poisoning. The toxicity from STX and its analogues (STXs) is suspected to be affected by nitrogen (N) availability. However, the toxicity-associated behavior and STX-biosynthesis gene responses of the toxic A. pacificum under N fluctuations have not been sufficiently investigated. In the present study, we identified the sxtI gene involved in sxt biosynthesis pathway and evaluated the effects of nitrate (NO3-) on STXs production and the expression of four sxt core genes (sxtA4, sxtG, sxtB, and sxtI). Quantification of total STXs levels in the cultures under different NO3- regimes showed that NO3- concentration influenced STXs production. In addition, the proportion and concentration of STXs varied depending on the NO3- concentration. Core sxt transcript abundance was also influenced by available NO3- in a time-dependent manner. Expressional levels and patterns of sxtI were correlated with those of sxtA and sxtB. The relationship between the toxins and sxt responses in A. pacificum under various NO3- regimes suggests the direct involvement of N in the STXs biosynthesis pathway. Understanding this link would provide a tool to understand the toxin dynamics of dinoflagellates following N shifts in marine environments.

Chelerythrine, a novel small molecule targeting IL-2, inhibits melanoma progression by blocking the interaction between IL-2 and its receptor.

AIMS: In this study, we investigated the inhibition of IL-2 activity and anticancer efficacy of chelerythrine (CHE), a natural small molecule that targets IL-2 and inhibits CD25 binding, and elucidated the mechanism underlying the action of CHE on immune cells. MAIN METHODS: CHE was discovered by competitive binding ELISA and SPR analysis. The effect of CHE on IL-2 activity was evaluated in CTLL-2, HEK-Blue reporter and immune cells, and in ex vivo generation of regulatory T cells (Treg cells). The antitumor activity of CHE was evaluated in B16F10 tumor-bearing C57BL/6 or BALB/c nude mice. KEY FINDINGS: We identified that CHE, which acts as an IL-2 inhibitor, selectively inhibits the interaction between IL-2 and IL-2Rα and directly binds to IL-2. CHE inhibited the proliferation and signaling of CTLL-2 cells and suppressed IL-2 activity in HEK-Blue reporter and immune cells. CHE prevented the conversion of naive CD4+ T cells into CD4+CD25+Foxp3+ Treg cells in response to IL-2. CHE reduced tumor growth in C57BL/6 mice but not in T-cell-deficient mice, upregulated the expression of IFN-γ and cytotoxic molecules, and limited Foxp3 expression. Furthermore, the combination of CHE and a PD-1 inhibitor synergistically increased antitumor activity in melanoma-bearing mice and almost completely regressed the implanted tumors. SIGNIFICANCE: We found that CHE, which targets IL-2 and inhibits its binding to CD25, exhibits T cell-mediated antitumor activity and that combination therapy with CHE and PD-1 inhibitor induced synergistic antitumor effects, suggesting that CHE may be a promising anticancer agent for melanoma monotherapy and combination therapy.

Saxitoxins-producing potential of the marine dinoflagellate Alexandrium affine and its environmental implications revealed by toxins and transcriptome profiling.

The marine dinoflagellate Alexandrium occurs widely in coastal waters, and some of them can produce saxitoxins (STXs) that cause paralytic shellfish poisoning (PSP). Alexandrium affine is a harmful algal bloom (HAB)-forming species off the coast of Asia; however, its ability to produce STXs has been controversial. In the present study, we detected STXs in A. affine Alex02 isolated from the southern coast of Korea. The total STXs equivalent (STXs eq) and profiles of Alex02 varied depending on the tested environmental conditions, including the temperature and nitrate concentrations. STXs toxicity levels of A. affine Alex02 (<0.8 STXs eq fmol cell-1) were significantly lower than those of toxic A. catenella Alex03 and A. pacificum Alex05. On a genetic basis, we identified all the STX biosynthesis sxt genes, except sxtX in A. affine, via large-scale transcriptome analysis. Interestingly, the two proteins, sxtA4 and sxtG, were similar in sequence and domain structure to those of other toxic dinoflagellates and cyanobacteria; however, their transcript levels were extremely low. Our results suggest that A. affine has the potential to produce STXs, while its toxicity is much lower or negligible, which is unlikely to cause PSP incidents in marine environments.