Lactobacillus reuteri AN417 cell-free culture supernatant as a novel antibacterial agent targeting oral pathogenic bacteria

Lactobacillus reuteri AN417 is a newly characterized probiotic strain. The activity of AN417 against oral pathogenic bacteria is unknown. We investigated the antibacterial activity of cell-free L. reuteri AN417 culture supernatant (LRS) against three oral pathogens: Porphyromonas gingivalis, Fusobacterium nucleatum, and Streptococcus mutans. P. gingivalis and F. nucleatum have been implicated in periodontal disease, whereas S. mutans causes dental caries. Exposing these oral pathogenic bacteria to LRS significantly reduced their growth rates, intracellular ATP levels, cell viability, and time-to-kill. The minimal inhibitory volume of LRS was 10% (v/v) against P. gingivalis, 20% (v/v) for F. nucleatum, and 30% (v/v) for S. mutans. LRS significantly reduced the integrity of biofilms and significantly suppressed the expression of various genes involved in P. gingivalis biofilm formation. The L. reuteri AN417 genome lacked genes encoding reuterin, reuteran, and reutericyclin, which are major antibacterial compounds produced in L. reuteri strains. LRS treated with lipase and α-amylase displayed decreased antibacterial activity against oral pathogens. These data suggest that the antibacterial substances in LRS are carbohydrates and/or fatty acid metabolites. Our results demonstrate that LRS has antimicrobial activity against dental pathogenic bacteria, highlighting its potential utility for the prevention and treatment of P. gingivalis periodontal disease.

Heat stress impairs oocyte maturation through ceramide-mediated apoptosis in pigs

Heat stress (HS) is an emerging issue that greatly impairs the reproductive performance of animals and humans. In particular, disruption of oocyte maturation due to HS is considered a major cause of impaired reproductive performance. HS is known to induce ceramide generation, which causes reactive oxygen species (ROS) production and mitochondrial dysfunction, thereby inducing apoptosis. Therefore, we investigated whether inhibition of ceramide generation ameliorates HS-induced apoptosis in porcine cumulus？oocyte complexes (COCs) using specific inhibitors of the de novo (fumonisin B1, FB1) and hydrolytic pathways (desipramine, Des) of ceramide formation. We investigated the effects of FB1 and Des supplementation under HS conditions (41.5 °C for 44 h) on in vitro maturation (IVM) of porcine COCs. After IVM, HS significantly reduced proportion of COCs exhibiting fully expanded cumulus cells and the rate of metaphase II in oocytes. After parthenogenetic activation (PA), HS significantly reduced the rates of cleavage and blastocyst formation with a lower total cell number and a higher percentage of apoptosis in blastocysts. However, FB1 or Des supplementation under HS avoided detrimental effects of HS on expansion of cumulus cells, nuclear maturation of oocytes, and embryonic development after PA including the rates of cleavage and blastocyst formation, total cell number, and the percentage of apoptosis in blastocysts. Furthermore, FB1 or Des addition under HS, compared with HS alone, significantly decreased ceramide generation, ROS production, cytochrome C expression, and apoptosis and increased mitochondrial membrane potential in COCs, reaching levels comparable with those of the control. Taken together, our results indicate that HS impaired oocyte maturation through ceramide-mediated apoptosis.

Mucilaginibacter mali sp. nov., isolated from rhizosphere soil of apple orchard

A novel Gram-negative bacterium, designated G2-14T, was isolated from rhizosphere soil sample collected from apple orchard in Chungju-si, Chungcheongbuk-do, Republic of Korea. Strain G2-14T was a strictly aerobic, non-spore-forming, non-motile and short-rod-shaped bacterium. Phylogenetic analysis based on 16S rRNA gene sequence showed that strain G2-14T was closely related to Mucilaginibacter myungsuensis HMD1056T (96.9？%) and Mucilaginibacter boryungensis BDR-9T (96.8？%). The major cellular fatty acids (>10？%) of strain G2-14T were summed feature 3 (C16:1ω6с and/or C16:1ω7с) and iso-C15:0. The predominant quinone and the major polar lipid were menaquinone-7 and phosphatidylethanolamine, respectively. Strain G2-14T produced acetic acid. The DNA G+C content based on whole genome sequences was 46.4 mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain G2-14T represents a novel species in the genus Mucilaginibacter, for which the name Mucilaginibacter mali sp. nov. is proposed. The type strain is G2-14T (=KCTC 72533T=NBRC 114179T).

Transcriptomic analysis of Chlorella sp. HS2 suggests the overflow of acetyl？CoA and NADPH cofactor induces high lipid accumulation and halotolerance

Previously, we isolated Chlorella sp. HS2 (referred hereupon as HS2) from a local tidal rock pool and demonstrated its halotolerance and high biomass productivity under different salinity conditions. To further understand acclimation responses of this alga under high salinity stress, we performed transcriptome analysis of triplicated culture samples grown in freshwater and marine conditions at both exponential and stationary growth phases. The results indicated that the transcripts involved in photosynthesis, TCA, and Calvin cycles were downregulated, whereas the upregulation of DNA repair mechanisms and an ABCB subfamily of eukaryotic type ABC transporter was observed at high salinity condition. In addition, while key enzymes associated with glycolysis pathway and triacylglycerol (TAG) synthesis were determined to be upregulated from early growth phase, salinity stress seemed to reduce the carbohydrate content of harvested biomass from 45.6 dw% to 14.7 dw% and nearly triple the total lipid content from 26.0 dw% to 62.0 dw%. These results suggest that the reallocation of storage carbon toward lipids played a significant role in conferring the viability of this alga under high salinity stress by remediating high level of cellular stress partially resulted from ROS generated in oxygen？evolving thylakoids as observed in a direct measure of photosystem activities.

WHAMM is essential for spindle formation and spindle actin polymerization in maturing mouse oocytes

WHAMM (WAS Protein Homolog Associated with Actin, Golgi Membranes, and Microtubules) is involved in Golgi membrane association, microtubule binding, and actin nucleation as a nucleation-promoting factor, which activates the actin-related protein 2/3 complex (the Arp2/3 complex). However, the role of WHAMM in mammalian oocyte maturation is poorly understood. The presence of WHAMM mRNA and protein during all stages of mouse oocyte maturation has been verified. It is mainly co-localized with the actin cage permeating the spindle during mouse oocyte maturation. Through the knockdown of WHAMM, we confirmed that it regulates spindle formation and affects the localization of the microtubule-organizing center (MTOC) during the early stages of spindle formation. Moreover, depletion of WHAMM impaired the formation of the spindle actin and chromosome alignment, which might be the cause of chromosomal aneuploidy and abnormal, asymmetric division. Treatment with brefeldin A (BFA), an inhibitor of vesicle transport from the endoplasmic reticulum (ER) to the Golgi apparatus, induced abnormal and dispersed localization of WHAMM. Taken together, these findings show that WHAMM is an essential component of the actin cytoskeleton machinery and plays a crucial role in oocyte maturation, presumably by controlling the formation of spindles with normal length by activating the formation of the spindle actin via the Arp2/3 complex.

Arenibacter arenosicollis sp. nov., isolated from a sand dune

A Gram-stain-negative, aerobic, non-spore-forming, non-motile and rod-shaped bacterial strain, designated BSSL-BM3T, was isolated from sand collected from a dune near the Yellow Sea, Republic of Korea, and subjected to a polyphasic taxonomic study. The neighbour-joining phylogenetic tree of 16S rRNA gene sequences showed that strain BSSL-BM3T fell within the clade comprising the type strains of Arenibacter species. Strain BSSL-BM3T exhibited 16S rRNA gene sequence similarity values of 98.0？99.0？% to the type strains of Arenibacter catalasegens , Arenibacter hampyeongensis , Arenibacter echinorum , Arenibacter palladensis and Arenibacter troitsensis and of 94.2？96.7？% to the type strains of the other Arenibacter species. The averagenucleotide identity and digitalDNA？DNA hybridization values between strain BSSL-BM3T and the type strains of A. catalasegens , A. hampyeongensis , A. echinorum , A. palladensis and A. troitsensis were 82.2？88.8？%？and 25.0？36.5？%, respectively. The DNA G+C content of strain BSSL-BM3T from genomic sequence data was 38.75？mol%. Strain BSSL-BM3T contained MK-6 as the predominant menaquinone and iso-C17？:？0 3-OH, summed feature 3 (C16？:？1 ？ω7c and/or C16？:？1 ？ω6c) and iso-C15？:？1 G as the major fatty acids. The major polar lipids of strain BSSL-BM3T were phosphatidylethanolamine and two unidentified lipids. Distinguishing phenotypic properties, along with the phylogenetic and genetic distinctiveness, revealed that strain BSSL-BM3T is separated from recognized Arenibacter species. On the basis of the data presented here, strain BSSL-BM3T is considered to represent a novel species of the genus Arenibacter , for which the name Arenibacter arenosicollis sp. nov. is proposed. The type strain is BSSL-BM3T (=KACC 21632T=NBRC 114502T).

Neobacillus endophyticus sp. nov., an endophytic bacterium isolated from Selaginella involvens roots

A Gram-stain-positive, facultatively anaerobic, rod-shaped, endospore-forming, oxidase-positive, and catalase-negative strain designated as BRMEA1T was isolated from the surface-sterilized Selaginella involvens roots. Growth of strain BRMEA1T was found to occur at pH 6.0？8.0 (optimum, pH 7.0), 15？50？°C (optimum, 25？30？°C) and in the absence of NaCl. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain BRMEA1T formed a lineage within the genus Neobacillus (family Bacillaceae ) and showed the highest sequence similarity to Neobacillus drentensis DSM 15600T (98.3？%) and Neobacillus fumarioli KCTC 13885T (98.2？%), and less than 98.2？% 16S rRNA gene sequence similarity to the other members of the genus Neobacillus . Whole-genome analysis of strain BRMEA1T comprised a circular chromosome (5？632？809？bp in size) with 38.5？mol% G+C content. Digital DNA？DNA hybridization analyses revealed that strain BRMEA1T showed 20.5 and 22.0% genomic DNA relatedness with the closest species, N. drentensis DSM 15600T and N. fumarioli KCTC 13885T, respectively. The whole-genome sequence of strain BRMEA1T showed the presence of 11 specific conserved signature indels for the genus Neobacillus . The major cellular fatty acids (>10？%) of strain BRMEA1T were found to be iso-C15？:？0 and anteiso-C15？:？0, while the major polar lipids were found to be diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. Polyphasic analysis results revealed that BRMEA1T represents a novel species of the genus Neobacillus , with the proposed name Neobacillus endophyticus sp. nov. The type strain is BRMEA1T (=KCTC 43208T=CCTCC AB 2020071T).

Deubiquitinase OTUD5 is a positive regulator of mTORC1 and mTORC2 signaling pathways

The mammalian Target of Rapamycin (mTOR) pathway regulates a variety of physiological processes, including cell growth and cancer progression. The regulatory mechanisms of these signals are extremely complex and comprise many feedback loops. Here, we identified the deubiquitinating enzyme ovarian tumor domain-containing protein 5 (OTUD5) as a novel positive regulator of the mTOR complex (mTORC) 1 and 2 signaling pathways. We demonstrated that OTUD5 stabilized β-transducin repeat-containing protein 1 (βTrCP1) proteins via its deubiquitinase (DUB) activity, leading to the degradation of Disheveled, Egl-10, and pleckstrin domain-containing mTOR-interacting protein (DEPTOR), which is an inhibitory protein of mTORC1 and 2. We also showed that mTOR directly phosphorylated OTUD5 and activated its DUB activity. RNA sequencing analysis revealed that OTUD5 regulates the downstream gene expression of mTOR. Additionally, OTUD5 depletion elicited several mTOR-related phenotypes such as decreased cell size and increased autophagy in mammalian cells as well as the suppression of a dRheb-induced curled wing phenotype by RNA interference of Duba, a fly ortholog of OTUD5, in Drosophila melanogaster. Furthermore, OTUD5 knockdown inhibited the proliferation of the cancer cell lines with mutations activating mTOR pathway. Our results suggested a positive feedback loop between OTUD5 and mTOR signaling pathway.

Process optimization for mass production of 2,3-butanediol by Bacillus subtilis CS13

Background: Bacillus subtilis CS13 was previously isolated for 2,3-butanediol (2,3-BD) and poly-γ-glutamic acid (γ-PGA) co-production. When culturing this strain without L-glutamic acid in the medium, 2,3-BD is the main metabolic product. 2,3-BD is an important substance and fuel with applications in the chemical, food, and pharmaceutical industries. However, the yield and productivity for the B. subtilis strain should be improved for more efficient production of 2,3-BD. Results: The medium composition, which contained 281.1 g/L sucrose, 21.9 g/L ammonium citrate, and 3.6 g/L MgSO4·7H2O, was optimized by response surface methodology for 2,3-BD production using B. subtilis CS13. The maximum amount of 2,3-BD (125.5？±？3.1 g/L) was obtained from the optimized medium after 96 h. The highest concentration and productivity of 2,3-BD were achieved simultaneously at an agitation speed of 500 rpm and aeration rate of 2 L/min in the batch cultures. A total of 132.4？±？4.4 g/L 2,3-BD was obtained with a productivity of 2.45？±？0.08 g/L/h and yield of 0.45 g2,3-BD/gsucrose by fed-batch fermentation. The meso-2,3-BD/2,3-BD ratio of the 2,3-BD produced by B. subtilis CS13 was 92.1%. Furthermore, 89.6？±？2.8 g/L 2,3-BD with a productivity of 2.13？±？0.07 g/L/h and yield of 0.42 g2,3-BD/gsugar was achieved using molasses as a carbon source. Conclusions: The production of 2,3-BD by B. subtilis CS13 showed a higher concentration, productivity, and yield compared to the reported generally recognized as safe 2,3-BD producers. These results suggest that B. subtilis CS13 is a promising strain for industrial-scale production of 2,3-BD.

iCSDB: an integrated database of CRISPR screens

High-throughput screening based on CRISPR-Cas9 libraries has become an attractive and powerful technique to identify target genes for functional studies. However, accessibility of public data is limited due to the lack of user-friendly utilities and up-to-date resources covering experiments from third parties. Here, we describe iCSDB, an integrated database of CRISPR screening experiments using human cell lines. We compiled two major sources of CRISPR-Cas9 screening: the DepMap portal and BioGRID ORCS. DepMap portal itself is an integrated database that includes three large-scale projects of CRISPR screening. We additionally aggregated CRISPR screens from BioGRID ORCS that is a collection of screening results from PubMed articles. Currently, iCSDB contains 1375 genome-wide screens across 976 human cell lines, covering 28 tissues and 70 cancer types. Importantly, the batch effects from different CRISPR libraries were removed and the screening scores were converted into a single metric to estimate the knockout efficiency. Clinical and molecular information were also integrated to help users to select cell lines of interest readily. Furthermore, we have implemented various interactive tools and viewers to facilitate users to choose, examine and compare the screen results both at the gene and guide RNA levels. iCSDB is available at https://www.kobic.re.kr/icsdb/.