B Cell Adhesion to Fibroblast-Like Synoviocytes Is Up-Regulated by Tumor Necrosis Factor-Alpha via Expression of Human Vascular Cell Adhesion Molecule-1 Mediated by B Cell-Activating Factor.

Fibroblast-like synoviocytes (FLSs) play a key role in the pathogenesis of rheumatoid arthritis (RA) by producing inflammatory cytokines and interacting with various immune cells, which contribute to cartilage destruction. RA-FLSs activated by tumor necrosis factor alpha (TNF-α), exacerbate joint damage by triggering the expression of various inflammatory molecules, including human vascular cell adhesion molecule-1 (hVCAM1) and B cell-activating factor (hBAFF), with a role in maturation and maintenance of B cells. Here, we investigated whether B cell interaction with FLSs could be associated with hVCAM1 expression by TNF-α through hBAFF, using WiL2-NS B cells and MH7A synovial cells. TNF-α enhanced the expression of hVCAM1 and hBAFF. B cell adhesion to FLSs was increased by treatment with TNF-α or hBAFF protein. hVCAM expression was up-regulated by transcriptional activation of the hVCAM1 promoter(-1549 to -54) in MH7A cells treated with hBAFF protein or overexpressed with hBAFF gene. In contrast, hVCAM1 expression was down-regulated by treatment with hBAFF-siRNA. JNK was activated by TNF-α treatment. Then, hVCAM1 expression and B cell adhesion to FLSs were reduced by the treatment with JNK inhibitor SP600125. Transcriptional activity of hVCAM1 by the stimulation with TNF-α was inhibited by the deletion of -1549 to -229 from the hVCAM1 promoter. hVCAM1 expression and B cell adhesion to FLSs were reduced by treatment with hVCAM1-siRNA. Taken together, these results suggest that B cell adhesion to FLSs is associated with TNF-α-induced up-regulation of hVCAM1 expression via hBAFF expression. Thus, the pathological progression of RA may be associated with hVCAM1-mediated interaction of synovial cells with B lymphocytes.

Whole-Genome Sequencing and Genomic Analysis of a Virulent Bacteriophage Infecting Bacillus cereus.

OBJECTIVE: Infants with a weak immune system are prone to infection with Bacillus cereus, which is commonly found in natural environments. With the aim of achieving better control of this pathogenic bacterium, in the present study we characterized a new bacteriophage, ΦBC01. METHODS: Bacteriophage particles were analyzed by transmission electron microscopy. Factors influencing adsorption were identified in a double-layer plaque assay. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was conducted to determine the size of major structural proteins. The complete genome of the phage was analyzed. RESULTS: Bacteriophage particles (105.3 ± 3.1 nm icosahedral head and 208.8 ± 3.6 nm contractile tail) were identified as Myoviridae. ΦBC01 was found to have host specificity to B. cereus. Major structural proteins of ΦBC01 showed 2 well-pronounced bands of 99 and 56 kDa. The 158,385-bp genome sequence of ΦBC01 was determined: 56 of the 239 open reading frames were annotated, indicating involvement in bacteriophage DNA manipulation, cell lysis, packaging, virion structure, and other functions. CONCLUSION: Because of characterization and genotyping of a new bacteriophage from soil samples containing earthworms, this study provides a baseline for the development of alternatives to antibiotics.

A2 milk consumption and its health benefits: an update.

Milk is a widely consumed nutrient-rich food containing protein variants such as casein A2 and A1. A1 differs from A2 in an amino acid at position 67 (Pro67 to His67). The breakdown of ß-casein yields ß-casomorphins (BCM), among which BCM-7 is extensively studied for its effects on the human body. Animal studies have shown that A1 ß-casein milk increases digestive transit time and enhances myeloperoxidase activity. Individuals with lactose intolerance prefer A2 milk to conventional A1 milk, as BCM-7 in A1 milk can lead to inflammation and discomfort in sensitive individuals. A2 milk, which contains A2 ß-casein, is believed to be more easily digestible than A1 ß-casein. Its popularity has grown owing to reports linking A1 casein to diseases such as type 1 diabetes, heart disease, and autism. A2 milk has gained popularity as an alternative to A1 milk, primarily because of its potential benefits for individuals with certain diseases. This review aims to provide an updated understanding of A2 milk consumption and its health benefits. This review aims to provide an updated understanding of A2 milk consumption and its health benefits.

Effects of Lactiplantibacillus plantarum FBT215 and prebiotics on the gut microbiota structure of mice.

Imbalanced intestinal microbiota is associated with diseases, including inflammatory bowel disease and obesity, and diet can alter the structure of the gut microbiota. In this study, the effects of dietary treatments including the potential probiotic Lactiplantibacillus plantarum FBT215 with/without prebiotics on the intestinal microbiota composition of mice were investigated. Lactiplantibacillus plantarum FBT215 administration significantly decreased the Firmicutes/Bacteroidetes ratio and increased the abundance of Muribaculum and Duncaniella. The diversity within and between groups was measured according to α and ß diversity metrics, respectively. The Shannon index of α diversity decreased significantly in all treatment groups except the probiotic group, although this group showed an increase in the Chao1 index. Principal coordinate analysis of ß diversity showed that the groups had different species distributions. Finally, gamma-aminobutyric acid (GABA) concentration increased in groups fed L. plantarum FBT215. These findings improve our understanding of the association between the gut microbiota structure and specific probiotic/prebiotic-containing diets.

Novel Podoviridae family bacteriophage infecting Weissella cibaria isolated from Kimchi.

The first complete genome sequence of a phage infecting Weissella cibaria (Weissella kimchii) is presented. The bacteriophage YS61 was isolated from kimchi, a Korean fermented vegetable dish. Bacteriophages are recognized as a serious problem in industrial fermentations; however, YS61 differed from many virulent phages associated with food fermentations since it was difficult to propagate and was very susceptible to resistance development. Sequence analysis revealed that YS61 resembles Podoviridae of the subfamily Picovirinae. Within the subfamily Picovirinae, the 29-like phages have been extensively studied, and their terminal protein-primed DNA replication is well characterized. Our data strongly suggest that YS61 also replicates by a protein-primed mechanism. Weissella phage YS61 is, however, markedly different from members of the Picovirinae with respect to genome size and morphology. Picovirinae are characterized by small (approximately 20-kb) genomes which contrasts with the 33,594-bp genome of YS61. Based on electron microscopy analysis, YS61 was classified as a member of the Podoviridae of morphotype C2, similar to the 29-like phages, but its capsid dimensions are significantly larger than those reported for these phages. The novelty of YS61 was also emphasized by the low number of open reading frames (ORFs) showing significant similarity to database sequences. We propose that the bacteriophage YS61 should represent a new subfamily within the family Podoviridae.

Probiotic Properties and Optimization of Gamma-Aminobutyric Acid Production by Lactiplantibacillus plantarum FBT215.

Gamma-aminobutyric acid (GABA) improves various physiological illnesses, including diabetes, hypertension, depression, memory lapse, and insomnia in humans. Therefore, interest in the commercial production of GABA is steadily increasing. Lactic acid bacteria (LAB) have widely been reported as a GABA producer and are safe for human consumption. In this study, GABA-producing LAB were preliminarily identified and quantified via GABase assay. The acid and bile tolerance of the L. plantarum FBT215 strain were evaluated. The one-factor-at-a-time (OFAT) strategy was applied to determine the optimal conditions for GABA production using HPLC. Response surface methodology (RSM) with Box-Behnken design was used to predict the optimum GABA production. The strain FBT215 was shown to be acid and bile tolerant. The optimization of GABA production via the OFAT strategy resulted in an average GABA concentration of 1688.65 ± 14.29 µg/ml, while it was 1812.16 ± 23.16 µg/ml when RSM was applied. In conclusion, this study provides the optimum culture conditions for GABA production by the strain FBT215 and indicates that L. plantarum FBT215 is potentially promising for commercial functional probiotics with health claims.

Gamma-aminobutyric acid fermentation in MRS-based medium by the fructophilic Lactiplantibacillus plantarum Y7.

Among the key metabolites produced by probiotic lactic acid bacteria (LAB), the use of gamma-aminobutyric acid (GABA), which alleviates hypertension, depression, and sleepiness in humans, is gaining popularity. Thus, GABA-producing LAB are sought after. GABA-producing LAB were preliminarily screened in acidified-MRS broth and quantified via GABase assays. The one-factor-at-a-time strategy was applied to determine the optimal conditions for GABA production. GABA production in reconstituted skim milk medium (RSM) and antibiotic susceptibility testing were performed to evaluate the potential of the strain as a yogurt starter. L. plantarum Y7 produced 4,856.86 ± 82.47 µg/mL of GABA at optimal culture conditions. Co-cultivation of Y7 and commercial Lactobacillus bulgaricus affected the amount of GABA production (6.85 ± 0.20 µg/mL) in RSM. Y7 was susceptible to ampicillin, erythromycin, and tetracycline. Therefore, L. plantarum Y7 represents a promising strain for GABA production in the food industry.

Large-Scale Production of Cronobacter sakazakii Bacteriophage Φ CS01 in Bioreactors via a Two-Stage Self-Cycling Process.

Cronobacter sakazakii is an opportunistic pathogenic bacterium found in powdered infant formula and is fatal to neonates. Antibiotic resistance has emerged owing to overuse of antibiotics. Therefore, demand for high-yield bacteriophages as an alternative to antibiotics has increased. Accordingly, we developed a modified mass-production method for bacteriophages by introducing a two-stage self-cycling (TSSC) process, which yielded high-concentration bacteriophage solutions by replenishing the nutritional medium at the beginning of each process, without additional challenge. pH of the culture medium was monitored in real-time during C. sakazakii growth and bacteriophage CS01 propagation, and the changes in various parameters were assessed. The pH of the culture medium dropped to 5.8 when the host bacteria reached the early log phase (OD540 = 0.3). After challenge, it decreased to 4.65 and then recovered to 4.94; therefore, we set the optimum pH to challenge the phage at 5.8 and that to harvest the phage at 4.94. We then compared phage production during the TSSC process in jar-type bioreactors and the batch culture process in shaker flasks. In the same volume of LB medium, the concentration of the phage titer solution obtained with the TSSC process was 24 times higher than that obtained with the batch culture process. Moreover, we stably obtained high concentrations of bacteriophage solutions for three cycles with the TSSC process. Overall, this modified TSSC process could simplify large-scale production of bacteriophage CS01 and reduce the unit cost of phage titer solution. These results could contribute to curing infants infected with antibiotic-resistant C. sakazakii.

Isolation, characterization, and genomic analysis of the novel T4-like bacteriophage ΦCJ20.

Pathogenic Escherichia coli infections have been consistently reported annually. The basic characteristics and genome of the newly isolated ΦCJ20 from swine feces was analyzed. To determine basic characteristics, dotting assays and double-layer agar assays were conducted. Bacteriophage particles were analyzed via transmission electron microscopy. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed to determine the sizes of major structural proteins. The complete genome of the phage was analyzed. Bacteriophage particles were identified as Myoviridae, with a head measuring 110.57 ± 1.89 nm and a contractile tail measuring 107.97 ± 3.20 nm and were found to infect E. coli. Major structural proteins of ΦCJ20 showed two well-pronounced bands of approximately 53.6 and 70.9 kDa. The genome size of ΦCJ20 was 169,884 bp, and 118 of 307 open reading frames were annotated. This study provides a baseline for the development of E. coli infection treatment strategies. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10068-021-00906-y.

Synovial Cell Migration is Associated with B Cell Activating Factor Expression Increased by TNFα or Decreased by KR33426.

Fibroblast-like synoviocytes (FLS) play a crucial role in initiating rheumatoid arthritis. B-cell activating factor (BAFF) plays a role in FLS survival as well as in B cell maturation and maintenance. Here, we investigated whether tumor necrosis factor (TNF)-α- induced BAFF expression controls FLS migration and whether BAFF expression in FLS could be regulated by KR33426 which is the inhibitor of BAFF binding to BAFF receptors (BAFF-R) by using MH7A synovial cells transfected with the SV40 T antigen. More TNF-α-treated cells migrated compared to the control. TNF-α increased BAFF expression in FLS, significantly. FLS migration was inhibited by the transfection with BAFF-siRNA. KR33426 also inhibited BAFF expression increased by TNF-α treatment in FLS as judged by western blotting, PCR, and transcriptional activity assay. Kinases including JNK, p38 and Erk were activated by TNF-α treatment. While JNK and p38 were inhibited by KR33426 treatment, no changes in Erk were observed. Transcription factors including p65, c-Fos, CREB and SP1 were enhanced by TNF-α treatment. Among them, c-Fos was inhibited by KR33426 treatment. Small interference(si)-RNA of c-fos decreased BAFF transcriptional activity. FLS migration induced by TNF-α was inhibited by the transfection with BAFF-siRNA. KR33426 increased Twist, Snail, Cadherin-11 and N-Cadherin. In contrast, KR33426 decreased E-cadherin and TNF-α-enhanced CCL2. Taken together, our results demonstrate that synovial cell migration via CCL2 expression could be regulated by BAFF expression which is decreased by KR33426 and c-Fos-siRNA. It suggests for the first time that the role of BAFF-siRNA on FLS migration might be matched in the effect of KR33426 on BAFF expression.

Curcumin-induced cell death depends on the level of autophagic flux in A172 and U87MG human glioblastoma cells.

Glioblastoma is the deadliest neoplasm with the worst 5-year survival rate among all human cancers. Autophagy promotes autophagic cell death or blocks the induction of apoptosis in eukaryotic cells. Here, we investigated whether varying levels of autophagic flux in glioblastoma lead to different efficacies of curcumin treatment using U87MG and A172 human glioblastoma cells. The number of LC3 puncta, the number of cells with LC3 puncta and the level of LC3 II, Atg5 and Atg7 protein were higher in U87MG cells compared with A172 cells. When the cells were incubated with curcumin for 24 or 48 h, the percentage of cell death was higher in A172 cells compared with U87MG cells. Although the level of LC3 was lower, that of curcumin-induced LC3 was higher, in A172 cells than in U87MG cells. The relative increases in cell death and LC3-mediated autophagy were greater under serum starvation in A172 cells compared with U87MG cells. Curcumin-induced A172 cell death was reduced by serum starvation. When both types of cells were transfected with LC3-GFP, the percentage of cell death was higher in A172 cells than that in U87MG cells. Taken together, the data demonstrate that curcumin-mediated tumor cell death is regulated by the basal level of autophagic flux in different glioblastoma cells. This suggests that prior to the use of various curcumin therapeutics, the level of basal or induced autophagic flux should be carefully examined in tumor cells for the best efficacy.

Genetic Analysis and Characterization of a Bacteriophage ØCJ19 Active against Enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is the major pathogenic E. coli that causes diarrhea and edema in post-weaning piglets. In this study, we describe the morphology and characteristics of ØCJ19, a bacteriophage that infects ETEC, and performed genetic analysis. Phage ØCJ19 belongs to the family Myoviridae. One-step growth curve showed a latent phase of 5 min and burst size of approximately 20 phage particles/infected cell. Phage infectivity was stable for 2 h between 4°C and 55°C, and the phage was stable between pH 3 and 11. Genetic analysis revealed that phage ØCJ19 has a total of 49,567 bases and 79 open reading frames (ORFs). The full genomic sequence of phage ØCJ19 showed the most similarity to an Escherichia phage, vB_EcoS_ESCO41. There were no genes encoding lysogeny, toxins, virulence factors, or antibiotic resistance in this phage, suggesting that this phage can be used safely as a biological agent to control ETEC. Comparative genomic analysis in terms of the tail fiber proteins could provide genetic insight into host recognition and the relationship with other coliphages. These results showed the possibility to improve food safety by applying phage ØCJ19 to foods of animal origin contaminated with ETEC and suggests that it could be the basis for establishing a safety management system in the animal husbandry.

Curcumin-Induced Autophagy Augments Its Antitumor Effect against A172 Human Glioblastoma Cells.

Glioblastoma is the most aggressive common brain tumor in adults. Curcumin, from Curcuma longa, is an effective antitumor agent. Although the same proteins control both autophagy and cell death, the molecular connections between them are complicated and autophagy may promote or inhibit cell death. We investigated whether curcumin affects autophagy, which regulates curcumin-mediated tumor cell death in A172 human glioblastoma cells. When A172 cells were incubated with 10 µM curcumin, autophagy increased in a time-dependent manner. Curcumin-induced cell death was reduced by co-incubation with the autophagy inhibitors 3-methyladenine (3-MA), hydroxychloroquine (HCQ), and LY294002. Curcumin-induced cell death was also inhibited by co-incubation with rapamycin, an autophagy inducer. When cells were incubated under serum-deprived medium, LC3-II amount was increased but the basal level of cell viability was reduced, leading to the inhibition of curcumin-induced cell death. Cell death was decreased by inhibiting curcumin-induced autophagy using small interference RNA (siRNA) of Atg5 or Beclin1. Therefore, curcumin-mediated tumor cell death is promoted by curcumin-induced autophagy, but not by an increase in the basal level of autophagy in rapamycin-treated or serum-deprived conditions. This suggests that the antitumor effects of curcumin are influenced differently by curcumin-induced autophagy and the prerequisite basal level of autophagy in cancer cells.

Characterization and Genomic Analysis of Novel Bacteriophage ΦCS01 Targeting Cronobacter sakazakii.

Cronobacter sakazakii is an opportunistic pathogen causing serious infections in neonates. In this study, a bacteriophage ΦCS01, which infects C. sakazakii, was isolated from swine feces and its morphology, growth parameters, and genomic analysis were investigated. Transmission electron microscopy revealed that ΦCS01 has a spherical head and is 65.74 nm in diameter with a 98.75 nm contracted tail, suggesting that it belongs to the family Myoviridae. The major viral proteins are approximately 71 kDa and 64 kDa in size. The latent period of ΦCS01 was shown to be 60 min, and the burst size was 90.7 pfu (plaque-forming units)/ infected cell. Bacteriophage ΦCS01was stable at 4-60°C for 1 h and lost infectivity after 1 h of heating at 70°C. Infectivity remained unaffected at pH 4-9 for 2 h, while the bacteriophage was inactivated at pH <3 or >10. The double-stranded ΦCS01 DNA genome consists of 48,195 base pairs, with 75 predicted open reading frames. Phylogenetic analysis is closely related to that of the previously reported C. sakazakii phage ESP2949-1. The newly isolated ΦCS01 shows infectivity in the host bacterium C. sakazakii, indicating that it may be a promising alternative to antibacterial agents for the removal of C. sakazakii from powdered infant formulas.

Isolation and molecular characterization of a cryptic plasmid from Bifidobacterium longum.

An isolate from the fecal samples of children was identified as Bifidobacterium longum. A plasmid isolated from it pBIFA24 was 4,892 bp with three open reading frames, ORFI, ORFII, and ORFIII. ORFI encoded a replication protein involved in a rolling-circle replication mechanism, and three sets of tandem repeat sequences featuring iteron structure were identified. Secondary structure prediction analysis of ORFII suggested it was a transmembrane protein. ORFIII showed high amino acid sequence identity with some mobilization proteins and contained an oriT sequence.

Detection and characterization of a lytic Pediococcus bacteriophage from the fermenting cucumber brine.

Of the twelve lytic bacteriophages recovered from five different fermenting cucumber tanks that were inoculated with Pediococcus sp. LA0281, a lytic phage, phips05, was characterized in the present study. The plaques were mostly clear and round-shaped on the lawn of starter strain, indicating lytic phage. Overall appearance indicated that it belongs to the Siphoviridae family or Bradley's group B1, with a small isometric head and a flexible noncontractile tail with swollen base plate. The average size was found to be 51.2 nm in head diameter and 11.6 nm wide x 129.6 nm long for the tail. The single-step growth kinetics curve showed that the eclipse and the latent period were 29 min and 34 min, respectively, and an average burst size was calculated to be 12 particles per infective center. The optimum proliferating temperature (35 degrees C) was slightly lower than that of cell growth (35 to 40 degrees C). The structural proteins revealed by SDS-PAGE consisted of one main protein of 33 kDa and three minor proteins of 85, 58, and 52 kDa. The phage genome was a linear double-stranded DNA without cohesive ends. Based on the single and double digestion patterns obtained by EcoRI, HindIII, and SalI, the physical map was constructed. The overall size of the phage genome was estimated to be 24.1 kb. The present report describes the presence of a lytic phage active against a commercial starter culture Pediococcus sp. LA0281 in cucumber fermentation, and a preliminary study characterizes the phage on bacterial successions in the process of starter-added cucumber fermentation.

Production of a fermented organic rice syrup with higher isomalto-oligosaccharide using Lactobacillus plantarum.

Isomalto-oligosaccharide (IMO) syrup was prepared from organic rice, in which Lactobacillus plantarum as a starter was inoculated to raise its purity and also produce a fermented rice beverage. Of the five strains of lactic acid bacteria tested, L. plantarum was preferentially selected in terms of a viable cells (7.3 × 108 colony forming unit (CFU/mL) and higher dry cell weight (13 mg/mL). The fermented syrup-based medium did not affect the growth of L. plantarum. As expected, the residual sugar content gradually decreased by 1.34% compared with the initial concentration. It was apparent that the residual sugars but not oligosaccharides were removed during the L. plantarum fermentation period. The production of lactic acid was the highest (8125.78 mg/kg) among the organic acids produced in the fermented IMO syrup.

A natural odor attraction between lactic acid bacteria and the nematode Caenorhabditis elegans.

Animal predators can track prey using their keen sense of smell. The bacteriovorous nematode Caenorhabditis elegans employs sensitive olfactory sensory neurons that express vertebrate-like odor receptors to locate bacteria. C. elegans displays odor-related behaviors such as attraction, aversion and adaptation, but the ecological significance of these behaviors is not known. Using a combination of food microbiology and genetics, we elucidate a possible predator-prey relationship between C. elegans and lactic acid bacteria (LAB) in rotting citrus fruit. LAB produces the volatile odor diacetyl as an oxidized by-product of fermentation in the presence of citrate. We show that C. elegans is attracted to LAB when grown on citrate media or Citrus medica L, commonly known as yuzu, a citrus fruit native to East Asia, and this attraction is mediated by the diacetyl odor receptor, ODR-10. We isolated a wild LAB strain and a wild C. elegans-related nematode from rotten yuzu, and demonstrate that the wild nematode was attracted to the diacetyl produced by LAB. These results not only identify an ecological function for a C. elegans olfactory behavior, but contribute to the growing understanding of ecological relationships between the microbial and metazoan worlds.

Inactivation of Salmonella on Eggshells by Chlorine Dioxide Gas.

Microbiological contamination of eggs should be prevented in the poultry industry, as poultry is one of the major reservoirs of human Salmonella. ClO2 gas has been reported to be an effective disinfectant in various industry fields, particularly the food industry. The aims of this study were to evaluate the antimicrobial effect of chlorine dioxide gas on two strains of Salmonella inoculated onto eggshells under various experimental conditions including concentrations, contact time, humidity, and percentage organic matter. As a result, it was shown that chlorine dioxide gas under wet conditions was more effective in inactivating Salmonella Enteritidis and Salmonella Gallinarum compared to that under dry conditions independently of the presence of organic matter (yeast extract). Under wet conditions, a greater than 4 log reduction in bacterial populations was achieved after 30 min of exposure to ClO2 each at 20 ppm, 40 ppm, and 80 ppm against S. Enteritidis; 40 ppm and 80 ppm against S. Gallinarum. These results suggest that chlorine dioxide gas is an effective agent for controlling Salmonella, the most prevalent contaminant in the egg industry.

Galactooligosaccharide and Sialyllactose Content in Commercial Lactose Powders from Goat and Cow Milk.

The most commonly used infant formulas contain lactose originating from cow milk. Goat milk has recently been claimed to be nutritionally more effective for infants than other milks. In baby foods, much emphasis is placed on the concentrations of intestinal microflora-promoting oligosaccharides, which are generally transferred into lactose from milk during crystallization process. Here we show that higher level of free sialic acid is present in goat lactose powder compared to cow lactose powder. Without proteinase K treatment, the amount of 3-sialyllactose and 6-sialyllactose were similar in goat and cow lactose powders. However, after proteolysis, 6-sialyllactose was present at higher levels in goat than in cow lactose powder. Galactooligosaccharides, a group of prebiotics, are present in milk in the form of glycoproteins. Galactooligosaccharide content was also higher in goat lactose powder than in cow lactose powder.