Effects of blackcurrant extract on indole and ammonia productions in an in vitro human fecal culture model.

Blackcurrant is available as a traditional medicine in Europe. However, the detailed effects of blackcurrant on the human gut microbiota remain unknown. In this study, we investigated the prebiotic effects of a blackcurrant extract using a human fecal culture model in six healthy subjects. Feces were individually inoculated into a medium with or without the blackcurrant extract and then fermented for 48 hr under anaerobic conditions. The results obtained from analysis of samples from the fermented medium demonstrated that after 48 hr of fermentation, the pH of the medium with the blackcurrant extract was significantly decreased (control, 6.62 ± 0.20; blackcurrant extract, 6.41 ± 0.33; p=0.0312). A 16S rRNA gene sequencing analysis of the microbiota of the fermented medium showed a significant increase in the relative abundance of Bifidobacteriaceae. In measuring the concentrations of putrefactive components in the fermented medium, we found that the blackcurrant extract significantly reduced ammonia levels and displayed a tendency toward reduced indole levels. Our results suggest that blackcurrant extract could be a potential ingredient for relief of putrefactive components in the gut.

Work engagement among older workers: a systematic review.

OBJECTIVES: Given current labor force conditions, including population aging, keeping older workers engaged in work and motivated is important. Aging may alter the effects that psychological and environmental factors have on work engagement. We conducted a systematic review to understand the features of work engagement among older workers. METHODS: A systematic search was conducted in July 2022 using 4 databases. The review included relevant articles that focused on participants aged 40 years and older. RESULTS: Fifty articles were selected for our review, which were grouped into 5 categories: (1) studies examining the relationship between chronological age and work engagement, (2) studies investigating the moderating effects of age on the relationship between job-related psychological factors and work environment factors and work engagement, (3) studies comparing the relationship of job-related psychological factors and work environment factors with work engagement across different age groups, (4) studies exploring the relationship between work engagement and retirement intentions or continued employment beyond retirement age, and (5) other studies discussing work engagement in the context of older workers. Most articles focused on workers in Europe and the United States and used observational study designs. CONCLUSIONS: Work engagement increases with age, and is mainly mediated by increased emotional regulation. In addition, age moderates the relationships between various job-related psychological and work-environmental factors and work engagement. Work engagement is associated with working beyond retirement age. Organizations should understand the characteristics of work engagement among older workers and make age-conscious efforts to support them in adapting to social changes.

Absolute quantification of bovine lactadherin to screen the anti-rotavirus activity of dairy ingredients.

The anti-rotavirus components in breast milk and infant formulas play an important role in the prevention of rotavirus infection. The present study examined whether the levels of phospholipids and bovine lactadherin, which are the major components and proteins of the milk fat globule membrane complex, are useful indices of the anti-rotavirus activity of dairy ingredients used in infant formulas. We compared the anti-rotavirus activity of 2 types of dairy ingredients enriched in the milk fat globule membrane complex: high-fat whey protein concentrate (high-fat WPC) and butter milk powder (BMP), using 50% inhibition concentration (IC50) and linear inhibition activity to determine levels of solid contents, total proteins, phospholipids, and bovine lactadherin. Here, we developed a quantification method using full-length isotope-labeled proteins to measure bovine lactadherin levels in these dairy ingredients. The evaluation of anti-rotavirus activity showed that the difference in IC50 was the smallest when the 2 dairy ingredients were compared at the bovine lactadherin level, among other indices in this study. Additionally, no significant difference was observed between the inhibition linearity of 2 dairy ingredients when evaluating only bovine lactadherin levels. These results indicated that the level of bovine lactadherin was more strongly associated with anti-rotavirus activity than the level of phospholipids. Our results suggest that bovine lactadherin levels can be used to estimate the anti-rotavirus activity of dairy ingredients and can be a criterion used in selecting ingredients for infant formulas.

The effects of denatured major bovine whey proteins on the digestive tract, assessed by Caco-2 cell differentiation and on viability of suckling mice.

Alpha-lactalbumin (α-LA) and ß-lactoglobulin (ß-LG) are contained in bovine milk whey. Chemical and physical treatments are known to alter the conformation of these proteins and we have previously reported that α-LA denatured with trifluoroethanol (TFE) and isolated from sterilized market milk inhibited the growth of rat crypt IEC-6 cells. In the present study, we aimed to evaluate the effects of TFE-treated α-LA and ß-LG on cell growth using cultured intestinal cells and on their safety using a suckling mouse model. First, we investigated the effect of the TFE-treated whey proteins on human colonic Caco-2 cells at various differentiation stages. In the undifferentiated stage, we assessed cell growth by a water-soluble tetrazolium-1 method. The native whey proteins enhanced cell proliferation, whereas the TFE-treated whey proteins strongly inhibited cell growth. We investigated cell barrier function in the post-differentiated stage by measuring transepithelial electrical resistance (TER). Not only native but also the TFE-treated whey proteins increased TER. Next, we evaluated whether the TFE-treated α-LA and ß-LG have adverse effects on healthy suckling mice. No mice given by the TFE-treated samples showed any adverse symptoms. We also performed a safety test using a human rotavirus infected gastrointestinal disease suckling mice model. Even the TFE-treated whey proteins appeared to prevent the development of diarrheal symptoms without any adverse effects. Although we cannot know the effect of long-term ingestion of denatured whey proteins, these results suggest that they have no adverse effects on differentiated intestinal cells and digestive tract, at least in short-term ingestion.

Screening for Components/Compounds with Anti-Rotavirus Activity: Detection of Interaction Between Viral Spike Proteins and Glycans.

Rotaviruses are the major etiologic agents of acute gastroenteritis. Viral attachment to the cell surface is crucial to initiate infection. The VP8∗ domain, the trypsinized cleavage fragment of the outermost spike protein VP4 of rotavirus, has a galectin-like structure required for binding to the cell surface. We used the evanescent-field fluorescence-assisted assay to understand the complex mechanism underlying the virus-glycan/glycoprotein interaction. Besides, we have described virus infection assays, neutralization assay, and pretreatment assay, using cell culture. These approaches using rotavirus particles will provide novel information that has been difficult to obtain from glycan microarray using recombinant VP8∗.

Treatment with myo-inositol attenuates binding of the carbohydrate-responsive element-binding protein to the ChREBP-ß and FASN genes in rat nonalcoholic fatty liver induced by high-fructose diet.

Dietary supplementation with the major lipotrope myo-inositol (MI) potently reduces triglyceride (TG) content and expression levels of the fatty acid synthesis genes, for example, fatty acid synthase (FASN), in rat nonalcoholic fatty liver induced by high-fructose diet. Fatty acid synthesis genes are regulated by the carbohydrate-responsive element-binding protein (ChREBP) that exists in 2 isoforms: ChREBP-α and ChREBP-ß. The gene encoding the latter isoform is more responsive to fructose. Because MI repressed the induction of fatty acid synthesis gene expression by high-fructose diet, we hypothesized that MI may reduce binding of ChREBP to the carbohydrate response elements (ChoREs) in the ChREBP-ß gene as well as in fatty acid synthesis genes in the liver. Rats were fed high-glucose, high-fructose, or high-fructose diets supplemented with MI (0.05% and 0.25%) for 2 weeks. Hepatic TG content and expression levels of the glucose-6-phosphate dehydrogenase, malic enzyme 1, FASN, acetyl-CoA carboxylase alpha, S14, and ChREBP-ß were remarkably elevated in rats fed with high fructose compared with the corresponding levels in high-glucose group. Notably, elevated values of these parameters in high-fructose group were reduced by MI. Similarly, high-fructose-induced ChREBP binding to the ChoREs of the ChREBP-ß and FASN genes was nominally decreased by MI. This study showed that treatment with MI reduced elevated TG content and expression of genes related to fatty acid synthesis, such as FASN and ChREBP-ß, in rat nonalcoholic fatty liver induced by high-fructose diet. Furthermore, MI treatment nominally decreased increased binding of ChREBP to the ChoREs of ChREBP-ß and FASN genes.

Evaluation of inactivated vaccines against equine group A rotaviruses by use of a suckling mouse model.

BACKGROUND: Equine group A rotaviruses (RVAs) cause diarrhea in suckling foals. The dominant RVAs circulating among horses worldwide, including Japan, are G3P[12] and/or G14P[12] genotypes. Inactivated vaccines containing a G3P[12] RVA are commercially available in some countries for prevention of diarrhea caused by equine RVAs. However, there is no reported evidence whether vaccines containing a G3P[12] RVA are effective against G14P[12] RVAs or whether using a G14P[12] RVA results in a more effective vaccine. This study used a suckling mouse model to evaluate the effectiveness of inactivated vaccines containing G3P[12] (G3 vaccine) or G14P[12] (G14 vaccine) RVAs against G3P[12] and G14P[12] RVAs. METHODS: Female mice were inoculated twice with G3 or G14 vaccines, and were then mated. After parturition, suckling mice were challenged with one of either two G3P[12] RVAs, two G14P[12] RVAs, or one G13P[18] RVA. After virus inoculation, suckling mice were observed for diarrhea, and the incidence rates of diarrhea in the vaccinated groups were compared with those in the non-vaccinated groups. RESULTS: Following G3P[12] RVA challenge, suckling mice in the G3 and G14 vaccinated groups had significantly lower rates of diarrhea incidence than did those in the non-vaccinated group, and the rates in the G3 vaccinated group tended to be lower than in the G14 vaccinated group. Following G14P[12] RVA challenge, suckling mice in the G14 vaccinated group had significantly lower rates of diarrhea incidence than did those in the non-vaccinated and G3 vaccinated groups. The G3 and G14 vaccines did not reduce the rate when challenged with the G13P[18] RVA. CONCLUSION: The mouse model showed that the G3 and G14 vaccines were both effective against G3P[12] RVAs, and that the G14 vaccine was effective against G14P[12] RVAs. These results suggest that at least a G14 RVA strain should be included in as a vaccine strain.

Daidzein reductase of Eggerthella sp. YY7918, its octameric subunit structure containing FMN/FAD/4Fe-4S, and its enantioselective production of R-dihydroisoflavones.

S-Equol is a metabolite of daidzein, a type of soy isoflavone, and three reductases are involved in the conversion of daidzein by specific intestinal bacteria. S-Equol is thought to prevent hormone-dependent diseases. We previously identified the equol producing gene cluster (eqlABC) of Eggerthella sp. YY7918. Daidzein reductase (DZNR), encoded by eqlA, catalyzes the reduction of daidzein to dihydrodaidzein (the first step of equol synthesis), which was confirmed using a recombinant enzyme produced in Escherichia coli. Here, we purified recombinant DZNR to homogeneity and analyzed its enzymological properties. DZNR contained FMN, FAD, and one 4Fe-4S cluster per 70-kDa subunit as enzymatic cofactors. DZNR reduced the CC bond between the C-2 and C-3 positions of daidzein, genistein, glycitein, and formononetin in the presence of NADPH. R-Dihydrodaidzein and R-dihydrogenistein were highly stereo-selectively produced from daidzein and genistein. The Km and kcat for daidzein were 11.9 µM and 6.7 s-1, and these values for genistein were 74.1 µM and 28.3 s-1, respectively. This enzyme showed similar kinetic parameters and wide substrate specificity for isoflavone molecules. Thus, this enzyme appears to be an isoflavone reductase. Gel filtration chromatography and chemical cross-linking analysis of the active form of DZNR suggested that the enzyme consists of an octameric subunit structure. We confirmed this by small-angle X-ray scattering and transmission electron microscopy at a magnification of ×200,000. DZNR formed a globular four-petal cloverleaf structure with a central vertical hole. The maximum particle size was 173 Å.

Association of Salmonella Serotypes with Quinolone Resistance in Broilers.

Fluoroquinolone is widely used for the treatment of bacterial diseases, and the emergence of quinolone resistance has become a serious concern in recent years, owing to an increase and inappropriate use of antimicrobials. Here, we attempted to understand the differences in the emergence frequency of quinolone-resistant bacterial variants in three Salmonella serotypes S. Infantis, S. Schwarzengrund, and S. Manhattan-which are mainly found in broiler industries in Japan. Emergence frequency tests for quinolone-resistant variants using enrofloxacin-containing agar plates and sequence analysis in the quinolone resistance-determining region (QRDR) of gyrA in DNA gyrase were performed. The results showed no significant difference in the emergence frequency among the three serotypes, and most of the resistant variants had mutations in the QRDR region. These findings suggest that differences in the serotypes tested are not associated with the emergence frequency of quinolone-resistant variants.

Capsular polysaccharide inhibits adhesion of Bifidobacterium longum 105-A to enterocyte-like Caco-2 cells and phagocytosis by macrophages.

BACKGROUND: Bifidobacterium longum 105-A produces markedly high amounts of capsular polysaccharides (CPS) and exopolysaccharides (EPS) that should play distinct roles in bacterial-host interactions. To identify the biological function of B. longum 105-A CPS/EPS, we carried out an informatics survey of the genome and identified the EPS-encoding genetic locus of B. longum 105-A that is responsible for the production of CPS/EPS. The role of CPS/EPS in the adaptation to gut tract environment and bacteria-gut cell interactions was investigated using the ΔcpsD mutant. RESULTS: A putative B. longum 105-A CPS/EPS gene cluster was shown to consist of 24 putative genes encoding a priming glycosyltransferase (cpsD), 7 glycosyltransferases, 4 CPS/EPS synthesis machinery proteins, and 3 dTDP-L-rhamnose synthesis enzymes. These enzymes should form a complex system that is involved in the biogenesis of CPS and/or EPS. To confirm this, we constructed a knockout mutant (ΔcpsD) by a double cross-over homologous recombination. Compared to wild-type, the ∆cpsD mutant showed a similar growth rate. However, it showed quicker sedimentation and formation of cell clusters in liquid culture. EPS was secreted by the ∆cpsD mutant, but had altered monosaccharide composition and molecular weight. Comparison of the morphology of B. longum 105-A wild-type and ∆cpsD by negative staining in light and electron microscopy revealed that the formation of fimbriae is drastically enhanced in the ∆cpsD mutant while the B. longum 105-A wild-type was coated by a thick capsule. The fimbriae expression in the ∆cpsD was closely associated with the disappearance of the CPS layer. The wild-type showed low pH tolerance, adaptation, and bile salt tolerance, but the ∆cpsD mutant had lost this survivability in gastric and duodenal environments. The ∆cpsD mutant was extensively able to bind to the human colon carcinoma Caco-2 cell line and was phagocytosed by murine macrophage RAW 264.7, whereas the wild-type did not bind to epithelial cells and totally resisted internalization by macrophages. CONCLUSIONS: Our results suggest that CPS/EPS production and fimbriae formation are negatively correlated and play key roles in the survival, attachment, and colonization of B. longum 105-A in the gut.

Effects of heat treatment on conformation and cell growth activity of alpha- lactalbumin and beta-lactoglobulin from market milk.

Heat processes, low temperature for long time (LTLT) pasteurization and ultra-heat treatment (UHT) sterilization, are essential for commercial market milk to improve the shelf life of raw milk and ensure microbial safety. We evaluated the effects of heat experience on the molecular properties of α-lactalbumin (α-LA) and ß-lactoglobulin (ß-LG) isolated from four types of market milk such as LTLT-A (66°C for 30 min), LTLT-B (65°C for 30 min), UHT-I (130°C for 2 s, indirect heating) and UHT-D (135°C for 2 s, direct heating) samples. We examined molecular conformations using circular dichroism spectrum measurement and cell growth activity using the WST-1 method for the proteins. α-LA isolated from each of these four types of market milk displayed no significant structural difference as compared to raw milk α-LA, while α-LA of UHT-I only inhibited cell growth of an intestinal epithelial cell line more potently than raw milk α-LA. In the case of ß-LG, only the UHT-I sample demonstrated a drastic change in structure, while it did not exhibit any cytotoxicity. We found that cell viability effects of α-LA and ß-LG are attributable to the type of UHT; indirect and direct. These findings indicate that the effect of heat treatment on whey proteins should carefully be investigated further.

Quercetin derivatives regulate melanosome transportation via EPI64 inhibition and elongate the cell shape of B16 melanoma cells.

4'-O-ß-D-glucopyranosyl-quercetin-3-O-ß-D-glucopyranosyl-(1→4)-ß-D-glucopyranoside (3C4'GQ), first isolated from Helminthostachys zeylanica root extract, was synthesized as a compound that stimulates intracellular melanogenesis. 3-O-methylquercetin (3MQ) and 3,4',7-O-trimethylquercetin (34'7TMQ) were synthesized as compounds that enhance extracellular melanin formation. The formation of dendrites and the expression of EBP50-PDZ interactor of 64 kDa (EPI64) relating to melanin transportation were investigated using B16 melanoma cells treated with 3C4'GQ, 3MQ, or 34'7TMQ in order to understand the mechanism underlying the observed activities. The influence of 3C4'GQ on the increase of intracellular melanin contents enhanced the expression of EPI64, exhibited no dendrite elongation activity, and inhibited melanin transportation. On the other hand, the increase of extracellular melanin content by 3MQ and 34'7TMQ inhibited the expression of EPI64 and formed elongated cells to stimulate melanin transportation.

Synthesized quercetin derivatives stimulate melanogenesis in B16 melanoma cells by influencing the expression of melanin biosynthesis proteins MITF and p38 MAPK.

In order to understand the effect of structure-activity relationships on melanogenesis using B16 melanoma cells, 19 quercetin derivatives were synthesized. Among the synthesized compounds, 3-O-methylquercetin (11) and 3',4',7-O-trimethylquercetin (14) increased melanin content more potently than the positive control theophylline, while exhibiting low cytotoxicity. Compound 11 exhibited less melanogenesis-stimulating activity than compound 14. However, 11 increased the expression of tyrosinase and tyrosinase-related protein 1 (TRP-1) to a greater extent than 14, thereby suggesting that melanogenesis in melanoma cells does not depend solely on the expression of the enzymes catalyzing melanin biosynthesis. Furthermore, 14 also stimulated the expression of the microphthalmia-associated transcription factor (MITF) and p-p38 mitogen activated protein kinase (MAPK), while they were not increased by 11. These results suggest that 11 may enhance the expression of tyrosinase and TRP-1 by regulating the proteasomal degradation of melanogenic enzymes and/or by activating other transcriptional factors regulating enzyme expression.

A preparation of cow's late colostrum fraction containing αs1-casein promoted the proliferation of cultured rat intestinal IEC-6 epithelial cells.

Colostrum is a complex mixture of bioactives that promotes neonate growth. Recently, we have found by in vivo study that skimmed, sterilized, and concentrated bovine late colostrum (SCBLC), obtained from a Holstein herd on days 6-7 after parturition, had an ability to maintain intestinal integrity. In the present study we investigated effects of SCBLC on rat intestinal IEC-6 cell proliferation in vitro. A fraction containing αs1-casein was found to have a robust stimulation effect as compared to other protein fractions from SCBLC and even the αs1-casein fraction from milk from other Holstein herds. Furthermore, the SCBLC αs1-casein molecule demonstrated not only slightly slower mobility on both SDS- and native-PAGE than other bovine milk αs1-caseins, but also a peculiar conformation reminiscent of moltenglobule in the circular dichroism spectrum. These findings may be of relevant to the competence of SCBLC to preserve intestinal integrity.

Comparison of the efficacy of alpha-lactalbumin from equine, bovine, and human milk in the growth of intestinal IEC-6 cells.

Native alpha-lactalbumins (α-LA) from equine, bovine, and human milk were not cytotoxic. However, after treatment with trifluoroethanol (TFE), all three α-LAs exhibited cytotoxicity. Toxic potencies were distinctly different among them. Equine α-LA was the most robust, bovine α-LA was moderate, and human α-LA was weak. There were no significant structural changes as between the native and the TFE-treated α-LAs.

An assay for detecting neutralization of rotavirus infection by quantitative determination of VP6 protein fluorescence intensity.

Previous rotavirus infection studies used the focus reduction assay extensively to evaluate cellular responses to viral infection, but this technique has a number of limitations. In this study, we developed a simplified, accurate rotavirus infection assay to evaluate the effects of inhibitory substances on rotavirus infection in vitro by measurement of the fluorescence intensities of stained cells.

The bovine lactophorin C-terminal fragment and PAS6/7 were both potent in the inhibition of human rotavirus replication in cultured epithelial cells and the prevention of experimental gastroenteritis.

Rotaviruses are the leading cause of severe dehydrating diarrhea in children worldwide. We have found that high-M(r) glycoprotein fraction (F1) from cow's milk whey has potent inhibitory activity against human rotavirus (HRV) in cell culture. The present study was undertaken to identify and characterize the components responsible for this inhibitory activity. F1 was initially heated at 95 degrees C for 30 min, rendering milk antibodies inert, subjected to ammonium sulfate fractionation, and then resolved by two-dimensional polyacrylamide gel electrophoresis. After electroelution, we found that a heat-stable milk protein lactophorin C-terminal fragment (LP16) and bovine milk fat globule membrane protein PAS6/7 strongly inhibited the replication of HRV MO strains in MA104 cells. Furthermore, we found that prophylactic oral administration of F1 once before inoculation of the HRV MO strain obviously prevented the development of diarrhea in vivo. These non-immunoglobulin components are a promising candidate for a prophylactic food additive against HRV infection.

In vitro and in vivo evaluation of the efficacy of bovine colostrum against human rotavirus infection.

We found that skimmed and concentrated bovine late colostrum (SCBLC) obtained from normal cows at 6-7 d after parturition exhibited high potency in inhibiting replication of human rotavirus (HRV) in vitro. Furthermore, prophylactic oral administration of SCBLC once before inoculation of HRV prevented the development of diarrhea in suckling mice in vivo. SCBLC from normal cows might be useful in the prevention of HRV-induced severe gastroenteritis in immunocompromised hosts.

The multiplicity of N-glycan structures of bovine milk 18 kda lactophorin (milk GlyCAM-1).

Lactophorin is a heat-stable phosphoglycoprotein, also known as milk glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1). Bovine 18 kDa lactophorin was purified by heparin affinity chromatography from cow's milk whey. Its N-glycans were obtained by proteomic techniques, including two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), followed by in-gel digestion with peptide-N(4)-(N-acetyl-beta-glucosaminyl)-asparagine amidase (PNGase F). The released N-glycans were derivatized with 2-aminopryridine (PA) and analyzed by matrix-assisted laser desorption ionization quadruple ion trap time of flight mass spectrometry (MALDI-QIT-TOF MS). Among the MS analyzed peaks, 15 peaks were found to be N-glycan molecules as detected by MS(2) analysis. These glycans consisted of mono-sialylated bi-, tri-, and tetra-antennary complex-type N-glycans carrying Gal-GlcNAc (LacNAc) or GalNAc-GlcNAc (LacdiNAc) with and without core-fucose.