An Exploratory Study on Seasonal Variation in the Gut Microbiota of Athletes: Insights from Japanese Handball Players.

Despite accumulating evidence that suggests a unique gut microbiota composition in athletes, a comprehensive understanding of this phenomenon is lacking. Furthermore, seasonal variation in the gut microbiota of athletes, particularly during the off-season, remains underexplored. This study aimed to compare the gut microbiotas between athletic subjects (AS) and non-athletic subjects (NS), and to investigate variations between athletic and off-season periods. The data were derived from an observational study involving Japanese male handball players. The results revealed a distinct gut microbiota composition in AS compared with NS, characterized by significantly higher alpha-diversity and a greater relative abundance of Faecalibacterium and Streptococcus. Moreover, a comparative analysis between athletic and off-season periods in AS demonstrated a significant change in alpha-diversity. Notably, AS exhibited significantly higher alpha-diversity than NS during the athletic season, but no significant difference was observed during the off-season. This study demonstrates the characteristics of the gut microbiota of Japanese handball players and highlights the potential for changes in alpha-diversity during the off-season. These findings contribute to our understanding of the dynamic nature of the gut microbiota of athletes throughout the season.

Synbiotics of Bifidobacterium breve MCC1274 and lactulose enhances production of tryptophan metabolites in fermented human fecal communities.

Probiotics and prebiotics have beneficial effects on host physiology via metabolites from the gut microbiota in addition to their own. Here, we used a pH-controlled single-batch fermenter as a human gut microbiota model. We conducted fecal fermentation with Bifidobacterium breve MCC1274 (probiotic), lactulose (prebiotic), or a combination of both (synbiotic) to evaluate their influence on the gut environment. Fecal inoculum without the probiotic and prebiotic was used as the control. Principal coordinate analysis (PCoA), based on the composition of gut microbiota, showed a significant difference among the groups. The relative abundance of Bifidobacterium was significantly higher in the synbiotic group, compared to that in the other three treatment groups. The relative abundance of Blautia was the highest in the control group among the four groups. CE-TOFMS and LC-TOFMS showed that the number of metabolites detected in the synbiotic group was the highest (352 in total); 29 of the 310 hydrophilic metabolites and 17 of the 107 lipophilic metabolites were significantly different among the four groups in the Kruskal-Wallis test. A clustering based on 46 metabolites indicated that tryptophan-metabolites such as indole-3-lactic acid (ILA), indole-3-ethanol, and indole-3-carboxaldehyde, were included in a sub cluster composed of metabolites enriched in the synbiotic group. Spermidine, a major polyamine, was enriched in the two groups supplemented with the probiotic whereas spermine was enriched only in the synbiotic group. Not all metabolites enriched in the probiotic and/or synbiotic groups were found in the monocultures of the probiotic strain with or without the prebiotics. This implies that some of the metabolites were produced through the interaction of the fecal microbiota with the inoculated probiotic strain. Co-abundance networking analysis indicated the differences in the correlations between the relative abundance of the fecal microbiota genus and the tryptophan metabolites in each group. There was a strong correlation between ldh4 gene abundance and ILA concentration in the fecal fermentation. The copy number of ldh4 gene was significantly higher in the groups with the probiotic than that in the control group. In conclusion, synbiotics could enhance the production of signaling molecules in the gut environment. Our results provide an insight into more effective administration of probiotics at the molecular level.

Do female bonobos (Pan paniscus) disperse at the onset of puberty? Hormonal and behavioral changes related to their dispersal timing.

Natal dispersal is a milestone in an animal's life history, but its timing in developmental trajectories may differ between species. Although the two Pan species exhibit a similar pattern of female-biased dispersal, female bonobos (P. paniscus) leave their natal groups at an earlier age than female chimpanzees (P. troglodytes). As a preliminary step to explore the dispersal strategies of female bonobos, this study aimed to determine the relations of sexual swelling development, behavioral and hormonal activation, and first ovulation relative to dispersal timing. We measured levels of urinary estrone conjugates (E1C) and pregnanediol glucuronide (PdG) from 14 nulliparous females in wild bonobo groups at Wamba in the Democratic Republic of the Congo, and recorded their copulations with mature males. When close to dispersal, female bonobos exhibited swelling of the sexual skin (labia minora and perianal region) that did not reach the mature stage. Urinary E1C levels and copulation rates increased slightly before dispersal and greatly increased after dispersal. Ovulatory or gestatory signs implied by daily hormone profiles were not detected until one to two years after dispersal. Our findings indicate that female bonobos disperse at an early pubertal stage before ovulatory cycling is established. This earlier dispersal than sexual maturation could allow female bonobos to postpone reproduction-related energy costs until they become familiar with their new group or gain more time finding the group more suitable for successful reproduction in the future before actually settling. Further demographic and genetic data from dispersal to reproduction will help clarify their dispersal strategies.

Do immigrant female bonobos prefer older resident females as important partners when integrating into a new group?

Intergroup transfer is a critical part of the life history of group-living species, with considerable variation in its timings and patterns among species. Immigrant female bonobos are documented to smoothly integrate into a new group through forming affiliative relationships with old, high-ranking resident females (Idani, Folia Primatol 57:83-95, 1991). However, only a few studies are available on immigration costs and strategies for female bonobos. Here, we compared social relationships of natal females (known to be 4.5-7.2 years old) and immigrant females (estimated to be 6.8-12.3 years old) from one bonobo group at Wamba in the Luo Scientific Reserve, Democratic Republic of the Congo. Similar to previous studies, resident females did not appear to spatially isolate immigrant females or act aggressively toward them. However, resident males were more frequently aggressive toward immigrant females than toward natal females. Both natal and immigrant females tended to groom high-ranking females more than middle- and low-ranking females, although immigrant females spent more time grooming unrelated females than natal females. Immigrant females did not exhibit rank-related partner preference for genito-genital rubbing and copulation. Although we did not control for age differences because of the small sample size, our results provide partial support for the hypothesis that old female bonobos are important partners for the successful integration of young females into an unfamiliar group. This strategy could explain why female bonobos disperse before reaching sexual maturity, which contrasts with the need for female chimpanzees to display sexual swellings and draw male interest as protection against aggression from resident females.

Two wild female bonobos adopted infants from a different social group at Wamba.

Adoption, the act of taking another individual's offspring and treating it as one's own, is rare but widely observed in various mammal species and may increase the survival of adoptees. Adoption may also benefit adoptive mothers, for example they might care for close kin to gain indirect fitness or to learn caregiving behaviours. Here, we report two cases of a wild bonobo adopting an infant from a different social group, the first report of cross-group adoption in great apes. In one case, the adoptive mother was already a mother of two dependent offspring. In the other case, the adoptive mother was an old parous female whose own offspring had already emigrated into a different social group. The adoptive mothers provided various maternal care to the adoptees, such as carrying, grooming, nursing, and sharing food. No aggression was observed by group members towards the out-group adoptees. In both cases, adoptees had no maternal kin-relationship with their adoptive mothers. Both adoptive mothers already had experience of rearing their own offspring. Instead, these cases of adoption may have been driven by other evolutionary adaptive traits of bonobos, such as their strong attraction to infants and high tolerance towards immatures and out-group individuals.

Age and sex differences in juvenile bonobos in party associations with their mothers at Wamba.

Mothers are the most fundamental individuals for the survival and development of their immature offspring. Sex-biased dispersal has an effect on strong and long-lasting social bonding between mothers and their philopatric offspring. Nepotistic relationships are often seen in species with male-biased dispersal but less frequently in species with female-biased dispersal. Bonobos are unique among female-biased dispersal primates in that the females are codominant with males, exert influence on the mating opportunities of their mature sons, and tolerate each other regardless of relatedness. Few studies on bonobos have examined sex differences in social relationships between juveniles and their mothers, and how this relates to mother-son and female-female cooperative relationships amongst adults. We investigated whether the association index of juveniles with their mothers changed with age depending on the sex of the juveniles in parties of bonobos at Wamba, the Democratic Republic of the Congo. We found that female bonobos had decreased party associations with their mothers prior to their emigration from the natal group, while male bonobos maintained relatively high party associations with their mothers. These results indicate that sex differences that emerge during the juvenile period in bonobos in their spatial relationships with their mothers differ from previous findings on chimpanzees, spider monkeys, and muriquis, in which philopatric sons tend to spend less time in proximity to their mothers and disperse earlier than, or at a similar age to, daughters. The contrasts between bonobos and other primate species suggest that the high social status of female bonobos relative to that of males may facilitate the extension of maternal support for their philopatric sons, and that high tolerance among unrelated females may enable the spatial independence of daughters from their mothers at an earlier stage.

Comparisons of between-group differentiation in male kinship between bonobos and chimpanzees.

Patterns of kinship among individuals in different groups have been rarely examined in animals. Two closest living relatives of humans, bonobos and chimpanzees share many characteristics of social systems including male philopatry, whereas one major difference between the two species is the nature of intergroup relationship. Intergroup relationship is basically antagonistic and males sometimes kill individuals of other groups in chimpanzees, whereas it is much more moderate in bonobos and copulations between individuals of different groups are often observed during intergroup encounters. Such behavioural differences may facilitate more frequent between-group male gene flow and greater between-group differentiation in male kinship in bonobos than in chimpanzees. Here we compared differences between average relatedness among males within groups and that among males of neighbouring groups, and between-group male genetic distance between bonobos and chimpanzees. Contrary to expectation, the differences between average relatedness among males within groups and that among males of neighbouring groups were significantly greater in bonobos than in chimpanzees. There were no significant differences in autosomal and Y-chromosomal between-group male genetic distance between the two species. Our results showed that intergroup male kinship is similarly or more differentiated in bonobos than in chimpanzees.

Heat-Killed Bifidobacterium breve B-3 Enhances Muscle Functions: Possible Involvement of Increases in Muscle Mass and Mitochondrial Biogenesis.

A previous clinical study on pre-obesity subjects revealed that Bifidobacterium breve B-3 shows anti-obesity effects and possibly increases muscle mass. Here, we investigated the effects of B-3 on muscle function, such as muscle strength and metabolism, and some signaling pathways in skeletal muscle. Male rodents were orally administered live B-3 (B-3L) or heat-killed B-3 (B-3HK) for 4 weeks. We found that administration of B-3 to rats tended to increase muscle mass and affect muscle metabolism, with stronger effects in the B-3HK group than in the B-3L group. B-3HK significantly increased muscle mass and activated Akt in the rat soleus. With regard to muscle metabolism, B-3HK significantly increased phosphorylated AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α and cytochrome c oxidase (CCO) gene expression in the rat soleus, suggesting an effect on the AMPK-PGC1α-mitochondrial biogenesis pathway. Furthermore, B-3HK promoted oxidative muscle fiber composition in the gastrocnemius. We also observed a significantly higher level of murine grip strength in the B-3HK group than in the control group. These findings suggest the potential of heat-killed B-3 in promoting muscle hypertrophy and modifying metabolic functions, possibly through the Akt and AMPK pathways, respectively.

Isolation and characterization of a novel oligomeric proanthocyanidin with significant anti-cancer activities from grape stems (Vitis vinifera).

Novel proanthocyanidin fractions from grape stem extracts were purified using Amberlite XAD-1180N, Sephadex-LH-20, Toyopearl HW40F and reverse phase high-performance liquid chromatography. Two key compounds were estimated as epigallocatechin-(epicatechin)7 gallate using electron-spray ionization time-of-flight mass spectrometry. Epigallocatechin-(epicatechin)7 gallate (compound 1) showed significant anti-cancer activity in PC-3 prostate cancer cells. In particular, compound 1 suppressed the gene expression of fatty acid-binding protein 5 (FABP5), which is involved in promoting cell proliferation and metastasis in prostate cancer cells.

Neonatal oral fluid as a transmission route for bifidobacteria to the infant gut immediately after birth.

Bifidobacteria are one of the most abundant bacterial groups in the infant gut microbiota and are closely associated with infant health and can potentially affect health in later life. However, the details regarding the source of bifidobacteria have yet to be completely elucidated. This study aimed to assess neonatal oral fluid (OF) as a transmission route for bifidobacteria to the infant gut during delivery. Neonatal OF and infant feces (IF) were collected immediately and one month after birth from 15 healthy vaginally delivered newborns. Bifidobacterium strains were isolated from OF and IF samples, and the similarity of strains between the OF-IF pairs was evaluated based on the average nucleotide identity (ANI) value. The 16S rRNA gene sequencing results revealed the presence of Bifidobacteriaceae at >1% relative abundance in all OF samples. Bifidobacterium strains were isolated from OF (9/15) and IF (11/15) samples, and those sharing high genomic homology (ANI values >99.5%) between the neonatal OF and IF samples were present in one-third of the OF-IF pairs. The results of this study indicate that viable bifidobacteria are present in neonatal OF and that OF at birth is a possible transmission route of bifidobacteria to the infant gut.

Bonobos' saliva remaining on the pith of terrestrial herbaceous vegetation can serve as non-invasive wild genetic resources.

Evaluating the genetic diversity of natural populations of endangered species is important for conservation. Although the genetic analysis of wildlife usually requires collecting DNA non-invasively, the variety of non-invasive DNA sampling methods is limited for each species. We present a method to obtain DNA of an endangered species, the bonobo (Pan paniscus), in which the pith of the terrestrial herbaceous vegetation (THV) that they consumed was newly utilized. We investigated the (1) frequency of encountering remnant saliva on three types of THV pith; (2) concentrations of DNA in the saliva samples by the real-time quantitative PCR; and (3) rates of positive PCR, accurate genotyping, and allelic drop out by analyzing two autosomal microsatellite loci (D7s817 and D9s910). The number of remnant saliva samples was recorded by following the bonobo groups on a daily basis. The frequency of encountering DNA samples was higher in saliva samples than in fecal samples. More than half of the saliva samples remaining on two types of THV pith provided sufficient concentrations of bonobo DNA (> 200 pg/µl). Rates of positive PCR and accurate genotyping were high, and allelic drop out rate was low when the amount of template DNA was above 200 pg per reaction. Our results suggest that the remnants of bonobo saliva on the pith of THV are a potential resource for obtaining DNA, and better than other kinds of samples from the perspective of the abundant sampling opportunities.

Paternity and kin structure among neighbouring groups in wild bonobos at Wamba.

Although both bonobos and chimpanzees are male-philopatric species, outcomes of male-male reproductive competition seem to be more closely associated with mating success in chimpanzees. This suggests that the extent of male reproductive skew is lower in bonobos. In addition, between-group male-male reproductive competition is more lethal in chimpanzees. This suggests that between-group differentiation in male kinship is lower in bonobos. We analysed the paternity of 17 offspring in two bonobo groups and estimated the relatedness of individuals among three neighbouring groups by using DNA extracted from non-invasive samples at Wamba, in the Democratic Republic of the Congo. The alpha males sired at least nine of 17 offspring. This supports a previous finding that the male reproductive skew is higher in bonobos than that in chimpanzees. Average relatedness among males within groups was significantly higher than that among males across groups, whereas there was no significant difference among females between within and across groups. These results are consistent with male philopatry, highly skewed reproductive success of males and female dispersal. Higher average relatedness among males within groups suggest that the differences in hostility towards males of different groups between bonobos and chimpanzees may be explained by factors other than kinship.

Fermented Rice Germ Extract Alleviates Morphological and Functional Damage to Murine Gastrocnemius Muscle by Inactivation of AMP-Activated Protein Kinase.

Sarcopenia, loss of muscle mass and function, is mainly observed in elderly people. In this study, we investigated whether fermented rice germ extract (FRGE) has some effects on the mouse gastrocnemius muscle by using behavioral and morphological analyses, Western blotting, and a murine model of immobilization-induced muscle atrophy. Daily oral FRGE administration increased muscle weight and strength. In addition, myofiber size in gastrocnemius muscle of FRGE-treated mice was increased as revealed by morphological quantification. Activation of AMP-activated protein kinase (AMPK) signaling, which inhibits protein synthesis and stimulates protein degradation in gastrocnemius muscle, was significantly attenuated in the FRGE-treated mice compared with control mice. Expression level of forkhead box 3a (FOXO3a) protein was also significantly decreased in the FRGE-treated group. Moreover, the decrease in mean myofiber cross-sectional area in immobilized hindlimb in vehicle-treated mice was inhibited by FRGE treatment in histological analysis. In conclusion, FRGE increased the strength and weight of gastrocnemius muscle and myofiber size, and reduced immobilization-induced muscle atrophy in mice. These findings indicated that FRGE might be beneficial in preventing motor dysfunction in a range of conditions, including sarcopenia.

Passionflower Extract Induces High-amplitude Rhythms without Phase Shifts in the Expression of Several Circadian Clock Genes in Vitro and in Vivo.

Circadian rhythms play key roles in the regulation of physiological and behavioral systems including wake-sleep cycles. We evaluated the effects of passionflower (aerial parts of Passiflora incarnata Linnaeus) extract (PFE) on circadian rhythms using NIH3T3 cells and mice. PFE (100 µg/mL) induced high-amplitude rhythms in the expression of period circadian protein (Per) 2, cryptochrome (Cry) 1, superoxide dismutase (SOD) 1, and glutathione peroxidase (GPx) in vitro from 12 h after a treatment with serum-rich medium. Isovitexin 2"-O-glucoside, isoschaftoside, and homoorientin, which were purified from PFE, also significantly enhanced Per2 mRNA expression at 20 h. An oral treatment with PFE (100 mg/kg/day) at zeitgeber time (ZT) 0 h for 15 days improved sleep latencies and sleeping times in the pentobarbital-induced sleep test in mice, similar to muscimol (0.2 mg/kg, i.p.). PFE induced high-amplitude rhythms without obvious phase shifts in serum corticosterone levels and the expression of Per1, Per2, and Cry1 in the liver as well as NIH3T3 cells. However, in the cerebrum, PFE enhanced the circadian expression of brain-muscle ARNT-like protein (Bmal) 1, circadian locomotor output cycles kaput (Clock), and Per1. Regarding this difference, we suggest the involvement of several neurotransmitters that influence the circadian rhythm. Indeed, PFE significantly increased dopamine levels at ZT 18 h, and then affected the mRNA expression of the synthetic and metabolic enzymes such as monoamine oxidase (MAO), catechol-O-methyltransferase (COMT), and glutamic acid decarboxylase (GAD). The results obtained show that PFE positively modulates circadian rhythms by inducing high-amplitude rhythms in the expression of several circadian clock genes.

Epicatechin oligomers longer than trimers have anti-cancer activities, but not the catechin counterparts.

Since procyanidins (oligomeric catechin or epicatechin) were reported to exhibit health benefits, much attention has been paid to the synthesis of these compounds, especially those that are longer than trimers. In the present study, syntheses of cinnamtannin A3 (epicatechin pentamer), A4 (epicatechin hexamer), catechin tetramer, pentamer, arecatannin A2 (epicatechin-epicatechin-epicatechin-catechin) and A3 (epicatechin-epicatechin-epicatechin-epicatechin-catechin) were achieved. The key reaction was a Lewis acid mediated equimolar condensation. The antitumor effects of these synthesized compounds against a human prostate cancer cell line (PC-3) were investigated. Among the tested compounds, cinnamtannin A3, A4 and arecatannin A3, which possess epicatechin oligomers longer than tetramers as the basic scaffold, showed significant activities for suppression of cell growth, invasion and FABP5 (fatty acid-binding protein 5) gene expression. Effects on cell cycle distribution showed that cell cycle arrest in the G2 phase was induced. Furthermore, these epicatechin oligomers suppressed significantly the expression of the cancer-promoting gene, FABP5, which is related to cell proliferation and metastasis in various cancer cells. Interestingly, the suppressive activities were associated with the degree of oligomerization of epicatechin. Thus, synthetic studies clearly demonstrate that epicatechin oligomers longer than trimers have significant anti-tumorigenic activities, but not the catechin counterparts.

Black ginger extract increases physical fitness performance and muscular endurance by improving inflammation and energy metabolism.

We previously reported that polymethoxyflavones (PMFs) in black ginger (Kaempferia parviflora) extract (KPE) increased energy production by activating AMP-activated protein kinase (AMPK) in C2C12 myoblasts. We herein evaluated the effects of KPE on physical fitness performance and muscular endurance in mice. Male mice were orally administered KPE for 4 weeks, and then forced swimming test, open-field test, inclined plane test, and wire hanging test were performed. KPE significantly increased the swimming time, motility after swimming, and grip strength. IL-6 and TNF-α mRNA expression levels were decreased in the soleus muscle, whereas peroxisome proliferator-activated receptor γ coactivator (PGC)-1α and glycogen synthase mRNA expression levels, mitochondrial number, and glycogen content were increased. These results were in agreement with those obtained for KPE and PMFs in C2C12. Therefore, the activation of AMPK by PMFs may be one of the mechanisms by which KPE improves physical fitness performance and muscular endurance.

Enhancement of energy production by black ginger extract containing polymethoxy flavonoids in myocytes through improving glucose, lactic acid and lipid metabolism.

Enhancement of muscular energy production is thought to improve locomotive functions and prevent metabolic syndromes including diabetes and lipidemia. Black ginger (Kaempferia parviflora) has been cultivated for traditional medicine in Thailand. Recent studies have shown that black ginger extract (KPE) activated brown adipocytes and lipolysis in white adipose tissue, which may cure obesity-related dysfunction of lipid metabolism. However, the effect of KPE on glucose and lipid utilization in muscle cells has not been examined yet. Hence, we evaluated the effect of KPE and its constituents on energy metabolism in pre-differentiated (p) and differentiated (d) C2C12 myoblasts. KPE (0.1-10 µg/ml) was added to pC2C12 cells in the differentiation process for a week or used to treat dC2C12 cells for 24 h. After culturing, parameters of glucose and lipid metabolism and mitochondrial biogenesis were assessed. In terms of the results, KPE enhanced the uptake of 2-deoxyglucose and lactic acid as well as the mRNA expression of glucose transporter (GLUT) 4 and monocarboxylate transporter (MCT) 1 in both types of cells. The expression of peroxisome proliferator-activated receptor γ coactivator (PGC)-1α was enhanced in pC2C12 cells. In addition, KPE enhanced the production of ATP and mitochondrial biogenesis. Polymethoxy flavonoids in KPE including 5-hydroxy-7-methoxyflavone, 5-hydroxy-3,7,4'-trimethoxyflavone and 5,7-dimethoxyflavone enhanced the expression of GLUT4 and PGC-1α. Moreover, KPE and 5,7-dimethoxyflavone enhanced the phosphorylation of 5'AMP-activated protein kinase (AMPK). In conclusion, KPE and its polymethoxy flavonoids were found to enhance energy metabolism in myocytes. KPE may improve the dysfunction of muscle metabolism that leads to metabolic syndrome and locomotive dysfunction.

Syntheses of procyanidin B2 and B3 gallate derivatives using equimolar condensation mediated by Yb(OTf)3 and their antitumor activities.

Synthesis of procyanidin B2 and B3 gallate derivatives, 3-O-gallate, 3"-O-gallate, and 3,3"-di-O-gallate, were synthesized using equimolar condensation mediated by Yb(OTf)3. Synthesized compounds showed significant antitumor effects against human prostate PC-3 cell lines. Their activities were weaker than well-known EGCG and prodelphinidin B3.

Estimation of the hydrophobicity of 2,4-diphenyl-1,3-oxazoline analogs and QSAR analysis of their ovicidal activity against Tetranychus [corrected] urticae.

Partition coefficients of six 2-phenyl-1,3-oxazoline congeners containing 2-I, 2-NO2, 2-CF3, 2,6-(CH3)2, 2,6-F2, and 2-F-6-Cl substitutions on the phenyl moiety were measured in a 1-octanol/water system using the flask-shaking method. The effect on the hydrophobicity (LogP) of substituents on the phenyl moiety of 2-phenyl-1,3-oxazolines linearly correlated with that of benzamide congeners. logP values of other 2-(substituted phenyl)-1,3-oxazoline analogs were empirically estimated from the corresponding substituted benzamides. The ovicidal activity of 2-(substituted phenyl)-4-phenyl-1,3-oxazoline analogs against the two-spotted spider mite Tetranychus [corrected] urticae was quantitatively analyzed using the classical QSAR (Hansch-Fujita) method. Results showed that ovicidal activity increases with hydrophobicity. The introduction of inductive electron-withdrawing groups at ortho-positions increased ovicidal activity, but addition of steric bulk was unfavorable. Substitution at either the meta- or para-position was detrimental to the acaricidal activity.