Association of toxin-producing Clostridioides difficile with piglet diarrhea and potential transmission to humans.

The pathogenicity of Clostridioides difficile in piglets remains controversial. It is unknown whether C. difficile control helps protect piglet health. To clarify the association between C. difficile presence and piglet diarrhea, isolates were obtained from piglets with and without diarrhea. In addition, to determine the genetic relationship of C. difficile from pigs and humans, we performed whole-genome sequencing (WGS) of C. difficile isolates. Diarrheal and non-diarrheal stool samples were collected from neonatal piglets from five farms in Japan in 2021. To clarify the relationship between C. difficile derived from pigs and those from human clinical cases, WGS of C. difficile isolates was performed. Toxin-positive C. difficile were significantly more prevalent in piglets with diarrhea, although the overall frequency of C. difficile did not differ between piglets with and without diarrhea. This observation indicates an association between toxin-positive C. difficile and diarrhea in piglets. However, further studies are needed to establish a direct causal relationship and to explore other contributing factors to diarrhea in piglets. WGS results showed that C. difficile sequence type (ST)11 including the hypervirulent PCR ribotype 078 isolates derived from Japanese pigs were closely related to ST11 of overseas strains (human clinical and animal-derived) and a Japanese human clinical strain. Toxin-positive C. difficile may cause diarrhea in piglets and hypervirulent C. difficile are spreading among pigs and human populations worldwide.

Combination of tea catechins and ornithine effectively activates the urea cycle: an in vitro and human pilot study.

PURPOSE: Accumulation of ammonia causes central and peripheral fatigue. This study aimed to investigate the synergistic effect of tea catechins and low-dose ornithine in activating the urea cycle to reduce blood ammonia levels during exercise. METHODS: We used hepatocyte-like cells derived from human-induced pluripotent stem (iPS) cells to assess the effect of tea catechins combined with ornithine on urea cycle activity. The urea production and expression of key genes involved in the metabolism of urea were investigated. We then examined the synergistic improvement in ammonia metabolism by tea catechins in combination with ornithine in a human pilot study. RESULTS: Tea catechins combined with ornithine increased urea cycle activity in hepatocyte-like cells derived from human iPS cells. Intake of 538.6 mg of tea catechins with 1592 mg of ornithine for 2 consecutive days during exercise loading suppressed the exercise-induced increase in the blood ammonia concentration as well as stabilized blood glucose levels. CONCLUSION: Controlling the levels of ammonia, a toxic waste produced in the body, is important in a variety of situations, including exercise. The present study suggests that a heterogeneous combination of polyphenols and amino acids efficiently suppresses elevated ammonia during exercise in humans by a mechanism that includes urea cycle activation. TRIAL REGISTRATION: This study was registered in the University Hospital Medical Information Network Clinical Trial Registry (No. UMIN000035484, dated January 8, 2019).

Antiviral Effect of Propylene Glycol against Envelope Viruses in Spray and Volatilized Forms.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly contagious and continues to spread worldwide. To avoid the spread of infection, it is important to control its transmission routes. However, as methods to prevent airborne infections are lacking, people are forced to take measures such as keeping distance from others or wearing masks. Here, we evaluate the antiviral activity of propylene glycol (PG), which is safe, odorless, and volatile. PG showed pronounced antiviral activity against the influenza virus (IAV) at concentrations above 55% in the liquid phase. Given its IAV inactivation mechanism, which involves increasing the fluidity of the viral membrane, PG is expected to have a broad effect on enveloped viruses. PG showed antiviral activity against SARS-CoV-2. We also developed a system to evaluate the antiviral effect of PG in spray and volatilized forms. PG was found to be effective against aerosol IAV in both forms; the effective PG concentration against IAV in the vapor phase was 87 ppmv (0.27 mg/L). These results demonstrate that PG is an effective means for viral inactivation in various situations for infection control. This technology is expected to control the spread of current and future infectious diseases capable of causing outbreaks and pandemics.

Tyrosine Is a Booster of Leucine-Induced Muscle Anabolic Response.

Leucine (Leu), an essential amino acid, is known to stimulate protein synthesis in the skeletal muscle via mTOR complex 1 (mTORC1) activation. However, the intrinsic contribution of other amino acids to Leu-mediated activation of mTORC1 signaling remains unexplored. This study aimed to identify amino acids that can promote mTORC1 activity in combination with Leu and to assess the effectiveness of these combinations in vitro and in vivo. We found that tyrosine (Tyr) enhanced Leu-induced phosphorylation of S6 kinase (S6K), an indicator of mTORC1 activity, although it exerted no such effect individually. This booster effect was observed in C2C12 cells, isolated murine muscle, and the skeletal muscles of mice orally administered the amino acids. To explore the molecular mechanisms underlying this Tyr-mediated booster effect, the expression of the intracellular Leu sensors, Sestrin1 and 2, was suppressed, and the cells were treated with Leu and Tyr. This suppression enabled Tyr alone to induce S6K phosphorylation and enhanced the booster effect, suggesting that Tyr possibly contributes to mTORC1 activation when Sestrin-GAP activity toward Rags 2 (GATOR2) is dissociated through Sestrin knockdown or the binding of Sestrins to Leu. Collectively, these results indicate that Tyr is a key regulator of Leu-mediated protein synthesis.

Protein-bound sialic acid in saliva contributes directly to salivary anti-influenza virus activity.

The oral cavity is an entrance for respiratory viruses, such as influenza. Recently, saliva has been shown to exert both antimicrobial and antiviral activities. Thus, saliva may be a biological factor that contributes to the prevention of influenza infection. However, the actual salivary anti-influenza A virus (IAV) activity in individuals and its determinant factors are unknown. By assessing individual variations in salivary anti-IAV activity in 92 people using an established new high-throughput system in this study, we found that the anti-IAV activity varied widely between individuals and showed a significant positive correlation with protein-bound sialic acid (BSA) level (ρ = 0.473; p < 0.001). Furthermore, the anti-IAV activity of saliva with enzymatically reduced BSA content was significantly lower. These results indicate that BSA is a direct regulator of salivary anti-IAV activity and is a determinant of individual differences. Additionally, after comparing the anti-IAV activity across the groups by age, anti-IAV activity in young people (aged 5-19 years) were lower than in adults aged 20-59 years and elderly people aged 60-79 years. Our study suggests that BSA levels in saliva may be important in preventing influenza infection.

Non-invasive human skin transcriptome analysis using mRNA in skin surface lipids.

Non-invasive acquisition of mRNA data from the skin can be extremely useful for understanding skin physiology and diseases. Inspired by the holocrine process, in which the sebaceous glands secrete cell contents into the sebum, we focused on the possible presence of mRNAs in skin surface lipids (SSLs). We found that measurable levels of human mRNAs exist in SSLs, where the sebum protects them from degradation by RNases. The AmpliSeq transcriptome analysis was modified to measure SSL-RNA levels, and our results revealed that the SSL-RNAs predominantly comprised mRNAs derived from sebaceous glands, the epidermis, and hair follicles. Analysis of SSL-RNAs non-invasively collected from patients with atopic dermatitis revealed increased expression of inflammation-related genes and decreased expression of terminal differentiation-related genes, consistent with the results of previous reports. Further, we found that lipid synthesis-related genes were downregulated in the sebaceous glands of patients with atopic dermatitis. These results indicate that the analysis of SSL-RNAs is a promising strategy to understand the pathophysiology of skin diseases.

Non-invasive evaluation of subjective sensitive skin by transcriptomics using mRNA in skin surface lipids.

Sensitive skin is a condition characterized by hypersensitivity to environmental stimuli, and its pathophysiology has not been fully elucidated. Questionnaires based on subjective symptoms, intervention tests, and measuring devices are used to diagnose sensitive skin; however, objective evaluation methods, including biomarkers, remain to be established. This study aimed to investigate the molecular profiles of self-reported sensitive skin, understand its pathophysiology and explore its biomarkers. Here, we analysed RNAs in skin surface lipids (SSL-RNAs), which can be obtained non-invasively by wiping the skin surface with an oil-blotting film, to compare the transcriptome profiles between questionnaire-based "sensitive" (n = 11) and "non-sensitive" (n = 10) skin participants. Exactly 417 differentially expressed genes in SSL-RNAs from individuals with sensitive skin were identified, of which C-C motif chemokine ligand 17 and interferon-γ pathways were elevated, while 50 olfactory receptor (OR) genes were downregulated. The expression of the detectable 101 OR genes was lower in individuals with sensitive skin compared to that in those with non-sensitive skin and was particularly associated with the subjective sensitivity among skin conditions. The receiver operating characteristic (ROC) curve demonstrated that the mean expression levels of OR genes in SSL-RNAs could discriminate subjective skin sensitivity with an area under the ROC curve of 0.836. SSL-RNA profiles suggest a mild inflammatory state in sensitive skin, and overall OR gene expression could be a potential indicator for sensitive skin.

Non-invasive diagnostic tool for Parkinson's disease by sebum RNA profile with machine learning.

Parkinson's disease (PD) is a progressive neurodegenerative disease presenting with motor and non-motor symptoms, including skin disorders (seborrheic dermatitis, bullous pemphigoid, and rosacea), skin pathological changes (decreased nerve endings and alpha-synuclein deposition), and metabolic changes of sebum. Recently, a transcriptome method using RNA in skin surface lipids (SSL-RNAs) which can be obtained non-invasively with an oil-blotting film was reported as a novel analytic method of sebum. Here we report transcriptome analyses using SSL-RNAs and the potential of these expression profiles with machine learning as diagnostic biomarkers for PD in double cohorts (PD [n = 15, 50], controls [n = 15, 50]). Differential expression analysis between the patients with PD and healthy controls identified more than 100 differentially expressed genes in the two cohorts. In each cohort, several genes related to oxidative phosphorylation were upregulated, and gene ontology analysis using differentially expressed genes revealed functional processes associated with PD. Furthermore, machine learning using the expression information obtained from the SSL-RNAs was able to efficiently discriminate patients with PD from healthy controls, with an area under the receiver operating characteristic curve of 0.806. This non-invasive gene expression profile of SSL-RNAs may contribute to early PD diagnosis based on the neurodegeneration background.

Investigation of the Oral Retention of Tea Catechins in Humans: An Exploratory Interventional Study.

Green tea catechin ingestion or gargling exhibit anti-viral activity against upper respiratory infection. We hypothesized that retention in the oral cavity could improve the anti-viral effects of catechins. The present study investigated the oral retention of catechins in humans and the effect of catechin beverage viscosity on oral retention. Two intervention studies with different test beverages, beverage-C (40 mL, containing 73.4 mg of catechins) and beverage-XT (40 mL, beverage-C containing 100 mg xanthan gum) were conducted in 20 healthy volunteers (mean age 38.7 years). Catechin concentrations were measured in buccal mucosa samples collected at 10 min, 40 min, and 60 min after ingesting test beverages, and the catechin variability of the tissue after intake was compared between test beverages. As a result, the mean (SEM) concentrations of EGCG were 99.9 (27.2), 58.2 (16.6), and 22.3 (5.7) ng/mg-mucosa at 10, 40, and 60 min, respectively, after ingestion of beverage-XT. Similarly, the catechin concentrations were 86.1 (20.3), 32.2 (5.3), and 27.8 (5.9) ng/mg-mucosa after ingestion of beverage-C. The total retention volume over 60 min tended to be slightly higher after ingestion of beverage-XT, though the difference was not statistically significant. Additional studies are needed to confirm the effect of xanthan gum on improving oral retention of catechins.

The Impact of Milk Fat Globule Membrane with Exercise on Age-Related Degeneration of Neuromuscular Junctions.

Morphological changes in neuromuscular junctions (NMJs), which are synapses formed between α-motor neurons and skeletal muscle fibers, are considered to be important in age-related motor dysfunction. We have previously shown that the intake of dietary milk fat globule membrane (MFGM) combined with exercise attenuates age-related NMJ alterations in the early phase of aging. However, it is unclear whether the effect of MFGM with exercise on age-related NMJ alterations persists into old age, and whether intervention from old age is still effective when age-related changes in NMJs have already occurred. In this study, 6- or 18-month-old mice were treated with a 1% MFGM diet and daily running wheel exercise until 23 or 24 months of age, respectively. MFGM treatment with exercise was effective in suppressing the progression of age-related NMJ alterations in old age, and even after age-related changes in NMJs had already occurred. Moreover, the effect of MFGM intake with exercise was not restricted to NMJs but extended to the structure and function of peripheral nerves. This study demonstrates that MFGM intake with exercise may be a novel approach for improving motor function in the elderly by suppressing age-related NMJ alterations.

Tape stripping method is useful for the quantification of antimicrobial peptides on the human skin surface including the stratum corneum.

Antimicrobial peptides (AMPs) play an important role in innate immunity in human skin. It is known that AMPs mainly function in the stratum corneum. Therefore, AMP concentrations in the stratum corneum need to be precisely measured to clarify functional and physiological importance of AMPs in cutaneous defence. Tape stripping (TS) is a well-established method by which components in the stratum corneum can be collected. However, the usefulness of the TS method for measuring AMP concentration in human skin remains unclear. Therefore, we compared it with another popular method, skin rinsing, which had been established as a method for measuring AMP concentration in human skin. When investigated on healthy medial forearm using RNase 7, which is one of the typical AMPs, as an index, there was a significant positive correlation between RNase 7 concentrations measured by the TS method at adjacent forearm sites, demonstrating the reproducibility of the TS method. Next, a significant positive correlation was detected in RNase 7 concentrations measured using the TS and the skin rinsing method, indicating that the TS method is comparable to the skin rinsing method. Thus, we speculate that the TS method is useful for measuring AMP concentration in human skin.

DOK7 Gene Therapy Enhances Neuromuscular Junction Innervation and Motor Function in Aged Mice.

Muscle denervation at the neuromuscular junction (NMJ), the essential synapse between motor neuron and skeletal muscle, is associated with age-related motor impairment. Therefore, improving muscle innervation at aged NMJs may be an effective therapeutic strategy for treating the impairment. We previously demonstrated that the muscle protein Dok-7 plays an essential role in NMJ formation, and, indeed, its forced expression in muscle enlarges NMJs. Moreover, therapeutic administration of an adeno-associated virus vector encoding human Dok-7 (DOK7 gene therapy) suppressed muscle denervation and enhanced motor activity in a mouse model of amyotrophic lateral sclerosis (ALS). Here, we show that DOK7 gene therapy significantly enhances motor function and muscle strength together with NMJ innervation in aged mice. Furthermore, the treated mice showed greatly increased compound muscle action potential (CMAP) amplitudes compared with the controls, suggesting enhanced neuromuscular transmission. Thus, therapies aimed at enhancing NMJ innervation have potential for treating age-related motor impairment.

Involvement of ammonia metabolism in the improvement of endurance performance by tea catechins in mice.

Blood ammonia increases during exercise, and it has been suggested that this increase is both a central and peripheral fatigue factor. Although green tea catechins (GTCs) are known to improve exercise endurance by enhancing lipid metabolism in skeletal muscle, little is known about the relationship between ammonia metabolism and the endurance-improving effect of GTCs. Here, we examined how ammonia affects endurance capacity and how GTCs affect ammonia metabolism in vivo in mice and how GTCs affect mouse skeletal muscle and liver in vitro. In mice, blood ammonia concentration was significantly negatively correlated with exercise endurance capacity, and hyperammonaemia was found to decrease whole-body fat expenditure and fatty acid oxidation-related gene expression in skeletal muscle. Repeated ingestion of GTCs combined with regular exercise training improved endurance capacity and the expression of urea cycle-related genes in liver. In C2C12 myotubes, hyperammonaemia suppressed mitochondrial respiration; however, pre-incubation with GTCs rescued this suppression. Together, our results demonstrate that hyperammonaemia decreases both mitochondrial respiration in myotubes and whole-body aerobic metabolism. Thus, GTC-mediated increases in ammonia metabolism in liver and resistance to ammonia-induced suppression of mitochondrial respiration in skeletal muscle may underlie the endurance-improving effect of GTCs.

5-Caffeoylquinic Acid Ameliorates Cognitive Decline and Reduces Aß Deposition by Modulating Aß Clearance Pathways in APP/PS2 Transgenic Mice.

The accumulation of amyloid ß (Aß) in the brain is a major pathological feature of Alzheimer's disease (AD). In our previous study, we demonstrated that coffee polyphenols (CPP) prevent cognitive dysfunction and Aß deposition in the brain of an APP/PS2 transgenic mouse AD model. The underlying mechanisms, however, remain to be elucidated. Here, we investigated the effects of the chronic administration of 5-caffeoylquinic acid (5-CQA), the most abundant component of CPP, on cognitive dysfunction in APP/PS2 mice to identify the role of CPP in Aß elimination. Relative to the untreated controls, the mice fed a 5-CQA-supplemented diet showed significant improvements in their cognitive function assessed by Y-maze and novel object recognition tests. Histochemical analysis revealed that 5-CQA substantially reduced Aß plaque formation and neuronal loss in the hippocampi. Moreover, 5-CQA upregulated the gene encoding low-density lipoprotein receptor-related protein 1, an Aß efflux receptor, and normalized the perivascular localization of aquaporin 4, which facilitates Aß clearance along the paravascular pathway. These results suggest that 5-CQA reduces Aß deposition in the brain by modulating the Aß clearance pathways and ameliorating cognitive decline and neuronal loss in APP/PS2 mice. Thus, 5-CQA may be effective in preventing cognitive dysfunction in AD.

Development of a cognitive function marker based on D-amino acid proportions using new chiral tandem LC-MS/MS systems.

The incidence of dementia, a clinical symptom characterized by severe cognitive decline, is increasing worldwide. Predictive biomarkers are therefore required for early identification and management. D-amino acids in the brain contribute to cognitive function and are suggested as useful biomarkers for diagnosing dementia risk. To clarify their relationship with human cognitive decline, we developed an identification method of chiral metabolomics for detecting slight differences in chiral amino acid amounts. Chiral tandem liquid chromatography-tandem mass spectrometry systems were applied for sensitive and selective amino acid species along with chiral species determination based on anion and zwitterion exchange mechanisms. In a comprehensive health cohort (cross-sectional study), we measured blood chiral amino acid levels from 305 women (65-80 years old) classified into Control, Mild-cognitive-Impairment (MCI), and Dementia groups using the Mini-Mental State Examination. MCI exhibited higher D-Pro (D-Pro/(D-Pro + L-Pro)) proportion vs the Control group, suggesting this proportion as a useful biomarker for MCI. Biomarker accuracy was improved in combination with D-Ser proportion. Receiver operating characteristics analysis of the Control vs. MCI proportion obtained area under the curve (0.80) with 70% sensitivity and 84% specificity at the optimal cutoff value (0.30). Thus, dementia monitoring can be improved by including trace D-amino acids measurements.

Coffee polyphenols prevent cognitive dysfunction and suppress amyloid ß plaques in APP/PS2 transgenic mouse.

Epidemiological studies have found that habitual coffee consumption may reduce the risk of Alzheimer's disease. Coffee contains numerous phenolic compounds (coffee polyphenols) such as chlorogenic acids. However, evidence demonstrating the contribution of chlorogenic acids to the prevention of cognitive dysfunction induced by Alzheimer's disease is limited. The present study investigated the effect of chlorogenic acids on the prevention of cognitive dysfunction in APP/PS2 transgenic mouse model of Alzheimer's disease. Five-week-old APP/PS2 mice were administered a diet supplemented with coffee polyphenols daily for 5 months. The memory and cognitive function of mice was determined using the novel object recognition test, Morris water maze test, and the step-through passive avoidance test. Immunohistochemical analysis revealed that chronic treatment with coffee polyphenols prevented cognitive dysfunction and significantly reduced the amount of amyloid ß (Aß) plaques in the hippocampus. Furthermore, we determined that 5-caffeoylquinic acid, one of the primary coffee polyphenols, did not inhibit Aß fibrillation; however, degraded Aß fibrils. In conclusion, our results demonstrate that coffee polyphenols prevent cognitive deficits and reduce Aß plaque deposition via disaggregation of Aß in the APP/PS2 mouse.

Continuous Supplementation of Milk Fat Globule Membrane with Habitual Exercise from a Young Age Improves Motor Coordination and Skeletal Muscle Function in Aged Mice.

Since the decline of physical performance gradually progresses with aging, continuous exercise with nutritional supplementation from a young age is a feasible and effective way to maintain a comfortable life until late old age. We examined the effects of continuous milk fat globule membrane (MFGM) supplementation combined with voluntary running exercise (VR) for prevention of aging-associated declines in physical performance in naturally aging mice. The MFGM with VR group showed a significantly attenuated age-related decline in motor coordination and suppression of the loss of muscle mass and strength. Compared with the control group, the MFGM with VR group showed significantly higher mRNA and protein expression for docking protein 7, which maintains neuromuscular junction (NMJ) integrity, in the quadriceps muscles. These results suggest that dietary MFGM and VR attenuate natural aging-related decline in motor coordination and muscle function by regulating NMJ integrity.

Dietary instructions focusing on meal-sequence and nutritional balance for prediabetes subjects: An exploratory, cluster-randomized, prospective, open-label, clinical trial.

BACKGROUND: Although lifestyle modifications are known to be effective in type 2 diabetes (T2D) as well as in prediabetes, adherence to a healthy diet is difficult for some, and interventions of lifestyle modifications need to be revised occasionally. Meal sequence has been gaining attention as a part of a healthy diet among T2D individuals to improve glycemia and body weight. In addition, a dietary instruction program, SMART Washoku®, which can help individuals to consume a more nutritionally balanced diet, has been developed. METHODS: The current exploratory trial was designed to examine the effects of dietary instructions focusing on meal sequence and nutritional balance in individuals with prediabetes in the Japanese national health check-up and guidance program. Participants were cluster-randomized into three groups: Group A, receiving a conventional health guidance program (n = 11); Group B, receiving health guidance with dietary instructions focusing on meal sequence (n = 18); and Group C, receiving health guidance with dietary instructions focusing on nutritional balance (n = 13). Participants received health guidance education and various measurements before and 6 months after the instructions. RESULTS: Body weight in Group B was significantly reduced compared to that in Group A, with similar adherence, while the effects on glycemia were similar between the two Groups. Body weight reduction was greater in Group C compared to that in Group A, although adherence in Group C was significantly lower than that in Group A. CONCLUSION: The group receiving health guidance with dietary instructions focusing on meal sequence exhibited similar adherence and greater reduction in body weight than the group receiving conventional health guidance.

TRPM8-mediated cutaneous stimulation modulates motor neuron activity during treadmill stepping in mice.

Motor units are generally recruited from the smallest to the largest following the size principle, while cutaneous stimulation has the potential to affect spinal motor control. We aimed to examine the effects of stimulating transient receptor potential channel sub-family M8 (TRPM8) combined with exercise on the modulation of spinal motor neuron (MN) excitability. Mice were topically administrated 1.5% icilin on the hindlimbs, followed by treadmill stepping. Spinal cord sections were immunostained with antibodies against c-fos and choline acetyltransferase. Icilin stimulation did not change the number of c-fos+ MNs, but increased the average soma size of the c-fos+ MNs during low-speed treadmill stepping. Furthermore, icilin stimulation combined with stepping increased c-fos+ cholinergic interneurons near the central canal, which are thought to modulate MN excitability. These findings suggest that TRPM8-mediated cutaneous stimulation with low-load exercise promotes preferential recruitment of large MNs and is potentially useful as a new training method for rehabilitation.

Guava leaf extract suppresses osteoarthritis progression in a rat anterior cruciate ligament transection model.

Guava leaf extract and ellagic acid, one of its polyphenolic components, inhibit the activity of a disintegrin and metalloproteinase with thrombospondin type 5 (ADAMTS-5), which is associated with aggrecan degeneration during the early stage of osteoarthritis (OA). To investigate the efficacy of guava leaf extract for preventing OA, we examined the effect of its dietary intake on cartilage destruction in anterior cruciate ligament-transected (ACLT) rats. Rats were randomly assigned to four groups: ACLT control rats fed with control diet, ACLT rats fed with diet containing 0.2% guava leaf extract, ACLT rats fed with diet containing 0.5% guava leaf extract, and sham-operated rats fed with control diet. Mankin's scores, an index of cartilage damage, were higher in rats that underwent ACLT. Guava leaf extract treatment dose-dependently led to lower Mankin's scores and higher concentrations of ellagic acid in the serum and synovial membrane. Ellagic acid levels in the synovial membrane negatively correlated with cartilage destruction scores. These results suggest that dietary guava leaf extract suppresses OA progression in ACLT rats through ellagic acid-mediated inhibition of early joint destruction.