Metabolomic analyses of plasma and liver of mice fed with immature Citrus tumida peel.

In this study, we investigated the effects of dietary supplementation of Citrus tumida hort. ex Tanaka on food intake, body and fat tissue weights, and metabolic profiles of plasma and liver in mice. Supplementation with 5% (w/w) of peels of immature C. tumida (PIC) for 4 weeks significantly suppressed body weight gain and decreased adipose tissue weight in epididymal, perirenal, and subcutaneous fats. Metabolome analyses showed that 2-hydroxyvaleric acid levels were reduced in the blood plasma of mice fed with PIC. PIC supplementation significantly elevated dipeptide (Thr-Asp, Ser-Glu, and Ala-Ala), glucuronic acid, and S-methylglutathione levels, and significantly reduced betaine aldehyde levels in the liver. In conclusion, PIC supplementation affects the metabolism of fatty acids, pectin, glutathione, and choline, showing potential beneficial effects for metabolic syndrome and obesity. PIC may be developed as a functional food and used in the treatment of these diseases.

Dietary intake of the citrus flavonoid hesperidin affects stress-resilience and brain kynurenine levels in a subchronic and mild social defeat stress model in mice.

Depressive disorders are partly caused by chronic inflammation through the kynurenine (KYN) pathway. Preventive intervention using anti-inflammatory reagents may be beneficial for alleviating the risk of depression. In this study, we focused on the Japanese local citrus plant, Citrus tumida hort. ex Tanaka (C. tumida; CT), which contains flavonoids such as hesperidin that have anti-inflammatory actions. The dietary intake of 5% immature peels of CT fruits slightly increased stress resilience in a subchronic and mild social defeat (sCSDS) model in mice. Moreover, the dietary intake of 0.1% hesperidin significantly increased stress resilience and suppressed KYN levels in the hippocampus and prefrontal cortex in these mice. In addition, KYN levels in the hippocampus and prefrontal cortex were significantly correlated with the susceptibility to stress. In conclusion, these results suggest that dietary hesperidin increases stress resilience by suppressing the augmentation of KYN signaling under sCSDS.

Development of Implantable Wireless Sensor Nodes for Animal Husbandry and MedTech Innovation.

In this paper, we report the development, evaluation, and application of ultra-small low-power wireless sensor nodes for advancing animal husbandry, as well as for innovation of medical technologies. A radio frequency identification (RFID) chip with hybrid interface and neglectable power consumption was introduced to enable switching of ON/OFF and measurement mode after implantation. A wireless power transmission system with a maximum efficiency of 70% and an access distance of up to 5 cm was developed to allow the sensor node to survive for a duration of several weeks from a few minutes' remote charge. The results of field tests using laboratory mice and a cow indicated the high accuracy of the collected biological data and bio-compatibility of the package. As a result of extensive application of the above technologies, a fully solid wireless pH sensor and a surgical navigation system using artificial magnetic field and a 3D MEMS magnetic sensor are introduced in this paper, and the preliminary experimental results are presented and discussed.

A Synthetic Analogue of Neopeltolide, 8,9-Dehydroneopeltolide, Is a Potent Anti-Austerity Agent against Starved Tumor Cells.

Neopeltolide, an antiproliferative marine macrolide, is known to specifically inhibit complex III of the mitochondrial electron transport chain (mETC). However, details of the biological mode-of-action(s) remain largely unknown. This work demonstrates potent cytotoxic activity of synthetic neopeltolide analogue, 8,9-dehydroneopeltolide (8,9-DNP), against starved human pancreatic adenocarcinoma PANC-1 cells and human non-small cell lung adenocarcinoma A549 cells. 8,9-DNP induced rapid dissipation of the mitochondrial membrane potential and depletion of intracellular ATP level in nutrient-deprived medium. Meanwhile, in spite of mTOR inhibition under starvation conditions, impairment of cytoprotective autophagy was observed as the lipidation of LC3-I to form LC3-II and the degradation of p62 were suppressed. Consequently, cells were severely deprived of energy sources and underwent necrotic cell death. The autophagic flux inhibited by 8,9-DNP could be restored by glucose, and this eventually rescued cells from necrotic death. Thus, 8,9-DNP is a potent anti-austerity agent that impairs mitochondrial ATP synthesis and cytoprotective autophagy in starved tumor cells.

[The Effect of Intravenous Gamma-globulin Reagents on the Measurement Results of (1➔3)-ß-D-glucan].

Serum (1→3) beta-D-glucan (BG) measurement is a useful test for systemic mycoses, and often used. On the other hand, various factors, including administration of intravenous immunoglobulins (IVIG) may cause false-positives. In the present study, we measured BG concentration of seven IVIG preparations with three lots respectively. BG levels varied with individual IVIG preparations (<3.0 - >300 pg/mL), and contamination from manufacturing processes was suspected. With serum BG concentration of clinical specimens obtained in Niigata University Medical & Dental Hospital, the difference between before and after administration of IVIG were calculated. The false-positive rate of BG due to IVIG administration was 9.8 %, and the positive predective value was reduced to 37.5%. Above all, administration of IVIG can complicate the BG test's interpretation, and caution is required.

Site-specific inhibitory mechanism for amyloid ß42 aggregation by catechol-type flavonoids targeting the Lys residues.

The aggregation of the 42-residue amyloid ß-protein (Aß42) is involved in the pathogenesis of Alzheimer disease (AD). Numerous flavonoids exhibit inhibitory activity against Aß42 aggregation, but their mechanism remains unclear in the molecular level. Here we propose the site-specific inhibitory mechanism of (+)-taxifolin, a catechol-type flavonoid, whose 3',4'-dihydroxyl groups of the B-ring plays a critical role. Addition of sodium periodate, an oxidant, strengthened suppression of Aß42 aggregation by (+)-taxifolin, whereas no inhibition was observed under anaerobic conditions, suggesting the inhibition to be associated with the oxidation to form o-quinone. Because formation of the Aß42-taxifolin adduct was suggested by mass spectrometry, Aß42 mutants substituted at Arg(5), Lys(16), and/or Lys(28) with norleucine (Nle) were prepared to identify the residues involved in the conjugate formation. (+)-Taxifolin did not suppress the aggregation of Aß42 mutants at Lys(16) and/or Lys(28) except for the mutant at Arg(5). In addition, the aggregation of Aß42 was inhibited by other catechol-type flavonoids, whereas that of K16Nle-Aß42 was not. In contrast, some non-catechol-type flavonoids suppressed the aggregation of K16Nle-Aß42 as well as Aß42. Furthermore, interaction of (+)-taxifolin with the ß-sheet region in Aß42 was not observed using solid-state NMR unlike curcumin of the non-catechol-type. These results demonstrate that catechol-type flavonoids could specifically suppress Aß42 aggregation by targeting Lys residues. Although the anti-AD activity of flavonoids has been ascribed to their antioxidative activity, the mechanism that the o-quinone reacts with Lys residues of Aß42 might be more intrinsic. The Lys residues could be targets for Alzheimer disease therapy.

Programmed cell death induced by (-)-8,9-dehydroneopeltolide in human promyelocytic leukemia HL-60 cells under energy stress conditions.

(+)-Neopeltolide is a marine macrolide natural product that exhibits potent antiproliferative activity against several human cancer cell lines. Previous study has established that this natural product primarily targets the complex III of the mitochondrial electron transport chain. However, the biochemical mode-of-actions of neopeltolide have not been investigated in detail. Here we report that (-)-8,9-dehydroneopeltolide (8,9-DNP), a more accessible synthetic analogue, shows potent cytotoxicity against human promyelocytic leukemia HL-60 cells preferentially under energy stress conditions. Nuclear morphology analysis, as well as DNA ladder assay, indicated that 8,9-DNP induced significant nuclear condensation/fragmentation and DNA fragmentation, and these events could be suppressed by preincubating the cells with a pan-caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD). Immunoblot analysis demonstrated the release of cytochrome c from the mitochondria and the cleavage of full-length caspase-3 and poly(ADP-ribose) polymerase (PARP). These results indicated that 8,9-DNP induced caspase-dependent apoptotic programmed cell death under energy stress conditions. It was also found that 8,9-DNP induced non-apoptotic cell death in the presence/absence of zVAD under energy stress conditions. Immunoblot analysis showed the intracytosolic release of apoptosis-inducing factor (AIF), although it did not further translocate to the nucleus. It appears most likely that, in the presence of zVAD, 8,9-DNP triggered necrotic cell death as a result of severe intracellular ATP depletion.

Siglec-15 protein regulates formation of functional osteoclasts in concert with DNAX-activating protein of 12 kDa (DAP12).

Osteoclasts are multinucleated giant cells that reside in osseous tissues and resorb bone. Signaling mediated by receptor activator of nuclear factor (NF)-κB (RANK) and its ligand leads to the nuclear factor of activated T cells 2/c1 (NFAT2 or NFATc1) expression, a critical step in the formation of functional osteoclasts. In addition, adaptor proteins harboring immunoreceptor tyrosine-based activation motifs, such as DNAX-activating protein of 12 kDa (DAP12), play essential roles. In this study, we identified the gene encoding the lectin Siglec-15 as NFAT2-inducible, and we found that the protein product links RANK ligand-RANK-NFAT2 and DAP12 signaling in mouse osteoclasts. Both the recognition of sialylated glycans by the Siglec-15 V-set domain and the association with DAP12 through its Lys-272 are essential for its function. When Siglec-15 expression was knocked down, fewer multinucleated cells developed, and those that did were morphologically contracted with disordered actin-ring structures. These changes were accompanied by significantly reduced bone resorption. Siglec-15 formed complexes with Syk through DAP12 in response to vitronectin. Furthermore, chimeric molecules consisting of the extracellular and transmembrane regions of Siglec-15 with a K272A mutation and the cytoplasmic region of DAP12 significantly restored bone resorption in cells with knocked down Siglec-15 expression. Together, these results suggested that the Siglec-15-DAP12-Syk-signaling cascade plays a critical role in functional osteoclast formation.

Non-toxic conformer of amyloid ß may suppress amyloid ß-induced toxicity in rat primary neurons: implications for a novel therapeutic strategy for Alzheimer's disease.

The 42-mer amyloid ß-protein (Aß42) oligomers cause neurotoxicity and cognitive impairment in Alzheimer's disease (AD). We previously identified the toxic conformer of Aß42 with a turn at positions 22-23 ("toxic" turn) to form oligomers and to induce toxicity in rat primary neurons, along with the non-toxic conformer with a turn at positions 25-26. G25P-Aß42 and E22V-Aß42 are non-toxic mutants that disfavor the "toxic" turn. Here we hypothesize that these non-toxic mutants of Aß42 could suppress Aß42-induced neurotoxicity, and examined their effects on the neurotoxicity, aggregation, and levels of the toxic conformer, which was evaluated by dot blotting using a monoclonal antibody (11A1) against the toxic conformer. G25P-Aß42 and E22V-Aß42 suppressed the neurotoxicity and aggregation of Aß42 as well as the formation of the toxic conformer. The neurotoxicity induced by Aß42 was also significantly reduced by the treatment of 11A1, but not of Aß-sequence specific antibodies (6E10 and 4G8). Since recent studies indicate that Aß oligomers contain parallel ß-sheet, the present results suggest that the non-toxic mutants of Aß42 without the "toxic" turn could prevent the propagation process of the toxic conformer of Aß42 resulting in suppression of the formation of the toxic oligomers. This could be a promising strategy for AD therapeutics.

Structure-activity relationship for (+)-taxifolin isolated from silymarin as an inhibitor of amyloid ß aggregation.

Silymarin, the seed extract of Silybium marianum, has preventive effects against Alzheimer's disease-like pathogenesis in vivo. We isolated (+)-taxifolin (4) from silymarin as an inhibitor of aggregation of the 42-residue amyloid ß-protein. Structure-activity relationship studies revealed the 3',4'-dihydroxyl groups to be critical to the anti-aggregative ability, whereas the 7-hydroxyl group and the stereochemistry at positions 2 and 3 were not important.

Abl-1-bridged tyrosine phosphorylation of VASP by Abelson kinase impairs association of VASP to focal adhesions and regulates leukaemic cell adhesion.

Mena [mammalian Ena (Enabled)]/VASP (vasodilator-stimulated phosphoprotein) proteins are the homologues of Drosophila Ena. In Drosophila, Ena is a substrate of the tyrosine kinase DAbl (Drosophila Abl). However, the link between Abl and the Mena/VASP family is not fully understood in mammals. We previously reported that Abi-1 (Abl interactor 1) promotes phosphorylation of Mena and BCAP (B-cell adaptor for phosphoinositide 3-kinase) by bridging the interaction between c-Abl and the substrate. In the present study we have identified VASP, another member of the Mena/VASP family, as an Abi-1-bridged substrate of Abl. VASP is phosphorylated by Abl when Abi-1 is co-expressed. We also found that VASP interacted with Abi-1 both in vitro and in vivo. VASP was tyrosine-phosphorylated in Bcr-Abl-positive leukaemic cells in an Abi-1-dependent manner. Co-expression of c-Abl and Abi-1 or the phosphomimetic Y39D mutation in VASP resulted in less accumulation of VASP at focal adhesions. VASP Y39D had a reduced affinity to the proline-rich region of zyxin. Interestingly, overexpression of both phosphomimetic and unphosphorylated forms of VASP, but not wild-type VASP, impaired adhesion of K562 cells to fibronectin. These results suggest that the phosphorylation and dephosphorylation cycle of VASP by the Abi-1-bridged mechanism regulates association of VASP with focal adhesions, which may regulate adhesion of Bcr-Abl-transformed leukaemic cells.

Dynamics of iron metabolism in patients with bloodstream infections: a time-course clinical study.

The close relationship between infectious diseases and iron metabolism is well known, but a more detailed understanding based on current knowledge may provide new insights into the diagnosis and treatment of infectious diseases, considering the growing threat of antibiotic-resistant bacteria. This study investigated adult patients with bloodstream infections, temporal changes, and relationships between blood levels of iron and related markers, including hepcidin and lipocalin-2 (LCN2). We included 144 samples from 48 patients (mean age 72 years, 50% male), with 30 diagnosed with sepsis. During the acute phase of infection, blood levels of hepcidin and LCN2 increased rapidly, whereas iron levels decreased, with values in 95.8% of cases below the normal range (40-188 µg/dL). Later, hepcidin and LCN2 decreased significantly during the recovery phase, and the decreased iron concentrations were restored. In the case of persistent inflammation, iron remained decreased. Acute LCN2 levels were significantly higher in patients with sepsis (p < 0.01). Hypoferremia induced by increased hepcidin would reduce iron in the environment of extracellular pathogens, and the increased LCN2 would inhibit siderophores, resulting in the prevention of the pathogen's iron acquisition in each manner during the acute phase of bloodstream infection.

Global Environmental Issues: Food and Agriculture Education to Address Food Loss and Waste, Aiming at a Sustainable Supply Chain.

It is estimated that during FY 2018, food waste of 25.31 million tons and food loss of 6 million tons occurred in Japan. Of this total amount, 3.24 million tons of waste was generated from business, including the food manufacturing, wholesale, retail, and restaurant industries, and 2.76 million tons of waste was generated from households. Thus, it is crucial to mitigate both upstream and downstream food loss. As consumption patterns greatly influence not only downstream but also upstream food loss, special emphasis must be placed on increasing consumers' awareness of this global issue, and several programs have been initiated to this end. Our research reveals that alongside awareness, enhancing consumers' skills is of vital importance to efficiently reduce downstream food loss. Additionally, business operators must invest in improving the skills of those in charge to minimize food loss in commercial setups. Educating both consumers and businesses with the requisite skills to lessen food loss is equally essential. The authors are developing impactful serious games to help impart such education. These include a vegetable supply chain game (Veggie Mart Game), where consumers are players, and a milk supply chain game (Milky-Chain Game), where businesses are players. This paper introduces the authors' current research on reduction of food loss.

Dietary Hesperidin Suppresses Lipopolysaccharide-Induced Inflammation in Male Mice.

Depressive disorders are partially attributed to chronic inflammation associated with the tryptophan (Trp)-kynurenine (Kyn) pathway. Recent evidence suggests that anti-inflammatory agents may reduce the risk of depression. The present study aimed to elucidate the potential of the citrus flavonoid hesperidin, which exhibits anti-inflammatory activity, in suppressing the Trp-Kyn pathway in the brain, using a lipopolysaccharide (LPS)-induced inflammation mouse model. Dietary hesperidin was found to suppress activation of the Trp-Kyn pathway in the prefrontal cortex. In addition, it reduced systemic LPS-induced signs of illness, such as low skin temperature and enhanced leukocyte count in the blood. However, dietary supplementation with hesperidin did not improve body weight loss, food intake, water intake, or splenic increases in leukocyte numbers in the LPS model. Collectively, the results suggest that dietary hesperidin can partially regulate central and peripheral events linked to inflammation in LPS mouse models.

Effect of Probiotic Bifidobacterium bifidum TMC3115 Supplementation on Psychosocial Stress Using a Sub-Chronic and Mild Social Defeat Stress in Mice.

With the accumulation of knowledge on the relation between psychological stress and gut microbiota, there is growing interest in controlling stress and/or mood disorders via probiotic supplementation. We aimed to examine the effect of probiotic Bifidobacterium bifidum TMC3115 (TMC3115) supplementation using a sub-chronic and mild social defeat stress murine model in this study. TM3115 supplementation maintained body weight gain and alleviated a polydipsia-like symptom induced by the stress. In the analyses of fecal and cecal bacterial profiles, expansions of Proteobacteria in stressed mice and increases in Actinobacteria and Bifidobacterium in mice supplemented with TMC3115 were observed. There was no marked difference in the diversity of cecal bacteria between the tested mice. Elevated serum levels of inflammatory markers such as tumor necrosis factor (TNF)-α and interleukin (IL)-6 were observed in the stressed mice, while TMC3115 only reduced the IL-6 level. These findings suggest that TMC3115 supplementation confers tolerance to psychosocial stress in the host through modulation of the gut microbiota and alleviation of stress-induced inflammatory responses. Furthermore, it may be expected to exert prevention and treatment of disorders related to peripheral IL-6, including depression.

Proteome analysis demonstrates involvement of endoplasmic reticulum stress response in human myocardium with subclinical left ventricular diastolic dysfunction.

AIM: Heart failure is increasing in Japan, in particular that with preserved ejection fraction (HFpEF) prevalent in older-aged patients. The purpose of this study was to investigate the pathophysiology during the early stage of left ventricular (LV) diastolic dysfunction by the quantitative proteome analysis of human myocardium. METHODS: Among 331 post-mortem autopsy patients, we selected 23 patients (aged 79 ± 9.6 years) with echocardiographic data and without major comorbidities, except hypertension. Cryopreserved autopsy tissue of the LV myocardium was subjected to proteome analysis. LV diastolic function was evaluated by echocardiographic data. Thirteen patients were classified into the impaired diastolic function (IDF) group, and 10 the normal cardiac function group. We performed comparative proteome analysis between the IDF and normal groups by isobaric tags for relative and absolute quantitation (iTRAQ) using nano-liquid chromatography-tandem mass spectrometry. RESULTS: The iTRAQ-based proteome analysis revealed 57 differentially expressed proteins in the IDF group. Molecular network analysis of differentially expressed proteins indicated that endoplasmic reticulum (ER) stress was a potentially important event. Furthermore, the expressions of proteins associated with the ER stress response, such as glucose-regulated protein 78 kDa, inositol-requiring kinase 1α and spliced X-box binding protein 1, were significantly decreased in the IDF group. CONCLUSIONS: This study suggested that reduced ER stress responses were involved during the early stage of LV diastolic dysfunction. Geriatr Gerontol Int ••; ••: ••-•• Geriatr Gerontol Int 2021; 21: 577-583.

A combination of unnatural phosphatidyl acceptor and tandem electrospray ionization mass spectrometry for tracing phospholipase D activity.

Phospholipase D (PLD) is a biocatalyst in the synthesis of bioactive compounds and a key enzyme in a variety of biological signal transductions. A combination of unnatural phosphatidyl acceptor, N,N,N-triethyl-N-2-hydroxyethylammonium bromide 6, as a substrate for PLD, and tandem electrospray ionization mass spectrometry (ESI MS) was found to provide information as to whether a given phospholipid serves as a substrate for the PLD-catalyzed reaction. Thus 2-(13'-hydroperoxy-octadecadienoyl)-1-palmitoylglycerophosphocholine 1, and its degradation products 2-(13'-oxo-octadecadienoyl)-1-palmitoylglycerophosphocholine 9 and 2-(13'-hydroxy-octadecadienoyl)-1-palmitoylglycerophosphocholine 11, in a mixture were found to be a substrate of the PLD-catalyzed transphosphatidylation. The sensitivity of this method was exemplified by the observation that PLD activity in cabbage leaves was detected using a small amount of crude crushed leaves with little pretreatment. This simple method can be used in screening for PLD activity and searching for inhibitors of the enzyme from various natural sources.

Dietary Intake of Immature Citrus tumida Hort. ex Tanaka Peels Suppressed Body Weight Gain and Fat Accumulation in a Mouse Model of Acute Obesity.

Citrus fruits have several potential benefits for maintaining our health. In this study, we investigated the anti-obesity effects of immature Citrus tumida hort. ex Tanaka (C. tumida) peels using an acute obesity mice model. C57BL/6J male mice were divided into 2 groups; HFD-LL subjected to a high fat diet (HFD) and constant light exposure (LL), and HFDC-LL subjected to a HFD containing immature peel powder of C. tumida (5% w/w) and LL. Dietary ingestion of immature C. tumida peels significantly suppressed body weight gain following decreased epidydimal, perirenal, and subcutaneous fat weights. Blood levels of triglyceride and total cholesterol in the HFDC-LL were significantly lower than those in the HFD-LL group; however, there was no significant difference in food or calorie intake between the 2 groups. These results suggested that immature C. tumida peels have a beneficial effect on the prevention of obesity and metabolic syndrome via its biochemical activities of lipid metabolism.

Effects of non-purified and semi-purified commercial diets on behaviors, plasma corticosterone levels, and cecum microbiome in C57BL/6J mice.

Diverse commercially available feeds are used in animal studies according to the purpose of the studies. We sought to understand the relationship between feed ingredients and their effects on animal physiology and behaviors. Here, we investigated how male laboratory mice (C57BL/6J ("B6") mice) were affected by chronic feeding with two commercially available diets, a non-purified diet (MF) and a semi-purified diet (AIN-93G). In B6 mice, both diets similarly induced spontaneous activities in the home cage and the open field box, anxiety in the elevated plus maze test, and depressive-like behaviors in tail-suspension and forced-swimming tests, and with both diets, similar data were obtained on calorie intake, water intake, body weight gain, and plasma corticosterone levels. By contrast, liver weight was significantly higher in MF-fed B6 mice than in AIN-93G-fed B6 mice. Furthermore, the cecum microbiome was drastically affected by the diets, and, specifically, Allobaculum was the major genus (43.4%) in the cecum microbiota of AIN-93G-fed mice but its abundance was reduced (to 3.8%) in the case of MF-fed mice. Future studies should address whether the differences in diet purity and cecum microbiota influence brain functions and behaviors in B6 mice.

Prevalence of human metapneumovirus and influenza virus infections among Japanese children during two successive winters.

BACKGROUND: Human metapneumoviruses (hMPVs) are recognized as a leading cause of respiratory infections in young infants in many countries. The objective of this study was to identify links between hMPV and influenza virus infections among children with influenza-like illness. METHOD: This study was conducted in 2 influenza seasons (2002-2003 and 2003-2004) at 2 pediatric outpatient clinics in Niigata city, Japan. Nasopharyngeal swabs or aspirates were collected from influenza-like illness patients, and hMPV and influenza were detected by reverse transcription-polymerase chain reaction (RT-PCR). A nucleotide sequence of 352 nucleotides segment of the F gene was performed. RESULTS: A total of 765 influenza viruses and 84 hMPV were identified from 1498 nasopharyngeal swabs or aspirates by virus isolation and RT-PCR, respectively. hMPV-positive rates in patients with influenza-like illness were 5.7 and 5.2% in the 2002-2003 and 2003-2004 seasons, respectively. Epidemic curves of influenza and hMPV patients showed similar patterns with peaks in February in 2 influenza seasons. hMPV infections occurred frequently in infants and school children. Approximately 46% of hMPV patients were coinfected with influenza A viruses, but those coinfected cases were not clinically distinct from the others. No coinfection with influenza B viruses was found. Phylogenetic analysis of the hMPV fusion gene sequences revealed that 2 distinct hMPV cocirculated and that completely identical strains in subgroup A were observed over 2 years. CONCLUSIONS: HMPV plays an important pathogenic role in patients with influenza-like illness in winter seasons, often in coinfections with influenza A viruses.