Theanine, a Tea-Leaf-Specific Amino Acid, Alleviates Stress through Modulation of Npas4 Expression in Group-Housed Older Mice.

Group rearing is a common housing condition, but group-housed older mice show increased adrenal hypertrophy, a marker of stress. However, the ingestion of theanine, an amino acid unique to tea leaves, suppressed stress. We aimed to elucidate the mechanism of theanine's stress-reducing effects using group-reared older mice. The expression of repressor element 1 silencing transcription factor (REST), which represses excitability-related genes, was increased in the hippocampus of group-reared older mice, whereas the expression of neuronal PAS domain protein 4 (Npas4), which is involved in the regulation of excitation and inhibition in the brain, was lower in the hippocampus of older group-reared mice than in same-aged two-to-a-house mice. That is, the expression patterns of REST and Npas4 were found to be just inversely correlated. On the other hand, the expression levels of the glucocorticoid receptor and DNA methyltransferase, which suppress Npas4 transcription, were higher in the older group-housed mice. In mice fed theanine, the stress response was reduced and Npas4 expression tended to be increased. These results suggest that Npas4 expression was suppressed by the increased expression of REST and Npas4 downregulators in the group-fed older mice, but that theanine avoids the decrease in Npas4 expression by suppressing the expression of Npas4 transcriptional repressors.

Possible correlated variation of GABAA receptor α3 expression with hippocampal cholinergic neurostimulating peptide precursor protein in the hippocampus.

Cholinergic neural activation from the medial septal nucleus to hippocampus plays a crucial role in episodic memory as a regulating system for glutamatergic neural activation in the hippocampus. As a candidate regulating factor for acetylcholine synthesis in the medial septal nucleus, hippocampal cholinergic neurostimulating peptide (HCNP) was purified from the soluble fraction of young adult rat hippocampus. HCNP is released from its precursor protein (HCNP-pp), also referred to as phosphatidylethanolamine-binding protein 1. We recently reported that HCNP-pp conditional knockout (KO) mice, in which the HCNP-pp gene was knocked out at 3 months of age by tamoxifen injection, display no significant behavioral abnormalities, whereas HCNP-pp KO mice have a diminished cholinergic projection to CA1 and a decreased of theta activity in CA1. In this study, to address whether HCNP-pp reduction in early life is associated with behavioral changes, we evaluated the behavior of HCNP-pp KO mice in which HCNP-pp was downregulated from an early phase (postnatal days 14-28). As unexpected, HCNP-pp KO mice had no behavioral deficits. However, a significant positive correlation between HCNP-pp and gamma-aminobutyric acid A (GABAA) receptor α3 subunit mRNA expression was found in individuals. This finding suggests involvement of HCNP-pp in regulating GABAA receptor α3 gene expression.

L-Arginine Exerts Excellent Anti-Stress Effects on Stress-Induced Shortened Lifespan, Cognitive Decline and Depression.

The anti-stress potential of dietary L-arginine (Arg) was assessed in psychosocially stress-loaded senescence-accelerated (SAMP10) mice. Although this strain of mouse is sensitive to stress, daily administration of Arg at 3 mg/kg significantly suppressed aging-related cognitive decline and behavioral depression at nine months of age and counteracted stress-induced shortened lifespan. To investigate the mechanism of the anti-stress effect of Arg in the brain, early changes in oxidative damage and gene expression levels were measured using SAMP10 mice that were stress-loaded for three days. Increased lipid peroxidation in the brains of stressed mice was significantly lowered by Arg intake. Several genes associated with oxidative stress response and neuronal excitotoxic cell death, including Nr4a1, Arc, and Cyr61, remarkably increased in response to psychosocial stress; however, their expression was significantly suppressed in mice that ingested Arg even under stress conditions. In contrast, the genes that maintain mitochondrial functions and neuronal survival, including Hba-a2 and Hbb-b2, were significantly increased in mice that ingested Arg. These results indicate that Arg reduces oxidative damage and enhances mitochondrial functions in the brain. We suggest that the daily intake of Arg plays important roles in reducing stress-induced brain damage and slowing aging.

Theanine, Antistress Amino Acid in Tea Leaves, Causes Hippocampal Metabolic Changes and Antidepressant Effects in Stress-Loaded Mice.

By comprehensively measuring changes in metabolites in the hippocampus of stress-loaded mice, we investigated the reasons for stress vulnerability and the effect of theanine, i.e., an abundant amino acid in tea leaves, on the metabolism. Stress sensitivity was higher in senescence-accelerated mouse prone 10 (SAMP10) mice than in normal ddY mice when these mice were loaded with stress on the basis of territorial consciousness in males. Group housing was used as the low-stress condition reference. Among the statistically altered metabolites, depression-related kynurenine and excitability-related histamine were significantly higher in SAMP10 mice than in ddY mice. In contrast, carnosine, which has antidepressant-like activity, and ornithine, which has antistress effects, were significantly lower in SAMP10 mice than in ddY mice. The ingestion of theanine, an excellent antistress amino acid, modulated the levels of kynurenine, histamine, and carnosine only in the stress-loaded SAMP10 mice and not in the group-housing mice. Depression-like behavior was suppressed in mice that had ingested theanine only under stress loading. Taken together, changes in these metabolites, such as kynurenine, histamine, carnosine, and ornithine, were suggested to be associated with the stress vulnerability and depression-like behavior of stressed SAMP10 mice. It was also shown that theanine action appears in the metabolism of mice only under stress loading.

Mutation in Sodium-Glucose Cotransporter 2 Results in Down-Regulation of Amyloid Beta (A4) Precursor-Like Protein 1 in Young Age, Which May Lead to Poor Memory Retention in Old Age.

Senescence-accelerated mouse prone 10 (SAMP10) exhibits cerebral atrophy and depression-like behavior. A line of SAMP10 with spontaneous mutation in the Slc5a2 gene encoding the sodium-glucose cotransporter (SGLT) 2 was named SAMP10/TaSlc-Slc5a2slc (SAMP10-ΔSglt2) and was identified as a renal diabetes model. In contrast, a line of SAMP10 with no mutation in SGLT2 (SAMP10/TaIdrSlc, SAMP10(+)) was recently established under a specific pathogen-free condition. Here, we examined the mutation effect in SGLT2 on brain function and longevity. No differences were found in the survival curve, depression-like behavior, and age-related brain atrophy between SAMP10-ΔSglt2 and SAMP10(+). However, memory retention was lower in SAMP10-ΔSglt2 mice than SAMP10(+). Amyloid beta (A4) precursor-like protein 1 (Aplp1) expression was significantly lower in the hippocampus of SAMP10-ΔSGLT2 than in SAMP10(+) at 2 months of age, but was similar at 12 months of age. CaM kinase-like vesicle association (Camkv) expression was remarkably lower in SAMP10(+). These genes have been reported to be involved in dendrite function. Amyloid precursor proteins have been reported to involve in maintaining homeostasis of glucose and insulin. These results suggest that mutation in SGLT2 results in down-regulation of Aplp1 in young age, which can lead to poor memory retention in old age.

Green Tea Catechins Trigger Immediate-Early Genes in the Hippocampus and Prevent Cognitive Decline and Lifespan Shortening.

Senescence-accelerated mouse prone 10 (SAMP10) mice, after ingesting green tea catechins (GT-catechin, 60 mg/kg), were found to have suppressed aging-related decline in brain function. The dose dependence of brain function on GT-catechin indicated that intake of 1 mg/kg or more suppressed cognitive decline and a shortened lifespan. Mice that ingested 1 mg/kg GT-catechin had the longest median survival, but the dose was less effective at suppressing cognitive decline. The optimal dose for improving memory acquisition was 60 mg/kg, and memory retention was higher in mice that ingested 30 mg/kg or more. To elucidate the mechanism by which cognitive decline is suppressed by GT-catechin, changes in gene expression in the hippocampus of SAMP10 mice one month after ingesting GT-catechin were analyzed. The results show that the expression of immediate-early genes such as nuclear receptor subfamily 4 (Nr4a), FBJ osteosarcoma oncogene (Fos), early growth response 1 (Egr1), neuronal PAS domain protein 4 (Npas4), and cysteine-rich protein 61 (Cyr61) was significantly increased. These results suggest that GT-catechin suppresses age-related cognitive decline via increased expression of immediate-early genes that are involved in long-term changes in plasticity of synapses and neuronal circuits.

Parametric analysis of RNA-seq expression data.

Various methods had been introduced for normalization and comparison of RNA-seq count data. However, they lacked objectivity because they based on ad hoc assumptions that were never verified their appropriateness. Here, we introduced a method that assumes parsimony models on data distribution; the assumptions were verified according to exploratory data analysis. As was expected, count data were lognormally distributed. The level of noise in recent data appeared to be much higher than those of microarrays. Still, the appropriate distribution model would improve certainty and accuracy of normalization, by finding out the reliable range of data. Primary cause of noise was not the principle of the methodology; that is, each read is a trial that which transcript is read. Rather, the cause would be overlooking of transcripts, and the overlooking occurred more often among lower range of data. To find out genes likely to be overlooked, number of replications would be more important than read depth, which will not prevent overlooking. Both signal and noise in the reliable range of data were distributed normally, showing the suitability to use generalized linear model to evaluate differences in expression levels. In the framework, normalized data can be compared and combined freely beyond studies.

Allantoin, a stress-related purine metabolite, can activate jasmonate signaling in a MYC2-regulated and abscisic acid-dependent manner.

Allantoin is a metabolic intermediate of purine catabolism that often accumulates in stressed plants. Recently, we used Arabidopsis knockout mutants (aln) of ALLANTOINASE to show that this purine metabolite activates abscisic acid (ABA) production, thereby stimulating stress-related gene expression and enhancing seedling tolerance to abiotic stress. A detailed re-examination of the microarray data of an aln mutant (aln-1) confirmed the increased expression of ABA-related genes and also revealed altered expression of genes involved in jasmonic acid (JA) responses, probably under the control of MYC2, a master switch in the JA signaling pathway. Consistent with the transcriptome profiles, the aln-1 mutant displayed increased JA levels and enhanced responses to mechanical wounding and exogenous JA. Moreover, aln mutants demonstrated modestly increased susceptibility to Pseudomonas syringae and Pectobacterium carotovorum, probably reflecting the antagonistic action of MYC2 on the defense against these bacterial phytopathogens. Exogenously administered allantoin elicited the expression of JA-responsive genes, including MYC2, in wild-type plants, supporting the idea that allantoin might be responsible for the observed JA-related phenotypes of aln mutants. However, mutants deficient in bioactive JA (jar1-1), insensitive to JA (myc2-3), or deficient in ABA (aba2-1 and bglu18) suppressed the effect of exogenous allantoin. The suppression was further confirmed in aln-1 jar1-1 and aln-1 bglu18 double mutants. These results indicate that allantoin can activate the MYC2-regulated JA signaling pathway through ABA production. Overall, this study suggests a possible connection of purine catabolism with stress hormone homeostasis and signaling, and highlights the potential importance of allantoin in these interactions.

Principal component analysis for designed experiments.

BACKGROUND: Principal component analysis is used to summarize matrix data, such as found in transcriptome, proteome or metabolome and medical examinations, into fewer dimensions by fitting the matrix to orthogonal axes. Although this methodology is frequently used in multivariate analyses, it has disadvantages when applied to experimental data. First, the identified principal components have poor generality; since the size and directions of the components are dependent on the particular data set, the components are valid only within the data set. Second, the method is sensitive to experimental noise and bias between sample groups. It cannot reflect the experimental design that is planned to manage the noise and bias; rather, it estimates the same weight and independence to all the samples in the matrix. Third, the resulting components are often difficult to interpret. To address these issues, several options were introduced to the methodology. First, the principal axes were identified using training data sets and shared across experiments. These training data reflect the design of experiments, and their preparation allows noise to be reduced and group bias to be removed. Second, the center of the rotation was determined in accordance with the experimental design. Third, the resulting components were scaled to unify their size unit. RESULTS: The effects of these options were observed in microarray experiments, and showed an improvement in the separation of groups and robustness to noise. The range of scaled scores was unaffected by the number of items. Additionally, unknown samples were appropriately classified using pre-arranged axes. Furthermore, these axes well reflected the characteristics of groups in the experiments. As was observed, the scaling of the components and sharing of axes enabled comparisons of the components beyond experiments. The use of training data reduced the effects of noise and bias in the data, facilitating the physical interpretation of the principal axes. CONCLUSIONS: Together, these introduced options result in improved generality and objectivity of the analytical results. The methodology has thus become more like a set of multiple regression analyses that find independent models that specify each of the axes.

Ascorbic acid deficiency affects genes for oxidation-reduction and lipid metabolism in livers from SMP30/GNL knockout mice.

BACKGROUND: We sought to elucidate the effect of an ascorbic acid (AA) deficiency on gene expression, because the water soluble antioxidant AA is an important bioactive substance in vivo. METHODS: We performed microarray analyses of the transcriptome in the liver from senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout (KO) mice, which are unable to synthesize AA in vivo. RESULTS: Our microarray analysis revealed that the AA deficiency increased gene expression related to the oxidation-reduction process, i.e., the nuclear factor, erythroid derived 2, like 2 (Nrf2) gene, which is a reactive oxygen species-sensitive transcriptional factor. Moreover, this AA deficiency increased the expression of genes for lipid metabolism including the cytochrome P450, family 7, subfamily a, polypeptide 1 (Cyp7a1), which is a late-limiting enzyme of the primary bile acid biosynthesis pathway. Although an AA deficiency increased the Cyp7a1 protein level, bile acid levels in the liver and gallbladder decreased. Since Cyp7a1 has a heme iron at the active site, AA must function as a reductant of the iron required for the continuous activation of Cyp7a1. CONCLUSIONS: This experimental evidence strongly supports a role for AA in the physiologic oxidation-reduction process and lipid metabolism including bile acid biosynthesis. GENERAL SIGNIFICANCE: Although many effects of AA supplementation have been reported, no microarray analysis of AA deficiency in vivo is available. Results from using this unique model of AA deficiency, the SMP30/GNL-KO mouse, now provide new information about formerly unknown AA functions that will implement further study of AA in vivo.

Distinctive features of lipoprotein profiles in stroke patients.

Classes of lipoproteins solubilize lipids in the blood, and their profiles are important for preventing atherosclerotic diseases. These can be identified by gel filtration HPLC, which has been analyzed in a manner that yields the same values as the de facto standard method, i.e., ultracentrifugation; however, previous studies have found that ultracentrifugation and its simplified alternatives, enzymatic methods, yield incorrect values. Here HPLC data of stroke patients and the controls were compared using data-driven analyses, without consideration for ultracentrifugation. The data well-separated patients from controls. In many patients, the level of HDL1 (a cholesterol scavenger) was low. The TG/cholesterol ratio of chylomicrons was found to be low in patients and high in the healthy elderly; the lower level may indicate a larger intake of animal fats. High levels of free glycerol in the elderly were hazardous, suggesting more dependence on lipids as an energy source. Statins had minimal effect on these factors. LDL cholesterol, the commonly-used risk indicator, was not a risk factor actually. Enzymatic methods failed to separate the patients from the control; hence, the existing guidelines for screening methods and medical treatment need to be revised. As an immediate step, glycerol would be an adaptable indicator.

Continuous mutation of SARS-CoV-2 during migration via three routes at the beginning of the pandemic.

Background: It remains unclear how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection started, spread worldwide, and mutated to result in the present variants. This difficulty can be attributed to the limitations associated with the analytical methodology for presenting the differences among genomic sequences. In this study, we critically analysed the early data to explain the start and spread of the pandemic. Methods: Objective analyses of the RNA sequences of earlier variants of SARS-CoV-2 (up to September 1, 2020, available in DDBJ and GISAID) were performed using Principal Component Analysis (PCA). The results were compared with information on the collection dates and location. The PCA was also conducted for 12 variants of interest to the WHO as of September 2021, and compared with earlier data. Results: The pandemic began in Wuhan, China. This strain was suspected to be related to other reported animal viruses; however, they had a minimal similarity. The strain then spreads via three routes while accumulating mutations. Several viral subgroups were identified along the routes, each with a large number of patients reported, indicating high infectivity to humans. These routes were only confirmed by the early data analysis, because newer variants would have more mutations, and would be preferentially be examined by PCA if they were included. On the original axes found in the early variants, the newer variants revealed that they retained previously acquired mutations, which helped to reveal the viral ancestors of the newer variants. The rate of mutation was found to be comparable to that of the influenza H1N1 virus, which causes recurrent seasonal epidemics. Another threat imposed by SARS-CoV-2 is that if the pandemic cannot be contained, new variants may emerge annually, preventing herd immunity.

Mutations in SARS-CoV-2 are on the increase against the acquired immunity.

Monovalent vaccines using mRNA or adenoviruses have provided substantial protection against the COVID-19 pandemic in many countries. However, viral mutations have hampered the efficacy of this approach. The Omicron variant, which appeared in Dec 2021, has caused a pandemic that has exerted pressure on the healthcare system worldwide. The COVID-19 vaccines are not very effective against this variant, resulting in an increased rate of infection and mortality. Owing to the rapidly increasing number of patients, few countries, such as Australia, New Zealand, and Taiwan, which aimed at zero-COVID cases, have discontinued their attempts to contain the spread of infection by imposing strict lockdowns, for example. Therefore, the administration of booster vaccinations has been initiated; however, there are concerns about their effectiveness, sustainability, and possible dangers. There is also the question of how a variant with such isolated mutations originated and whether this is likely to continue in the future. Here, we compare the mutations in the Omicron variant with others by direct PCA to consider questions pertaining to their evolution and characterisation. The Omicron variant, like the other variants, has mutated in humans. The accumulated mutations overwhelmed the acquired immunity and caused a pandemic. Similar mutations are likely to occur in the future. Additionally, the variants infecting animals were investigated; they rapidly mutated in animals and varied from the human strains. These animal-adapted strains are probably not highly infectious or pathogenic to humans. Hence, the possibility of using these strains as vaccines will be discussed.

Human lipoproteins comprise at least 12 different classes that are lognormally distributed.

This study presents the results of HPLC, a gentler and rapid separation method in comparison with the conventional ultracentrifugation, for 55 human serum samples. The elution patterns were analysed parametrically, and the attribute of each class was confirmed biochemically. Human samples contained 12 classes of lipoproteins, each of which may consist primarily of proteins. There are three classes of VLDLs. The level of each class was distributed lognormally, and the standard amount and the 95% range were estimated. Some lipoprotein classes with a narrow range could become ideal indicators of specific diseases. This lognormal character suggests that the levels are controlled by the synergy of multiple factors; multiple undesirable lifestyle habits may drastically increase the levels of specific lipoprotein classes. Lipoproteins in medical samples have been measured by enzymatic methods that coincide with conventional ultracentrifugation; however, the high gravity and time required for ultracentrifugation can cause sample degradation. Actually, the enzymatic methods measured the levels of several mixed classes. The targets of enzymatic methods have to be revised.

Neurotrophin-3 is involved in the formation of apical dendritic bundles in cortical layer 2 of the rat.

Apical dendritic bundles from pyramidal neurons are a prominent feature of cortical neuropil but with significant area specializations. Here, we investigate mechanisms of bundle formation, focusing on layer (L) 2 bundles in rat granular retrosplenial cortex (GRS), a limbic area implicated in spatial memory. By using microarrays, we first searched for genes highly and specifically expressed in GRS L2 at postnatal day (P) 3 versus GRS L2 at P12 (respectively, before and after bundle formation), versus GRS L5 (at P3), and versus L2 in barrel field cortex (BF) (at P3). Several genes, including neurotrophin-3 (NT-3), were identified as transiently and specifically expressed in GRS L2. Three of these were cloned and confirmed by in situ hybridization. To test that NT-3-mediated events are causally involved in bundle formation, we used in utero electroporation to overexpress NT-3 in other cortical areas. This produced prominent bundles of dendrites originating from L2 neurons in BF, where L2 bundles are normally absent. Intracellular biocytin fills, after physiological recording in vitro, revealed increased dendritic branching in L1 of BF. The controlled ectopic induction of dendritic bundles identifies a new role for NT-3 and a new in vivo model for investigating dendritic bundles and their formation.

SARS-CoV-2 mutations among minks show reduced lethality and infectivity to humans.

SARS-CoV-2 infection in minks has become a serious problem, as the virus may mutate and reinfect humans; some countries have decided to cull minks. Here, the virus sequencing data in minks were analysed and compared to those of human-virus. Although the mink-virus maintained the characteristics of human-virus, some variants rapidly mutated, adapting to minks. Some mink-derived variants infected humans, which accounted for 40% of the total SARS-CoV-2 cases in the Netherlands. These variants appear to be less lethal and infective compared to those in humans. Variants that have mutated further among minks were not found in humans. Such mink-viruses might be suitable for vaccination for humans, such as in the case of the smallpox virus, which is less infective and toxic to humans.

Effect of control measures on the pattern of COVID-19 Epidemics in Japan.

BACKGROUND: COVID-19 has spread worldwide since its emergence in 2019. In contrast to many other countries with epidemics, Japan differed in that it avoided lockdowns and instead asked people for self-control. A travel campaign was conducted with a sizable budget, but the number of PCR tests was severely limited. These choices may have influenced the course of the epidemic. METHODS: The increase or decrease in the classes of SARS-CoV-2 variants was estimated by analyzing the published sequences with an objective multivariate analysis. This approach observes the samples in multiple directions, digesting complex differences into simpler forms. The results were compared over time with the number of confirmed cases, PCR tests, and overseas visitors. The kinetics of infection were analyzed using the logarithmic growth rate. RESULTS: The declared states of emergency failed to alter the movement of the growth rate. Three epidemic peaks were caused by domestically mutated variants. In other countries, there are few cases in which multiple variants have peaked. However, due to the relaxation of immigration restrictions, several infective variants have been imported from abroad and are currently competing for expansion, creating the fourth peak. By April 2021, these foreign variants exceeded 80%. The chaotic situation in Japan will continue for some time, in part because no effort has been made to identify asymptomatic carriers, and details of the vaccination program are undecided.

Principal component analysis of coronaviruses reveals their diversity and seasonal and pandemic potential.

Coronaviruses and influenza viruses have similarities and differences. In order to comprehensively compare them, their genome sequencing data were examined by principal component analysis. Coronaviruses had fewer variations than a subclass of influenza viruses. In addition, differences among coronaviruses that infect a variety of hosts were also small. These characteristics may have facilitated the infection of different hosts. Although many of the coronaviruses were conservative, those repeatedly found among humans showed annual changes. If SARS-CoV-2 changes its genome like the Influenza H type, it will repeatedly spread every few years. In addition, the coronavirus family has many other candidates for new pandemics.

Theanine, the Main Amino Acid in Tea, Prevents Stress-Induced Brain Atrophy by Modifying Early Stress Responses.

Chronic stress can impair the health of human brains. An important strategy that may prevent the accumulation of stress may be the consumption of functional foods. When senescence-accelerated mice prone 10 (SAMP10), a stress-sensitive strain, were loaded with stress using imposed male mouse territoriality, brain volume decreased. However, in mice that ingested theanine (6 mg/kg), the main amino acid in tea leaves, brain atrophy was suppressed, even under stress. On the other hand, brain atrophy was not clearly observed in a mouse strain that aged normally (Slc:ddY). The expression level of the transcription factor Npas4 (neuronal PAS domain protein 4), which regulates the formation and maintenance of inhibitory synapses in response to excitatory synaptic activity, decreased in the hippocampus and prefrontal cortex of stressed SAMP10 mice, but increased in mice that ingested theanine. Lipocalin 2 (Lcn2), the expression of which increased in response to stress, was significantly high in the hippocampus and prefrontal cortex of stressed SAMP10 mice, but not in mice that ingested theanine. These data suggest that Npas4 and Lcn2 are involved in the brain atrophy and stress vulnerability of SAMP10 mice, which are prevented by the consumption of theanine, causing changes in the expression of these genes.