Silencing dentate newborn neurons alters excitatory/inhibitory balance and impairs behavioral inhibition and flexibility.

Adult neurogenesis confers the hippocampus with unparalleled neural plasticity, essential for intricate cognitive functions. The specific influence of sparse newborn neurons (NBNs) in modulating neural activities and subsequently steering behavior, however, remains obscure. Using an engineered NBN-tetanus toxin mouse model (NBN-TeTX), we noninvasively silenced NBNs, elucidating their crucial role in impulse inhibition and cognitive flexibility as evidenced through Morris water maze reversal learning and Go/Nogo task in operant learning. Task-based functional MRI (tb-fMRI) paired with operant learning revealed dorsal hippocampal hyperactivation during the Nogo task in male NBN-TeTX mice, suggesting that hippocampal hyperexcitability might underlie the observed behavioral deficits. Additionally, resting-state fMRI (rs-fMRI) exhibited enhanced functional connectivity between the dorsal and ventral dentate gyrus following NBN silencing. Further investigations into the activities of PV+ interneurons and mossy cells highlighted the indispensability of NBNs in maintaining the hippocampal excitation/inhibition balance. Our findings emphasize that the neural plasticity driven by NBNs extensively modulates the hippocampus, sculpting inhibitory control and cognitive flexibility.

Protein-Balanced Dietary Habits Benefit Cognitive Function in Japanese Older Adults.

Since daily dietary habits can affect cognitive function, dietary patterns such as the Mediterranean-DASH Intervention for Neurodegenerative Delay diet have been proposed as interventions to slow cognitive decline. However, because dietary habits vary widely among different food cultures, it is necessary to establish dietary pattern intervention methods that are appropriate for each population. Therefore, in this study, the dietary patterns of elderly Japanese individuals were classified using cluster analysis, and their relationship with cognitive function was investigated. We then modeled the dietary patterns and applied them to another cohort of elderly Japanese individuals to determine whether differences in dietary patterns could predict cognitive decline. One hundred and fifty older adults ≥ 65 years of age in the community were recruited. Their daily food intake and cognitive function were measured using the brief-type self-administered diet history questionnaire and Montreal Cognitive Assessment, respectively. K-means cluster analysis identified a high-carbohydrate (HC) dietary pattern with high cereal intake and a protein-balanced (PB) dietary pattern with high intake of legumes, vegetables, seafood, meat, and eggs. Cognitive function was significantly higher in the PB group than in the HC group. Furthermore, to classify the new data into HC and PB patterns, a classification model was created by discriminant analysis using food groups with significantly different intakes among dietary patterns. Next, we recruited 267 new older adults ≥ 65 years of age and measured food intake and cognitive function assessed using the memory performance index score. Individuals with cognitive decline were identified and their detailed cognitive functions were assessed using the neurocognitive index score. Cognitive function was significantly impaired in the HC pattern in both the general elderly and cognitively impaired cohorts. These findings suggest that a dietary pattern of low carbohydrate and high protein intake is associated with good cognitive function in elderly Japanese individuals. Classification by these dietary patterns can predict cognitive reservation in community-dwelling older adults.

Effects of Lactiplantibacillus plantarum OLL2712 on Memory Function in Older Adults with Declining Memory: A Randomized Placebo-Controlled Trial.

The use of probiotics is expected to be an intervention in neurodegenerative conditions that cause dementia owing to their ability to modulate neuroinflammatory responses via the microbiome-gut-brain axis. Therefore, we selected Lactiplantibacillus plantarum OLL2712 (OLL2712), the optimal anti-inflammatory lactic acid bacteria strain with high IL-10-inducing activity in immune cells, and aimed to verify its protective effects on memory function in older adults. A 12-week, randomized, double-blind, placebo-controlled trial was performed with older adults over the age of 65 years with declining memory. The participants consumed either powder containing heat-treated OLL2712 cells or placebo. Memory function was assessed using a computer-assisted cognitive test, Cognitrax. Daily dietary nutrient intake was assessed using the Brief-type Self-administered Diet History Questionnaire (BDHQ). The composition of the gut microbiota was analyzed by fecal DNA extraction and 16S rDNA sequencing. Data from 78 participants who completed the entire procedure were analyzed, and significant improvements in composite memory and visual memory scores were observed in the active group, after accounting for the effect of daily nutritional intake (p = 0.044 and p = 0.021, respectively). In addition, the active group had a lower abundance ratio of Lachnoclostridium, Monoglobus, and Oscillibacter genera, which have been reported to be involved in inflammation. The present study suggests that OLL2712 ingestion has protective effects against memory function decline in older adults.

Spatial Characteristics of the Efflux Pump MexB Determine Inhibitor Binding.

The multidrug efflux transporters MexB and MexY in Pseudomonas aeruginosa and AcrB in Escherichia coli contribute to these organisms' multidrug resistance. Efflux pump inhibitor (EPI) ABI-PP inhibits MexB and AcrB, but not MexY. We previously determined the structure of ABI-PP bound to the hydrophobic trap (the inhibitor-binding pit) of AcrB and MexB. The insensitivity of MexY to ABI-PP was attributed to a bulky tryptophan (Trp). AcrB(Phe178Trp) became uninhibited by ABI-PP, while MexY(Trp177Phe) resensitized MexY for ABI-PP. Interestingly, ABI-PP was able to inhibit MexB(Phe178Trp). Thus, it is not clear which bulky amino acid mutations are critical for inhibitor binding in MexB. Here, we investigated the pit of MexB in more detail, and elucidated which Trp mutation locations in the pit were hindering ABI-PP binding, but did not affect the function of the efflux pumps. Mutating positions 139, 277, 279, and 612 to tryptophan eliminated the inhibitory effect. However, the tryptophan mutation at position 571 did not cause any effect. These results show that the effectiveness of EPIs is greatly affected by mutations in different locations, and that binding of EPIs is partly attributed by spatial characteristics. These results should be taken into account for new inhibitor and drug discovery.

Consumption of Oleic Acid on the Preservation of Cognitive Functions in Japanese Elderly Individuals.

We recruited 154 community-dwelling elderly individuals and conducted a cohort study to find out the nutrient intake that is suitable for maintaining cognitive function in Japanese elders. Cognitive function was evaluated by the two functional tests, the Montreal Cognitive Assessment (MoCA) and Wechsler Memory Scale-Delayed Recall (WMS-DR), and daily nutrient intake was estimated from a Brief-type Self-administered Diet History Questionnaire (BDHQ). By a multiple regression analysis, among the four major nutrients (protein, fat, carbohydrate and ash), we detected a significant correlation between the score of cognitive functions assessed by both MoCA and WMS-DR and daily consumption of fat (p = 0.0317 and p = 0.0111, respectively). Among categories of fatty acid, we found a significant correlation between the score of both MoCA and WMS-DR and consumption of monounsaturated fatty acid (MUFA) (p = 0.0157 and p = 0.0136, respectively). Finally, among MUFAs, we observed a significant correlation between the score of both MoCA and WMS-DR and consumption of oleic acid (p = 0.0405 and p = 0.0165, respectively). From these observations, we can propose that daily consumption of fat, especially in oleic acid, has a beneficial effect against cognitive decline in community-dwelling Japanese elderly individuals.

Anserine, HClO-scavenger, protected against cognitive decline in individuals with mild cognitive impairment.

Neuroinflammation has been recognized as a promising target when considering strategies for treating AD. In particular, it has been shown that neutrophils and MPO-mediated neuroinflammatory responses with the production of HClO play a role in the progression of AD. In this study, we aimed to evaluate the effects of anserine, a scavenger of HClO, on the protection of cognitive declines in persons with MCI. Fifty-eight elderly volunteers were screened, and 36 MCI individuals were assigned either to an active arm, who received 500 mg anserine per day, or a placebo arm, for 12-weeks. To assess cognitive function, we performed MMSE at baseline and after the ingestion. The data of the MMSE for 30 subjects who completed the follow-up tests were analyzed. A significant difference was detected in the change score of MMSE between the active arm (1.9 ± 2.0; n = 15) and the placebo arm (0 ± 2.8; n = 15) (p = 0.036). After the correction with the daily intake of anserine, the significance was elevated (p = 0.0176). Our results suggest that anserine protects elderly persons with MCI from cognitive declines by suppressing MPO-mediated neuroinflammatory responses.

Mutations in the Phenicol Exporter Gene fexA Impact Resistance Levels in Three Bacterial Hosts According to Susceptibility Testing and Protein Modeling.

The prototype fexA gene confers combined resistance to chloramphenicol and florfenicol. However, fexA variants mediating resistance only to chloramphenicol have been identified, such as in the case of a Staphylococcus aureus isolate recovered from poultry meat illegally imported to Germany. The effects of the individual mutations detected in the fexA sequence of this isolate were investigated in this study. A total of 11 fexA variants, including prototype fexA and variants containing the different previously described mutations either alone or in different combinations, were generated by on-chip gene synthesis and site-directed mutagenesis. The constructs were inserted into a shuttle vector and transformed into three recipient strains (Escherichia coli, Staphylococcus aureus, and Salmonella Typhimurium). Subsequently, minimal inhibitory concentrations (MIC) of florfenicol and chloramphenicol were determined. In addition, protein modeling was used to predict the structural effects of the mutations. The lack of florfenicol-resistance mediating properties of the fexA variants could be attributed to the presence of a C110T and/or G98C mutation. Transformants carrying fexA variants containing either of these mutations, or both, showed a reduction of florfenicol MICs compared to those transformants carrying prototype fexA or any of the other variants. The significance of these mutations was supported by the generated protein models, indicating a substitution toward more voluminous amino-acids in the substrate-binding site of FexA. The remaining mutations, A391G and C961A, did not result in lower florfenicol-resistance compared to prototype fexA.

Effects of Matcha Green Tea Powder on Cognitive Functions of Community-Dwelling Elderly Individuals.

Matcha Green Tea Powder contains a variety of active ingredients beneficial to health, such as tea catechins, lutein and vitamin K. It is also known that these ingredients confer benefits upon cognitive functions of elderly people. Therefore, we aimed to investigate the relationship between a daily supplementation of Matcha and the change in cognitive functions of community-dwelling elderly people. A randomized, double-blind, placebo-controlled 12-week trial was performed. Sixty-one participants were recruited and randomly assigned to receive test drink containing 3g powder from fresh Matcha or placebo powder per day. Changes in cognitive function were assessed utilizing a psychometric test battery. Daily food intake was assessed by a Brief-type Self-administered Diet History Questionnaire (BDHQ). In the gender-specific analysis, a significant cognitive enhancement was observed in the Montreal Cognitive Assessment (MoCA) score in the active group of women. In dietary analysis, we found a significant inverse correlation between consumption of vitamin K in daily diet, excluding test drinks, and change in MoCA. The present study suggests that daily supplementation of Matcha Green Tea Powder has protective effects against cognitive decline in community-dwelling elderly women.

Relationship between elevated impulsivity and cognitive declines in elderly community-dwelling individuals.

Impulse control disorders are recognized as one of the behavioral and psychological symptoms of dementia (BPSD). Majority of studies on the treatment of BPSD related to impulsivity have rather focused on the aggression and agitation. In particular, it has not been investigated how cognitive declines are associated with impulsivity in community-dwelling elderly people. Here, we have measured the cognitive and memory functions and impulsivity of 212 elderly community-dwelling people using a psychometric test battery and analyzed the correlation between their level of impulsivity and cognitive functions by multiple regression analysis. We found an elevation of impulsivity, which was evaluated by the Barratt Impulsiveness Scale-11, closely related to decline of cognitive functions, which were evaluated by the Montreal Cognitive Assessment and the Mini-Mental State Examination, and Logical Memory function, which were evaluated by the Wechsler Memory Scale-Delayed Recall. Then we have divided them into groups based on the severity of cognitive decline and conducted an analysis of each group, the result of which showed that as this tendency was particularly noticeable in the suspected dementia group. Therefore, we have concluded that heightened impulsivity is negatively associated with cognitive and memory functions in community-dwelling elderly people.

Epidemiological features after emergency declaration in Hokkaido and report of 15 cases of COVID-19 including 3 cases requiring mechanical ventilation.

The ongoing spread of coronavirus disease (COVID-19) is a worldwide crisis. Hokkaido Prefecture in Japan promptly declared a state of emergency following the rapid increase of COVID-19 cases, and the policy became an example to mitigate the spread of COVID-19. We herein report 15 cases of COVID-19 including 3 cases requiring mechanical ventilation. Based on review of our cases, among patients over 50 years of age with underlying diseases such as hypertension and diabetes mellitus, and those who required oxygen administration tended to deteriorate. These cases highlight the importance of understanding the background and clinical course of severe cases to predict prognosis.

Unified Total Synthesis of (-)-Enigmazole A and (-)-15-O-Methylenigmazole A.

The total synthesis of cytotoxic marine phosphomacrolides, (-)-enigmazole A and (-)-15-O-methylenigmazole A, is described in detail. The 2,6-cis-substituted tetrahydropyran ring was efficiently elaborated by using a tandem olefin cross-metathesis/intramolecular oxa-Michael addition reaction. The 18-membered macrolactone skeleton was forged via a Au-catalyzed propargylic benzoate rearrangement/macrocyclic ring-closing metathesis sequence. Late-stage diversification of a common intermediate enabled unified total synthesis of (-)-enigmazole A and (-)-15-O-methylenigmazole A.

Hyper BOLD Activation in Dorsal Raphe Nucleus of APP/PS1 Alzheimer's Disease Mouse during Reward-Oriented Drinking Test under Thirsty Conditions.

Alzheimer's disease (AD), a neurodegenerative disease, causes behavioural abnormalities such as disinhibition, impulsivity, and hyperphagia. Preclinical studies using AD model mice have investigated these phenotypes by measuring brain activity in awake, behaving mice. In this study, we monitored the behavioural alterations of impulsivity and hyperphagia in middle-aged AD model mice. As a behavioural readout, we trained the mice to accept a water-reward under thirsty conditions. To analyse brain activity, we developed a measure for licking behaviour combined with visualisation of whole brain activity using awake fMRI. In a water-reward learning task, the AD model mice showed significant hyperactivity of the dorsal raphe nucleus in thirsty conditions. In summary, we successfully visualised altered brain activity in AD model mice during reward-oriented behaviour for the first time using awake fMRI. This may help in understanding the causes of behavioural alterations in AD patients.

GABA-Induced GnRH Release Triggers Chordate Metamorphosis.

Metamorphosis, a widespread life history strategy in metazoans, allows dispersal and use of different ecological niches through a dramatic body change from a larval stage [1, 2]. Despite its conservation and importance, the molecular mechanisms underlying its initiation and progression have been characterized in only a few animal models. In this study, through pharmacological and gene functional analyses, we identified neurotransmitters responsible for metamorphosis of the ascidian Ciona. Ciona metamorphosis converts swimming tadpole larvae into vase-like, sessile adults. Here, we show that the neurotransmitter GABA is a key regulator of metamorphosis. We found that gonadotropin-releasing hormone (GnRH) is a downstream neuropeptide of GABA. Although GABA is generally thought of as an inhibitory neurotransmitter, we found that it positively regulates secretion of GnRH through the metabotropic GABA receptor during Ciona metamorphosis. GnRH is necessary for reproductive maturation in vertebrates, and GABA is an important excitatory regulator of GnRH in the hypothalamus during puberty [3, 4]. Our findings reveal another role of the GABA-GnRH axis in the regulation of post-embryonic development in chordates.

Crystal structures of multidrug efflux pump MexB bound with high-molecular-mass compounds.

RND-type multidrug efflux pumps have two voluminous multisite drug-binding pockets named the proximal and distal binding pocket. High- and low-molecular-mass drugs bind to these proximal and distal pocket, respectively. Here, we report the crystal structures of MexB of Pseudomonas aeruginosa bound with high-molecular-mass compounds. Contrary to the expectations, lauryl maltose neopentyl glycol (LMNG, MW 1,005), which is a surfactant larger than the proximal pocket-binding drugs, was found to bind to the distal pocket: one of the two hydrophobic alkyl chains was inserted into the hydrophobic pit, which is the binding site of the efflux pump inhibitor ABI-PP. LMNG is a substrate of the MexAB-OprM system and competitively inhibits the export of other substrates by this system. However, LMNG does not inhibit the export of other substrates by the inhibitor-binding-pit mutant F178W, which retains the export activity of LMNG. The crystal structure of this mutant suggested that the alkyl chain of LMNG could no longer be inserted into the pit because of steric hindrance. We also determined the crystal structure of MexB containing the high-molecular-mass compound neopentyl glycol derivative C7NG (MW 1,028), the binding site of which overlapped with LMNG in the distal pocket, indicating that whether a substrate binds to the distal or proximal pockets is controlled not only by its molecular weight but also by its individual molecular characteristic.

Crystal structure of the multidrug resistance regulator RamR complexed with bile acids.

During infection, Salmonella senses and responds to harsh environments within the host. Persistence in a bile-rich environment is important for Salmonella to infect the small intestine or gallbladder and the multidrug efflux system AcrAB-TolC is required for bile resistance. The genes encoding this system are mainly regulated by the ramRA locus, which is composed of the divergently transcribed ramA and ramR genes. The acrAB and tolC genes are transcriptionally activated by RamA, whose encoding gene is itself transcriptionally repressed by RamR. RamR recognizes multiple drugs; however, the identity of the environmental signals to which it responds is unclear. Here, we describe the crystal structures of RamR in complexes with bile components, including cholic acid and chenodeoxycholic acid, determined at resolutions of 2.0 and 1.8 Å, respectively. Both cholic and chenodeoxycholic acids form four hydrogen bonds with Tyr59, Thr85, Ser137 and Asp152 of RamR, instead of π-π interactions with Phe155, a residue that is important for the recognition of multiple compounds including berberine, crystal violet, dequalinium, ethidium bromide and rhodamine 6 G. Binding of these compounds to RamR reduces its DNA-binding affinity, resulting in the increased transcription of ramA and acrAB-tolC. Our results reveal that Salmonella senses bile acid components through RamR and then upregulates the expression of RamA, which can lead to induction of acrAB-tolC expression with resulting tolerance to bile-rich environments.

Novel erythrocyte clumps revealed by an orphan gene Newtic1 in circulating blood and regenerating limbs of the adult newt.

The newt, a group of urodele amphibians, has outstanding ability to repeatedly regenerate various body parts, even in the terrestrial life-stage. In this animal, when the limb is amputated, a cell mass named the blastema appears on the stump and eventually gives rise to a new functional limb. Erythrocytes (red blood cells) in most non-mammalian vertebrates, including the newt, preserve their nucleus throughout their life-span, although physiological roles of such nucleated erythrocytes, other than oxygen delivery, are not known. Here we report novel behavior of erythrocytes in the newt. We identified an orphan gene Newtic1, whose transcripts significantly increased in the blastema. Newtic1 was expressed in a subset of erythrocytes that formed a novel clump (EryC). EryC formed a complex with monocytes and was circulating throughout the body. When the limb was amputated, EryCs were newly generated in the stump and accumulated into a distal portion of the growing blastema. Our data suggested that the newt erythrocytes carried multiple secretory molecules including growth factors and matrix metalloproteases, and were capable of delivering these molecules into the blastema as a form of EryCs. This study provides insight into regulations and roles of nucleated erythrocytes, that are independent of oxygen delivery.

Total Synthesis of (-)-Enigmazole†A.

Total synthesis of (-)-enigmazole A, a marine macrolide natural product with cytotoxic activity, has been accomplished. The tetrahydropyran moiety was constructed by means of a domino olefin cross-metathesis/intramolecular oxa-Michael addition of a δ-hydroxy olefin. After coupling of advanced intermediates, the macrocycle was formed through gold-catalyzed rearrangement of a propargylic benzoate, followed by ring-closing metathesis of the resultant α,ß-unsaturated ketone.

Acid-Tolerant Monomeric GFP from Olindias formosa.

The fluorescent protein (FP) color palette has greatly contributed to the visualization of molecular and cellular processes. However, most FPs lose fluorescence at a pH lower than their neutral pKa (∼6), and this has hampered their application in acidic organelles (pH ∼4.5-6.0). Currently, several cyan- and red-colored acid-tolerant FPs are available; however, there are few reports of acid-tolerant green FPs (GFPs) that are practically applicable to bioimaging. Here, we developed the acid-tolerant monomeric GFP "Gamillus" from the jellyfish Olindias formosa, with excellent brightness, maturation speed, and photostability. Results from X-ray crystallography and point mutagenesis suggest that across a broad pH range the acid tolerance is attributed to stabilization of deprotonation in the chromophore phenyl ring by forming a unique trans configuration. We demonstrate that Gamillus can serve as a molecular tag suitable for imaging in acidic organelles through autophagy-mediated molecular tracking to lysosomes.

Multiple entry pathways within the efflux transporter AcrB contribute to multidrug recognition.

AcrB is the major multidrug exporter in Escherichia coli. Although several substrate-entrances have been identified, the specificity of these various transport paths remains unclear. Here we present evidence for a substrate channel (channel 3)  from the central cavity of the AcrB trimer, which is connected directly to the deep pocket without first passing the switch-loop and the proximal pocket . Planar aromatic cations, such as ethidium, prefer channel 3 to channels 1 and 2. The efflux through channel 3 increases by targeted mutations and is not in competition with the export of drugs such as minocycline and erythromycin through channels 1 and 2. A switch-loop mutant, in which the pathway from the proximal to the deep pocket is hindered, can export only channel 3-utilizing drugs. The usage of multiple entrances thus contributes to the recognition and transport of a wide range of drugs with different physicochemical properties.

Crystallographic Analysis of Drug and Inhibitor-Binding Structure of RND-Type Multidrug Exporter AcrB in Physiologically Relevant Asymmetric Crystals.

Xenobiotic extruding pumps have recently been known to be widely distributed in living organisms from mammalian to bacteria as a host-defense mechanism in cellular level. These pumps not only confer multidrug resistance of cancer cells and pathogenic bacteria but also cause hereditary diseases through the mutation. Our purposes are to elucidate the molecular structures and mechanisms of these xenobiotic exporters.We had succeeded to determine the crystal structure of bacterial major multidrug exporter AcrB at 3.5 Å resolution (Murakami et al., Nature 419:587-593, 2002) and elucidated the structural bases of substrate recognition that the pump recognize the places and thus act as a "membrane vacuum cleaner." After that we also determined the crystal structure of the drug-binding form of AcrB in space group C2 in which asymmetric unit contains structurally asymmetric homo-trimer of AcrB (Murakami et al., Nature 443:173-179, 2006; Nakashima et al., Nature 480:565-569, 2011; Nakashima et al., Nature 500:120-126, 2013). Analyses revealed the existence of a specific mechanism to recognize numerous substrates that the multisite binding is the base of multidrug recognition rather than induced-fit, and functional-rotation mechanism in which three monomers undergo a strictly coordinated sequential conformational change cycle of access, binding, and extrusion. Determination of physiological asymmetric AcrB structure was crucially important to understand these transport mechanisms.