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Crystallographic studies show that high-molecular-mass drugs bind to the bacterial multidrug transporter AcrB at a previously unseen ‘proximal’ binding pocket before peristaltic transfer to the known ‘distal’ pocket, whereas low-molecular-mass drugs bind directly to the distal pocket. Two sites for AcrB drug transporter The crystal structure of the bacterial multidrug efflux transporter AcrB, bound to various antibiotics, has been determined. Rifampicin and erythromycin bind to a previously unseen binding pocket. These high-molecular-weight drugs bind first to the proximal binding pocket and then to the distal pocket after a series of conformational changes. Low-molecular-weight drugs, such as doxorubicin and minocycline, bind directly to the distal binding pocket. AcrB and its homologues are the principal multidrug transporters in Gram-negative bacteria 1 , 2 , 3 , 4 , 5 , 6 and are important in antibiotic drug tolerance 7 , 8 . AcrB is a homotrimer that acts as a tripartite complex 9 , 10 with the outer membrane channel TolC 11 , 12 and the membrane fusion protein AcrA 13 , 14 . Minocycline and doxorubicin have been shown to bind to the phenylalanine cluster region of the binding monomer 15 . Here we report the crystal structures of AcrB bound to the high-molecular-mass drugs rifampicin and erythromycin. These drugs bind to the access monomer, and the binding sites are located in the proximal multisite binding pocket, which is separated from the phenylalanine cluster region (distal pocket) by the Phe-617 loop. Our structures indicate that there are two discrete multisite binding pockets along the intramolecular channel. High-molecular-mass drugs first bind to the proximal pocket in the access state and are then forced into the distal pocket in the binding state by a peristaltic mechanism involving subdomain movements that include a shift of the Phe-617 loop. By contrast, low-molecular-mass drugs, such as minocycline and doxorubicin, travel through the proximal pocket without specific binding and immediately bind to the distal pocket. The presence of two discrete, high-volume multisite binding pockets contributes to the remarkably broad substrate recognition of AcrB.

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.

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. Multidrug transporters possess several drug binding sites. Here the authors describe a transport path specific for planar aromatic cations in the E. coli multi-drug transporter AcrB.

The first inhibitor-bound X-ray crystal structures of the bacterial multidrug efflux transporter AcrB and its homologue MexB are presented, with the inhibitor shown to bind the transporter through a narrow hydrophobic pit, thereby preventing rotation of AcrB and MexB monomers. Bacterial multidrug exporter structures Inhibitors of bacterial multidrug efflux transporters are necessary to combat bacterial multidrug resistance, but no clinically useful inhibitors are currently available. The multidrug efflux transporter AcrB and its homologues facilitate the multidrug resistance of many Gram-negative pathogens, and in this paper Akihito Yamaguchi and colleagues describe the first X-ray crystal structures of inhibitor-bound AcrB and its homologue MexB. The inhibitor, a pyridopyrimidine derivative, binds in a narrow hydrophobic 'pit' and inhibits the functional rotation of the AcrB/MexB monomers. These inhibitor-bound structures may facilitate the development of new inhibitors of this family of multidrug efflux transporters, which could be used in conjunction with existing antibiotics to help make them more effective. The multidrug efflux transporter AcrB and its homologues are important in the multidrug resistance of Gram-negative pathogens 1 , 2 . However, despite efforts to develop efflux inhibitors 3 , clinically useful inhibitors are not available at present 4 , 5 . Pyridopyrimidine derivatives are AcrB- and MexB-specific inhibitors that do not inhibit MexY 6 , 7 ; MexB and MexY are principal multidrug exporters in Pseudomonas aeruginosa 8 , 9 , 10 . We have previously determined the crystal structure of AcrB in the absence and presence of antibiotics 11 , 12 , 13 . Drugs were shown to be exported by a functionally rotating mechanism 12 through tandem proximal and distal multisite drug-binding pockets 13 . Here we describe the first inhibitor-bound structures of AcrB and MexB, in which these proteins are bound by a pyridopyrimidine derivative. The pyridopyrimidine derivative binds tightly to a narrow pit composed of a phenylalanine cluster located in the distal pocket and sterically hinders the functional rotation. This pit is a hydrophobic trap that branches off from the substrate-translocation channel. Phe 178 is located at the edge of this trap in AcrB and MexB and contributes to the tight binding of the inhibitor molecule through a π–π interaction with the pyridopyrimidine ring. The voluminous side chain of Trp 177 located at the corresponding position in MexY prevents inhibitor binding. The structure of the hydrophobic trap described in this study will contribute to the development of universal inhibitors of MexB and MexY in P. aeruginosa .

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.

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.

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.

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 3 g 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.

Most vertebrate retinas contain a single type of rod for scotopic vision and multiple types of cones for photopic and color vision. The retinas of certain amphibian species uniquely contain two types of rods: red rods, which express rhodopsin, and green rods, which express a blue-sensitive cone pigment (M1/SWS2 group). Spontaneous activation of rhodopsin induced by thermal isomerization of the retinal chromophore has been suggested to contribute to the rod’s background noise, which limits the visual threshold for scotopic vision. Therefore, rhodopsin must exhibit low thermal isomerization rate compared with cone visual pigments to adapt to scotopic condition. In this study, we determined whether amphibian blue-sensitive cone pigments in green rods exhibit low thermal isomerization rates to act as rhodopsin-like pigments for scotopic vision. Anura blue-sensitive cone pigments exhibit low thermal isomerization rates similar to rhodopsin, whereas Urodela pigments exhibit high rates like other vertebrate cone pigments present in cones. Furthermore, by mutational analysis, we identified a key amino acid residue, Thr47, that is responsible for the low thermal isomerization rates of Anura blue-sensitive cone pigments. These results strongly suggest that, through this mutation, anurans acquired special blue-sensitive cone pigments in their green rods, which could form the molecular basis for scotopic color vision with normal red rods containing green-sensitive rhodopsin.

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.