Identification and characterization of a novel class of self-sufficient cytochrome P450 hydroxylase involved in cyclohexanecarboxylate degradation in Paraburkholderia terrae strain KU-64.

Cytochrome P450 monooxygenases play important roles in metabolism. Here, we report the identification and biochemical characterization of P450CHC, a novel self-sufficient cytochrome P450, from cyclohexanecarboxylate-degrading Paraburkholderia terrae KU-64. P450CHC was found to comprise a [2Fe-2S] ferredoxin domain, NAD(P)H-dependent FAD-containing reductase domain, FCD domain, and cytochrome P450 domain (in that order from the N terminus). Reverse transcription-polymerase chain reaction results indicated that the P450CHC-encoding chcA gene was inducible by cyclohexanecarboxylate. chcA overexpression in Escherichia coli and recombinant protein purification enabled functional characterization of P450CHC as a catalytically self-sufficient cytochrome P450 that hydroxylates cyclohexanecarboxylate. Kinetic analysis indicated that P450CHC largely preferred NADH (Km = 0.011 m m) over NADPH (Km = 0.21 m m). The Kd, Km, and kcat values for cyclohexanecarboxylate were 0.083 m m, 0.084 m m, and 15.9 s-1, respectively. The genetic and biochemical analyses indicated that the physiological role of P450CHC is initial hydroxylation in the cyclohexanecarboxylate degradation pathway.

Cloning, expression, and characterization of Baeyer-Villiger monooxygenases from eukaryotic Exophiala jeanselmei strain KUFI-6N.

The fungus Exophiala jeanselmei strain KUFI-6N produces a unique cycloalkanone monooxygenase (ExCAMO) that displays an uncommon substrate spectrum of Baeyer-Villiger oxidation of 4-10-membered ring ketones. In this study, we aimed to identify and sequence the gene encoding ExCAMO from KUFI-6N and overexpress the gene in Escherichia coli. We found that the primary structure of ExCAMO is most closely related to the cycloalkanone monooxygenase from Cylindrocarpon radicicola ATCC 11011, with 54.2% amino acid identity. ExCAMO was functionally expressed in E. coli and its substrate spectrum and kinetic parameters were investigated. Substrate profiling indicated that ExCAMO is unusual among known Baeyer-Villiger monooxygenases owing to its ability to accept a variety of substrates, including C4-C12 membered ring ketones. ExCAMO has high affinity and catalytic efficiency toward cycloalkanones, the highest being toward cyclohexanone. Five other genes encoding Baeyer-Villiger monooxygenases were also cloned and expressed in E. coli.

Relationship between depression and movement quality in normal young adults.

[Purpose] Somatic symptoms, such as hyposomnia and anorexia, can affect depression and result in decreased movement quality; however, the relationship between movement quality and somatic symptoms is unclear. The present study investigated the characteristics of this relationship using observable posture and movements. [Participants and Methods] Twenty healthy young adults (mean age 20.9 ± 0.4 years) participated in the study. First, we administered the Self-rating Depression Scale questionnaire to the participants. Second, a qualified physical therapist assessed the movement quality of the volunteers using the Body Awareness Rating Scale-Movement Quality and Experience. We classified the participants into high- and low-score groups based on their Self-rating Depression Scale scores and then analyzed the Body Awareness Rating Scale-Movement Quality and Experience scores in both groups. [Results] There was a significant difference in the movement quality between the Self-rating Depression Scale high- and low-score groups. The Self-rating Depression Scale total score ranged from 20 to 80, with higher scores indicating a greater severity of depression. The low-score group had higher scores for the Body Awareness Rating Scale-Movement Quality and Experience than the high-score group in all items. [Conclusion] The present study showed that movement quality was related to symptoms of depression, thereby suggesting that early detection and treatment interventions for depression are possible by an assessment of movement quality.

The modulation of motor control by imitating non-biological motions: a study about motor resonance.

[Purpose] Sensorimotor experience modulates motor resonance, such as motor interference, which occurs when observing others' movements; however, it is unclear how motor resonance is modulated by intentionally imitating others' movements. This study examined the effects of imitation experience on subsequent motor resonance. [Subjects and Methods] Twenty-seven healthy participants performed horizontal arm movements while observing non-biological, incongruent (vertical) movements of a visual stimulus (triangle object) in pre- and post-test procedures. Thirteen participants in the imitation group imitated vertical movements (non-biological motion) of the triangle object between pre- and post-test procedures and fourteen participants in the non-imitation group observed that. [Results] Variance in the executed movements was measured as an index of motor resonance. Although there was no significant difference in the non-imitation group, there was a significantly smaller variance for post-test compared to pre-test in the imitation group. [Conclusion] Motor resonance was inhibited by intentionally imitating non-biological motions. Imitating movements different from one's own motor property might inhibit subsequent motor resonance. This finding might be applied to selectively using motor resonance as a form of rehabilitation.

Top 10 research questions to promote physical activity research in people with binge eating disorder.

Despite emerging evidence illustrating the benefits of physical activity for people with binge eating disorder, engaging this population in physical activity is challenging. The International Organization of Physical Therapists in Mental Health (IOPTMH) set out to summarize, appraise, and strengthen the direction of physical activity endeavors. This process led to the identification of 10 important research questions which are discussed. Addressing these 10 research questions is critical for developing evidence-based approaches for promoting and sustaining an active lifestyle in people with binge eating disorder.

Temperature dependence of lasing characteristics of irregular-shaped-microparticle ZnO laser.

We investigate the temperature dependence of the lasing characteristics (lasing peak energy spontaneous emission factor ß, and lasing threshold) of an irregular-shaped-ZnO-microparticle laser. The shift of the lasing peak energy with temperature is very small in the range of 120-300 K, thus, indicating that the peak is determined mainly by the resonance energy position of a given cavity mode, and not by the gain spectral peak. On the other hand, ß and lasing threshold are strongly dependent on temperature; ß reaches a maximum at a particular temperature, whereas the lasing threshold exhibits a minimum. In comparison with the theoretical calculations, it is found that ß and lasing threshold are optimum at the temperature at which the spontaneous emission spectral peak is in resonance with the peak of the cavity mode.

Discrete-mode ZnO microparticle random laser.

A random laser incorporating irregular-shaped ZnO microparticles exhibits a small number of lasing lines with stable lasing intensities and negligibly low background emission. This unique feature is in direct contrast to that observed for a conventional ZnO nanoparticle film random laser. Gain competition between the discrete laser modes also occurs in this microparticle random laser. These lasing characteristics are because of the intra-particle confinement of the laser cavity modes in each ZnO microparticle.

Cross-Sectional Analysis of Fall-Related Factors with a Focus on Fall Prevention Self-Efficacy and Self-Cognition of Physical Performance among Community-Dwelling Older Adults.

This study aimed to determine how fall prevention self-efficacy and degree of deviation in self-cognition of physical performance, which have recently received attention for their potential to explain falls in combination with a wide variety of fall-related factors, as well as affect falls. Older adults using day-care services (n = 27 with six men, mean age: 81.41 ± 7.43 years) were included in this study. Fall history in the past year, the modified fall efficacy scale (MFES), and physical performance and cognition errors were examined by evaluating the functional reach test (FRT), the stepping over test, and the timed up and go test (TUG), along with a questionnaire. In the fall (n = 14) and non-fall (n = 13) groups, logistic regression analysis using Bayesian statistical methods was used to identify factors associated with falls. The odds ratios for the MFES ranged from 0.97 to 1.0, while those of cognition-error items ranged from 3.1 to 170.72. These findings suggested that deviation in self-cognition of physical performance, particularly overestimation of timed cognitive ability, was a factor with more explanatory power for fall history. Future studies should analyze differences by disease and age group, which were not clarified in this study, to identify more detailed fall risk factors.

Cloning of two gene clusters involved in the catabolism of 2,4-dinitrophenol by Paraburkholderia sp. strain KU-46 and characterization of the initial DnpAB enzymes and a two-component monooxygenases DnpC1C2.

Little is currently known about the metabolism of the industrial pollutant 2,4-dinitrophenol (DNP), particularly among gram-negative bacteria. In this study, we identified two non-contiguous genetic loci spanning 22 kb of Paraburkholderia (formerly Burkholderia) sp. strain KU-46. Additionally, we characterized four key initial genes (dnpA, dnpB, and dnpC1C2) responsible for DNP degradation, providing molecular and biochemical evidence for the degradation of DNP via the formation of 4-nitrophenol (NP), a pathway that is unique among DNP utilizing bacteria. Reverse transcription polymerase chain reaction (PCR) analysis indicated that dnpA, which encodes the initial hydride transferase, and dnpB which encodes a nitrite-eliminating enzyme, were induced by DNP and organized in an operon. Moreover, we purified DnpA and DnpB from recombinant Escherichia coli to demonstrate their effect on the transformation of DNP to NP through the formation of a hydride-Meisenheimer complex of DNP, designated as H--DNP. The function of DnpB appears new since all homologs of the DnpB sequences in the protein database are annotated as putative nitrate ABC transporter substrate-binding proteins. The gene cluster responsible for the degradation of DNP after NP formation was designated dnpC1C2DXFER, and DnpC1 and DnpC2 were functionally characterized as the FAD reductase and oxygenase components of the two-component DNP monooxygenase, respectively. By elucidating the hqdA1A2BCD gene cluster, we are now able to delineate the final degradation pathway of hydroquinone to ß-ketoadipate before it enters the tricarboxylic acid cycle.

Factors Affecting the Life Satisfaction of Older People with Care Needs Who Live at Home.

The number of older people is increasing rapidly, and the number of older people with care needs who live at home is also increasing in Japan. Maintaining their life satisfaction has been a primary challenge. This study aimed to identify factors affecting the life satisfaction of older people with care needs. The study was conducted among older people using homecare services; 126 participants (mean age, 79.33 ± 7.51 years, 54 male) were included in the analysis. Logistic regression analysis with adjustment for age, sex, and economic status was conducted with life satisfaction as the objective variable and the Japanese version of occupational gaps questionnaire (OGQ-J), sense of coherence, functional independence measure, and environmental factors as explanatory variables. The variables that significantly affected life satisfaction were the OGQ-J (p = 0.0352, OR 0.90, 95% CI 0.81-0.99) and environmental factors (p = 0.0083, OR 4.41, 95% CI 1.52-14.11). This study's results indicate the importance of focusing on environmental factors and facilitating the participation of older people with care needs in activities they want to do to maintain and improve their life satisfaction.

The association between the clinical severity of heart failure and docosahexaenoic acid accumulation in hypertrophic cardiomyopathy.

OBJECTIVE: Hypertrophic cardiomyopathy (HCM) is a common genetic disease with diverse morphology, symptoms, and prognosis. Hypertrophied myocardium metabolism has not been explored in detail. We assessed the association between myocardium lipid metabolism and clinical severity of heart failure (HF) in HCM using imaging mass spectrometry (IMS). RESULTS: We studied 16 endomyocardial biopsy (EMB) specimens from patients with HCM. Analysis was conducted using desorption electrospray ionization IMS. The samples were assigned into two cohorts according to the period of heart biopsy (cohort 1, n = 9 and cohort 2, n = 7). In each cohort, samples were divided into two groups according to the clinical severity of HF in HCM: clinically severe and clinically mild groups. Signals showing a significant difference between the two groups were analyzed by volcano plot. In cohort 1, the volcano plot identified four signals; the intensity in the clinically severe group was more than twice that of the mild group. Out of the four signals, docosahexaenoic acid (DHA) showed significant differences in intensity between the two groups in cohort 2 (10,575.8 ± 2750.3 vs. 19,839.3 ± 4803.2, P = 0.025). The intensity of DHA was significantly higher in EMB samples from the clinically severe HCM group than in those from the mild group.

A methodology for creating mutants of G-protein coupled receptors stabilized in active state by combining statistical thermodynamics and evolutionary molecular engineering.

We challenged the stabilization of a G-protein coupled receptor (GPCR) in the active state solely by multiple amino-acid mutations without the agonist binding. For many GPCRs, the free energy of the active state is higher than that of the inactive state. When the inactive state is stabilized through the lowering of its free energy, the apparent midpoint temperature of thermal denaturation Tm exhibits a significant increase. However, this is not always the case for the stabilization of the active state. We constructed a modified version of our methodology combining statistical thermodynamics and evolutionary molecular engineering, which was recently developed for the inactive state. First, several residues to be mutated are determined using our statistical-thermodynamics theory. Second, a gene (mutant) library is constructed using Escherichia coli cells to efficiently explore most of the mutational space. Third, for the mutant screening, the mutants prepared in accordance with the library are expressed in engineered Saccharomyces cerevisiae YB14 cells which can grow only when a GPCR mutant stabilized in the active state has signaling function. For the adenosine A2A receptor tested, the methodology enabled us to sort out two triple mutants and a double mutant. It was experimentally corroborated that all the mutants exhibit much higher binding affinity for G protein than the wild type. Analyses indicated that the mutations make the structural characteristics shift toward those of the active state. However, only slight increases in Tm resulted from the mutations, suggesting the unsuitability of Tm to the stability measure for the active state.

Identification and characterization of a chc gene cluster responsible for the aromatization pathway of cyclohexanecarboxylate degradation in Sinomonas cyclohexanicum ATCC 51369.

Cyclohexanecarboxylate (CHCA) is formed by oxidative microbial degradation of n-alkylcycloparaffins and anaerobic degradation of benzoate, and also known to be a synthetic intermediate or the starter unit of biosynthesis of cellular constituents and secondary metabolites. Although two degradation pathways have been proposed, genetic information has been limited to the ß-oxidation-like pathway. In this study, we identified a gene cluster, designated chcC1XTC2B1B2RAaAbAc, that is responsible for the CHCA aromatization pathway in Sinomonas (formerly Corynebacterium) cyclohexanicum strain ATCC 51369. Reverse transcription-PCR analysis indicated that the chc gene cluster is inducible by CHCA and that it consists of two transcriptional units, chcC1XTC2B1B2R and chcAaAbAc. Overexpression of the various genes in Escherichia coli, and purification of the recombinant proteins led to the functional characterization of ChcAaAbAc as subunits of a cytochrome P450 system responsible for CHCA hydroxylation; ChcB1 and ChcB2 as trans-4-hydroxyCHCA and cis-4-hydroxyCHCA dehydrogenases, respectively; ChcC1 was identified as a 4-oxoCHCA desaturase containing a covalently bound FAD; and ChcC2 was identified as a 4-oxocyclohexenecarboxylate desaturase. The binding constant of ChcAa for CHCA was found to be 0.37 mM. Kinetic parameters established for ChcB1 indicated that it has a high catalytic efficiency towards 4-oxoCHCA compared to trans- or cis-4-hydroxyCHCA. The Km and Kcat values of ChcC1 for 4-oxoCHCA were 0.39 mM and 44 s-1, respectively. Taken together with previous work on the identification of a pobA gene encoding a 4-hydroxybenzoate hydroxylase, we have now localized the remaining set of genes for the final degradation of protocatechuate before entry into the tricarboxylic acid cycle.

Complete Genome Sequence of Mameliella alba Strain KU6B, a Cyclohexylamine-Utilizing Marine Bacterium.

Here, we report the complete genome sequence of Mameliella alba strain KU6B, a bacterium newly isolated from seawater of Boso Peninsula in Japan that is capable of utilizing cyclohexylamine. The complete genome contained a 5,386,988-bp circular chromosome and three circular plasmids of 256,516, 112,434, and 76,727 bp.

A novel piperidine degradation mechanism in a newly isolated piperidine degrader Pseudomonas sp. strain KU43P.

The degradation pathways in microorganisms for piperidine, a secondary amine with various applications, are not yet fully understood, especially in non-Mycobacterium species. In this study, we have identified a piperidine-degrading isolate (KU43P) from a soil sample collected in a cultivation field in Osaka, Japan, and characterized its mechanisms of piperidine degradation, thereby furthering current understanding of the process. The genome of isolate KU43P consists of a 5,869,691-bp circular chromosome with 62.67% GC content and with 5,294 predicted protein-coding genes, 77 tRNA genes, and 22 rRNA genes. 16S rRNA gene sequence analysis and average nucleotide identity analysis suggest that the isolate is a novel species of the Pseudomonas putida group in the genus Pseudomonas. The genomic region encoding the piperidine degradation pathway, designated as the pip gene cluster, was identified using transposon mutagenesis and reverse transcription polymerase chain reaction. Deletion analyses of pipA, which encodes a glutamine synthetase (GS)-like protein, and pipBa, which encodes a cytochrome P450 monooxygenase, indicate that pipA and pipBa are involved in piperidine metabolism and suggest that pipA is involved in the first step of the piperidine metabolic pathway. Escherichia coli whole cells overexpressing PipA converted piperidine and glutamate to γ-glutamylpiperidide, and crude cell extract enzyme assays of PipA showed that this reaction requires ATP and Mg2+. These results clearly show that pipA encodes γ-glutamylpiperidide synthetase and that piperidine is first glutamylated and then hydroxylated in the piperidine degradation pathway of Pseudomonas sp. strain KU43P. This study has filled a void in the general knowledge of the microbial degradation of amine compounds.

Complete Genome Sequence of Pseudomonas sp. Strain KUIN-1, a Model Strain for Studies on the Production of Cell-Free Ice Nucleation Proteins.

Pseudomonas sp. (formerly Pseudomonas fluorescens) strain KUIN-1 is an ice-nucleating bacterium that was isolated from the leaves of field beans (Phaseolus vulgaris L.). This microorganism can release cell-free ice nucleation proteins and shows cold shock-induced freezing tolerance. Here, we report the 6,028,589-bp complete genome sequence of Pseudomonas sp. KUIN-1.

Isolation of marine xylene-utilizing bacteria and characterization of Halioxenophilus aromaticivorans gen. nov., sp. nov. and its xylene degradation gene cluster.

Seven xylene-utilizing bacterial strains were isolated from seawater collected off the coast of Japan. Analysis of 16S rRNA gene sequences indicated that six isolates were most closely related to the marine bacterial genera Alteromonas, Marinobacter or Aestuariibacter. The sequence of the remaining strain, KU68FT, showed low similarity to the 16S rRNA gene sequences of known bacteria with validly published names, the most similar species being Maricurvus nonylphenolicus strain KU41ET (92.6% identity). On the basis of physiological, chemotaxonomic and phylogenetic data, strain KU68FT is suggested to represent a novel species of a new genus in the family Cellvibrionaceae of the order Cellvibrionales within the Gammaproteobacteria, for which the name Halioxenophilus aromaticivorans gen. nov., sp. nov. is proposed. The type strain of Halioxenophilus aromaticivorans is KU68FT (=JCM 19134T = KCTC 32387T). PCR and sequence analysis revealed that strain KU68FT possesses an entire set of genes encoding the enzymes for the upper xylene methyl-monooxygenase pathway, xylCMABN, resembling the gene set of the terrestrial Pseudomonas putida strain mt-2.

Top 10 research questions to promote physical activity in bipolar disorders: A consensus statement from the International Organization of Physical Therapists in Mental Health.

BACKGROUND: Research has only recently started to consider the importance and applicability of physical activity (PA) for people with bipolar disorder (BD). The aim of the current study is to highlight 10 pertinent PA research questions in people with BD. METHODS: The International Organization of Physical Therapy in Mental Health executed a consultation with all National organizations (n=13) to identify the most salient questions to guide future research on PA in BD. RESULTS: We identified the following 10 questions: (1) What are the benefits of PA for people with BD? (2) What are the most prominent safety issues for PA prescription in BD? (3) What is the optimal PA prescription for people with BD? (4) What are the key barriers to PA among people with BD? (5) What are the most effective motivational strategies for ensuring PA adoption and maintenance in BD? (6) How do we translate PA research into community practice? (7) If one treatment goal is increased physical activity, what type of professionals are needed as part of a multidisciplinary team? (8) How do we incorporate PA as a vital sign in clinical practice? (9) How can we prevent sedentary behavior in BD? (10) What is the most appropriate PA assessment method? LIMITATIONS: We did not consult people with BD. CONCLUSIONS: Addressing these questions is critical for developing evidence-based approaches for promoting and sustaining an active lifestyle in BD. Ultimately, achieving this will reduce the burden of cardiovascular disease and improve the quality of life of this population.

What are the top 10 physical activity research questions in schizophrenia?

PURPOSE: Research has only recently started to consider the applicability of physical activity (PA) for people with schizophrenia. Although there is increasing evidence for the benefits of physical activity, this population remains generally physically inactive and sedentary. The aim of the current study is to highlight 10 pertinent physical activity research questions in people with schizophrenia. METHOD: The International Organisation of Physical Therapy in Mental Health (IOPTMH) executed a consultation of its National Organisations (n = 13) to identify the most salient questions relevant to guide clinical practise on physical activity in people with schizophrenia. RESULTS: We identified the following 10 questions: (1) What are the benefits of physical activity for people with schizophrenia? (2) What are the mechanisms of the physical activity effects in people with schizophrenia? (3) What are the most prominent safety issues for physical activity prescription in people with schizophrenia? (4) What is the most optimal physical activity prescription for people with schizophrenia? (5) What are the key barriers for engaging people with schizophrenia in physical activity? (6) What are the most effective motivational interventions for physical activity adoption and maintenance in people with schizophrenia? (7) How do we translate physical activity research into clinical and community practise? (8) How can we ensure integration of physical therapists within the multidisciplinary mental health treatment team? (9) How can we prevent sedentary behaviour in people with schizophrenia? (10) What is the most appropriate physical activity assessment method in clinical practise? CONCLUSIONS: Addressing these questions is critical for developing evidence-based approaches for promoting and sustaining an active lifestyle in people with schizophrenia. Ultimately, achieving this will improve the quality of life of this population. Implications for Rehabilitation Investigation of behaviour change interventions for people with schizophrenia is critical. A low cost, easy to use, clinical, valid physical activity questionnaire is urgently needed.