Characterization of the molecular properties of KtrC, a second RCK domain that regulates a Ktr channel in Bacillus subtilis

RCK (regulating conductance of K+) domains are common regulatory domains that control the activity of eukaryotic and prokaryotic K+ channels and transporters. In bacteria these domains play roles in osmoregulation, regulation of turgor and membrane potential and in pH homeostasis. Whole-genome sequencing unveiled RCK gene redundancy, however the biological role of this redundancy is not well understood. In Bacillus subtilis, there are two closely related RCK domain proteins (KtrA and KtrC) that regulate the activity of the Ktr cation channels. KtrA has been well characterized but little is known about KtrC. We have characterized the structural and biochemical proprieties of KtrC and conclude that KtrC binds ATP and ADP, just like KtrA. However, in solution KtrC exist in a dynamic equilibrium between octamers and non-octameric species that is dependent on the bound ligand, with ATP destabilizing the octameric ring relative to ADP. Accordingly, KtrC-ADP crystal structures reveal closed octameric rings similar to those in KtrA, while KtrC-ATP adopts an open assembly with RCK domains forming a super-helix. In addition, both KtrC-ATP and -ADP octamers are stabilized by the signaling molecule cyclic-di-AMP, which binds to KtrC with high affinity. In contrast, c-di-AMP binds with 100-fold lower affinity to KtrA. Despite these differences we show with an E. coli complementation assay that KtrC and KtrA are interchangeable and able to form functional transporters with both KtrB and KtrD. The distinctive properties of KtrC, in particular ligand-dependent assembly/disassembly, suggest that this protein has a specific physiological role that is distinct from KtrA.Work was supported by Fundação Luso-Americana para o Desenvolvimento through the FLAD Life Science 2020 award entitled “Bacterial K+ transporters are potential antimicrobial targets: mechanisms of transport and regulation” and by FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT - Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project POCI-01-0145-FEDER-029863 (PTDC/BIA-BQM/29863/2017) and of project "Institute for Research and Innovation in Health Sciences" (POCI-01-0145-FEDER-007274). RR was supported by FCT fellowship (SFRH/BPD/111525/2015), CMT-D was supported by FCT fellowship (SFRH/BD/123761/2016 ).

Fluorometric Liposome Screen for Inhibitors of a Physiologically Important Bacterial Ion Channel

The bacterial K+ homeostasis machinery is widely conserved across bacterial species, and different from that in animals. Dysfunction in components of the machinery has an impact on intracellular turgor, membrane potential, adaptation to changes in both extracellular pH and osmolarity, and in virulence. Using a fluorescence-based liposome flux assay, we have performed a high-throughput screen to identify novel inhibitors of the KtrAB ion channel complex from Bacillus subtilis, a component of the K+ homeostasis machinery that is also present in many bacterial pathogens. The screen identified 41 compounds that inhibited K+ flux and that clustered into eight chemical groups. Many of the identified inhibitors were found to target KtrAB with an in vitro potency in the low µM range. We investigated the mechanisms of inhibition and found that most molecules affected either the membrane component of the channel, KtrB alone or the full KtrAB complex without a preference for the functional conformation of the channel, thus broadening their inhibitory action. A urea derivative molecule that inhibited the membrane component of KtrAB affected cell viability in conditions in which KtrAB activity is essential. With this proof-of-concept study, we demonstrate that targeting components of the K+ homeostasis machinery has the potential as a new antibacterial strategy and that the fluorescence-based flux assay is a robust tool for screening chemical libraries.This work was supported by FEDER funds through COMPETE 2020-POCI, Portugal 2020, and FCT – Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior: POCI-01-0145-FEDER-029863 (PTDC/BIA-BQM/29863/2017), and by “Fundação Luso-Americana para o Desenvolvimento” FLAD Life Science 2020 awarded to JM-C. We acknowledge FCT fellowship SFRH/BPD/105672/2015 and contract DL 57/2016/CP1355/CT0026 awarded to AF, fellowship SFRH/BPD/107785/2015 to AP, and fellowship SFRH/BD/123761/2016 to CT-D

Effects of orofacial myofunctional therapy on masticatory function in individuals submitted to orthognathic surgery: a randomized trial

Abstract Objectives: The esthetic and functional results of orthognathic surgery of severe dentofacial deformities are predictable, however there are differences regarding the effects on stomatognathic system. The aim was to investigate the effects of orofacial myofunctional therapy (OMT) on the masticatory function in individuals with dentofacial deformity submitted to orthognathic surgery (OGS). Material and Methods: Forty-eight individuals (18-40 years) were evaluated, 14 undergoing OMT (treated group-TG), 10 without this treatment (untreated group-UTG) and 24 in a control group with normal occlusion; for clinical aspects the data of an individual was missed (n=46). Chewing was performed using the Expanded protocol of orofacial myofunctional evaluation with scores (OMES-E). Muscle tone and mobility were also analyzed before (P0), three (P1) and six months (P2) after OGS. Surface electromyography of the masseter and temporalis muscles was performed, considering the parameters amplitude and duration of act and cycle, and the number of masticatory cycles. The OMT consisted of ten therapeutic sessions along the postoperative period. The results were compared using parametric and non-parametric tests. Results: TG showed higher scores in P1 and P2 than P0; for the masticatory type the scores in P2 were significantly higher than P0. In addition, the proportion of individuals with adequate tone of lower lip and adequate tongue mobility for TG increased significantly from P1 and P2 in relation to P0. The EMG results showed a decrease in act and cycle duration in P2 in relation to P0 and P1 for the TG; furthermore the values were close to controls. An increase in the number of cycles from P0 to P2 was also observed, indicating faster chewing, which may be attributed to an improvement of balanced occlusion associated with OMT. Conclusion: There were positive effects of OMT on the clinical and electromyography aspects of chewing in individual submitted to orthognathic surgery

Chemical composition and antibacterial activity of essential oils from the medicinal plant Mentha cervina L. grown in Portugal

Mentha cervina is a medicinal plant traditionally used in Portugal in folk medicine, in different gastric disorders and inflammations of the respiratory tract. In order to validate those traditional uses, M. cervina essential oils (EOs) were characterized by GC and GC–MS and their antimicrobial activity was tested against 23 bacterial strains (including multiresistant strains). The EOs were dominated by the monoterpenes pulegone (52–75%), isomenthone (8–24%), limonene (4–6%), and menthone (1–2%). The antibacterial activity of these EOs was compared to that of the main components standards. The most effective antibacterial activity was expressed by the EOs against the Gram-negative bacteria, Escherichia coli and Acinetobacter baumanni, with MIC values of 1 mg/ml. The EOs complex mixtures were more active than the individual aromatic components supporting the hypothesis that the EOs antibacterial activity is a function of the synergistic effect of their different aromatic components. These results show the potential role of M. cervina EOs as antibacterial agents and validate the traditional use of this plant

Tuberculosis control: decentralization, local planning and management specificities

The goal was to analyze, according to the perception of health managers, the practices that guide tuberculosis control actions in cities in the metropolitan region of João Pessoa - PB, Brazil. This qualitative study involved eight professionals in management functions. Testimonies were collected through semi-structured interviews between May and June 2009 and organized through content analysis. Despite the acknowledged benefits of tuberculosis control action decentralization, local planning indicates the predominance of a bureaucratic model that is restricted to negotiation and supplies. Local programming is centered on the coordinator, which shows a command line and vertical management that lead to the fragmentation of the work process. Management action should follow an innovative and transformative route that surpasses bureaucratic barriers and faces the biggest challenge it is proposed: to balance professional interrelations with a view to improving health work performance.Se buscó analizar, según la percepción de los gestores de salud, las prácticas que orientan las acciones de control de la tuberculosis en municipios de la región metropolitana de Joao Pesoa, estado de Paraíba. Se trata de un estudio cualitativo que envolvió ocho profesionales que ejercían cargos de gestión. Las declaraciones fueron recolectadas por medio de entrevistas semiestructuradas, entre mayo y julio de 2009, y organizadas mediante análisis de contenido. A pesar de que se reconozcan los beneficios de la descentralización de las acciones de control de la tuberculosis, la planificación local señala la predominancia del modelo burocrático restricto a negociación y suministro de insumos. La programación local se centra en la figura del coordinador, retratando una línea de comando y gestión vertical que induce a la fragmentación del proceso de trabajo. La tarea de administrar debe explorar un camino innovador y transformador, que ultrapase las barreras burocráticas y alcance el mayor desafío que le es impuesto: equilibrar las interrelaciones profesionales con la finalidad de perfeccionar el desempeño del trabajo en salud.Buscou-se analisar, segundo a percepção dos gestores de saúde, as práticas que norteiam as ações de controle da tuberculose, em municípios da região metropolitana de João Pessoa, PB. Trata-se de estudo qualitativo que envolveu oito profissionais que exerciam cargos de gestão. Os depoimentos foram coletados por meio de entrevista semiestruturada, entre maio e julho de 2009, e organizados mediante análise de conteúdo. Embora se reconheça os benefícios da descentralização das ações de controle da tuberculose, o planejamento local sinaliza a predominância de modelo burocrático restrito à negociação e provisão de insumos. A programação local centra-se na figura do coordenador, retratando uma linha de comando e gestão vertical que induzem à fragmentação do processo de trabalho. A tarefa de gerenciar deve trilhar caminho inovador e transformador que ultrapasse as barreiras burocráticas e alcance o maior desafio que lhe é imposto: equilibrar as inter-relações profissionais no intuito de aperfeiçoar o desempenho do trabalho em saúde

Tauroursodeoxycholic Acid Improves Motor Symptoms in a Mouse Model of Parkinson's Disease

Parkinson's disease (PD) is characterized by severe motor symptoms, and currently there is no treatment that retards disease progression or reverses damage prior to the time of clinical diagnosis. Tauroursodeoxycholic acid (TUDCA) is neuroprotective in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD; however, its effect in PD motor symptoms has never been addressed. In the present work, an extensive behavior analysis was performed to better characterize the MPTP model of PD and to evaluate the effects of TUDCA in the prevention/improvement of mice phenotype. MPTP induced significant alterations in general motor performance paradigms, including increased latency in the motor swimming, adhesive removal and pole tests, as well as altered gait, foot dragging, and tremors. TUDCA administration, either before or after MPTP, significantly reduced the swimming latency, improved gait quality, and decreased foot dragging. Importantly, TUDCA was also effective in the prevention of typical parkinsonian symptoms such as spontaneous activity, ability to initiate movement and tremors. Accordingly, TUDCA prevented MPTP-induced decrease of dopaminergic fibers and ATP levels, mitochondrial dysfunction and neuroinflammation. Overall, MPTP-injected mice presented motor symptoms that are aggravated throughout time, resembling human parkinsonism, whereas PD motor symptoms were absent or mild in TUDCA-treated animals, and no aggravation was observed in any parameter. The thorough demonstration of improvement of PD symptoms together with the demonstration of the pathways triggered by TUDCA supports a subsequent clinical trial in humans and future validation of the application of this bile acid in PD.National funds, through the Foundation for Science and Technology (Portugal) (FCT), under the scope of the projects PTDC/NEU-NMC/0248/2012, UID/DTP/04138/2013 and POCI-01-0145-FEDER-007038, and post-doctoral grants SFRH/BPD72891/2010 (to A.I.R.), SFRH/BPD/95855/2013 (to M.J.N.), SFRH/BPD/98023/2013 (to A.N.C.), SFRH/BPD/91562/2012 (to A.S.F.) and UMINHO/BI/248/2016 (to S.D.S.). This work has also been developed under the scope of the project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Program (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER), and by FEDER funds, through the Competitiveness Factors Operational Program (COMPETE)info:eu-repo/semantics/publishedVersio