Isoprenoid biosynthesis inhibition disrupts Rab5 localization and food vacuolar integrity in Plasmodium falciparum

The antimalarial agent fosmidomycin is a validated inhibitor of the nonmevalonate isoprenoid biosynthesis (methylerythritol 4-phosphate [MEP]) pathway in the malaria parasite, Plasmodium falciparum. Since multiple classes of prenyltransferase inhibitors kill P. falciparum, we hypothesized that protein prenylation was one of the essential functions of this pathway. We found that MEP pathway inhibition with fosmidomycin reduces protein prenylation, confirming that de novo isoprenoid biosynthesis produces the isoprenyl substrates for protein prenylation. One important group of prenylated proteins is small GTPases, such as Rab family members, which mediate cellular vesicular trafficking. We have found that Rab5 proteins dramatically mislocalize upon fosmidomycin treatment, consistent with a loss of protein prenylation. Fosmidomycin treatment caused marked defects in food vacuolar morphology and integrity, consistent with a defect in Rab-mediated vesicular trafficking. These results provide insights to the biological functions of isoprenoids in malaria parasites and may assist the rational selection of secondary agents that will be useful in combination therapy with new isoprenoid biosynthesis inhibitors

The Antibacterial and Anti-biofilm Activity of Metal Complexes Incorporating 3,6,9- Trioxaundecanedioate and 1,10-Phenanthroline Ligands in Clinical Isolates of Pseudomonas Aeruginosa from Irish Cystic Fibrosis Patients

Chronic infections of Pseudomonas aeruginosa in the lungs of cystic fibrosis (CF) patients are problematic in Ireland where inherited CF is prevalent. The bacteria\u27s capacity to form a biofilm in its pathogenesis is highly virulent and leads to decreased susceptibility to most antibiotic treatments. Herein, we present the activity profiles of the Cu(II), Mn(II) and Ag(I) tdda-phen chelate complexes {[Cu(3,6,9-tdda)(phen)2].3H2O.EtOH}n (Cu-tdda-phen), {[Mn(3,6,9-tdda)(phen)2].3H2O.EtOH}n (Mn-tdda-phen) and [Ag2(3,6,9-tdda)(phen)4].EtOH (Ag-tdda-phen) (tddaH2 = 3,6,9-trioxaundecanedioic acid; phen = 1,10-phenanthroline) towards clinical isolates of P. aeruginosa derived from Irish CF patients in comparison to two reference laboratory strains (ATCC 27853 and PAO1). The effects of the metal-tdda-phen complexes and gentamicin on planktonic growth, biofilm formation (pre-treatment) and mature biofilm (post-treatment) alone and in combination were investigated. The effects of the metal-tdda-phen complexes on the individual biofilm components; exopolysaccharide, extracellular DNA (eDNA), pyocyanin and pyoverdine are also presented. All three metal-tdda-phen complexes showed comparable and often superior activity to gentamicin in the CF strains, compared to their activities in the laboratory strains, with respect to both biofilm formation and established biofilms. Combination studies presented synergistic activity between all three complexes and gentamicin, particularly for the post-treatment of established mature biofilms, and was supported by the reduction of the individual biofilm components examined

In Vivo Activity of Metal Complexes Containing 1,10-Phenanthroline and 3,6,9-Trioxaundecanedioate Ligands against Pseudomonas aeruginosa Infection in Galleria mellonella Larvae

Drug-resistant Pseudomonas aeruginosa is rapidly developing resulting in a serious global threat. Immunocompromised patients are specifically at risk, especially those with cystic fibrosis (CF). Novel metal complexes incorporating 1,10-phenanthroline (phen) ligands have previously demonstrated antibacterial and anti-biofilm effects against resistant P. aeruginosa from CF patients in vitro. Herein, we present the in vivo efficacy of {[Cu(3,6,9-tdda)(phen)2]·3H2O·EtOH}n (Cu-tdda-phen), {[Mn(3,6,9-tdda)(phen)2]·3H2O·EtOH}n (Mn-tdda-phen) and [Ag2(3,6,9-tdda)(phen)4]·EtOH (Ag-tdda-phen) (tddaH2 = 3,6,9-trioxaundecanedioic acid). Individual treatments of these metal-tdda-phen complexes and in combination with the established antibiotic gentamicin were evaluated in vivo in larvae of Galleria mellonella infected with clinical isolates and laboratory strains of P. aeruginosa. G. mellonella were able to tolerate all test complexes up to 10 µg/larva. In addition, the immune response was affected by stimulation of immune cells (hemocytes) and genes that encode for immune-related peptides, specifically transferrin and inducible metallo-proteinase inhibitor. The amalgamation of metal-tdda-phen complexes and gentamicin further intensified this response at lower concentrations, clearing a P. aeruginosa infection that were previously resistant to gentamicin alone. Therefore this work highlights the anti-pseudomonal capabilities of metal-tdda-phen complexes alone and combined with gentamicin in an in vivo model

Primary care research priorities in low- and middle-income countries

PURPOSE To identify and prioritize the needs for new research evidence for primary health care (PHC) in low-and middle-income countries (LMICs) about organization, models of care, and financing of PHC. METHODS Three-round expert panel consultation of LMIC PHC practitioners and academics sampled from global networks, via web-based surveys. Iterative literature review conducted in parallel. Round 1 (pre–Delphi survey) elicited possible research questions to address knowledge gaps about organization and models of care and about financing. Round 2 invited panelists to rate the importance of each question, and in round 3 panelists provided priority ranking. RESULTS One hundred forty-one practitioners and academics from 50 LMICs from all global regions participated and identified 744 knowledge gaps critical to improving PHC organization and 479 for financing. Four priority areas emerged: effective transition of primary and secondary services, horizontal integration within a multidisciplinary team and intersectoral referral, integration of private and public sectors, and ways to support successfully functioning PHC professionals. Financial evidence priorities were mechanisms to drive investment into PHC, redress inequities, increase service quality, and determine the minimum necessary budget for good PHC. CONCLUSIONS This novel approach toward PHC needs in LMICs, informed by local academics and professionals, created an expansive and prioritized list of critical knowledge gaps in PHC organization and financing. It resulted in research questions, offering valuable guidance to global supporters of primary care evaluation and implementation. Its source and context specificity, informed by LMIC practitioners and academics, should increase the likelihood of local relevance and eventual success in implementing research findings

Primary care financing: a systematic assessment of research priorities in low- and middle-income countries

Introduction: Financing of primary healthcare (PHC) is the key to the provision of equitable universal care. We aimed to identify and prioritise the perceived needs of PHC practitioners and researchers for new research in low- and middle-income countries (LMIC) about financing of PHC. Methods: Three-round expert panel consultation using web-based surveys of LMIC PHC practitioners, academics and policy-makers sampled from global networks. Iterative literature review conducted in parallel. First round (PreDelphi survey) elicited possible research questions to address knowledge gaps about financing. Responses were independently coded, collapsed and synthesised to two lists of questions. Round 2 (Delphi Round 1) invited panellists to rate importance of each question. In Round 3 (Delphi Round 2), panellists ranked questions in order of importance. Results: A diverse range of PHC practitioners, academics and policy-makers in LMIC representing all global regions identified 479 knowledge gaps as potentially critical to improving PHC financing. Round 2 provided 31 synthesised questions on financing for rating. The top 16 were ranked in Round 3e to produce four prioritised research questions. Conclusions: This novel exercise created an expansive and prioritised list of critical knowledge gaps in PHC financing research questions. This offers valuable guidance to global supporters of primary care evaluation and implementation, including research funders and academics seeking research priorities. The source and context specificity of this research, informed by LMIC practitioners and academics on a global and local basis, should increase the likelihood of local relevance and eventual success in implementing the findings

Research gaps in the organisation of primary healthcare in low-income and middle-income countries and ways to address them: a mixed-methods approach

Introduction Since the Alma-Ata Declaration 40 years ago, primary healthcare (PHC) has made great advances, but there is insufficient research on models of care and outcomes—particularly for low-income and middle-income countries (LMICs). Systematic efforts to identify these gaps and develop evidence-based strategies for improvement in LMICs has been lacking. We report on a global effort to identify and prioritise the knowledge needs of PHC practitioners and researchers in LMICs about PHC organisation. Methods Three-round modified Delphi using web-based surveys. PHC practitioners and academics and policy-makers from LMICs sampled from global networks. First round (pre-Delphi survey) collated possible research questions to address knowledge gaps about organisation. Responses were independently coded, collapsed and synthesised. Round 2 (Delphi round 1) invited panellists to rate importance of each question. In round 3 (Delphi round 2), panellists ranked questions into final order of importance. Literature review conducted on 36 questions and gap map generated. Results Diverse range of practitioners and academics in LMICs from all global regions generated 744 questions for PHC organisation. In round 2, 36 synthesised questions on organisation were rated. In round 3, the top 16 questions were ranked to yield four prioritised questions in each area. Literature reviews confirmed gap in evidence on prioritised questions in LMICs. Conclusion In line with the 2018 Astana Declaration, this mixed-methods study has produced a unique list of essential gaps in our knowledge of how best to organise PHC, priority-ordered by LMIC expert informants capable of shaping their mitigation. Research teams in LMIC have developed implementation plans to answer the top four ranked research questions

Shoreline armoring removal: assessment of restoration effectiveness in the Salish Sea

Shoreline armoring removal is becoming a common restoration technique in the nearshore of the Salish Sea, yet we lack a comprehensive understanding of the ecological benefits obtained, and how such an understanding could be used to inform management recommendations and educate diverse audiences. To address this knowledge gap, we studied effects of shoreline armor removal at 10 sites, expanding the spatial framework of what was previously known by collaborating across academic (University of Washington), agency (Washington Department of Fish and Wildlife), and citizen science groups. Each site had three beach types of: (1) restored beaches with armoring removed 1-11 years ago, with a mean of four years, (2) armored beaches altered by bulkheads or riprap, and (3) un-armored reference beaches with more natural conditions. We sampled eight metrics of physical and biological conditions, focusing on supratidal and upper intertidal elevations most affected by armoring and targeted by restoration actions: beach wrack, wrack invertebrates, sediments, terrestrial insects, riparian vegetation and logs, beach profiles, forage fish habitat, and stable isotopes of beach-hopper amphipods to reveal ratios of marine and terrestrial food sources. These metrics spanned the functions of beach stability, ecological diversity, and food web support for juvenile salmon and birds. Results indicated that some beach metrics restore quickly, such as wrack accumulation, while others take longer, such as log accumulation. Sediment sizes at restored beaches approximated those of reference beaches, and were appropriate for forage fish spawning. In general, terrestrial-associated metrics were greater at reference beaches, although there was evidence that insect diversity and logs with plant growth increased when beaches were restored greater than four years. This implies that restored beach functions increase through time, providing improved support for forage fish, salmon, and birds

The relationship between adverse neighborhood socioeconomic context and HIV continuum of care outcomes in a diverse HIV clinic cohort in the Southern United States

Retention in care and viral suppression are critical to delaying HIV progression and reducing transmission. Neighborhood socioeconomic context (NSEC) may affect HIV care receipt. We therefore assessed NSEC's impact on retention and viral suppression in a diverse HIV clinical cohort. HIV-positive adults with ≥1 visit at the Vanderbilt Comprehensive Care Clinic and 5-digit ZIP code tabulation area (ZCTA) information between 2008 and 2012 contributed. NSEC z-score indices used neighborhood-level socioeconomic indicators for poverty, education, labor-force participation, proportion of males, median age, and proportion of residents of black race by ZCTA. Retention was defined as ≥2 HIV care visits per calendar year, >90 days apart. Viral suppression was defined as an HIV-1 RNA <200 copies/mL at last measurement per calendar year. Modified Poisson regression was used to estimate risk ratios (RR) and 95% confidence intervals (CI). Among 2272 and 2541 adults included for retention and viral suppression analyses, respectively, median age and CD4 count at enrollment were approximately 38 (1st and 3rd quartile: 30, 44) years and 351 (176, 540) cells/μL, respectively, while 24% were female, and 39% were black. Across 243 ZCTAs, median NSEC z-score was 0.09 (-0.66, 0.48). Overall, 79% of person-time contributed was retained and 74% was virally suppressed. In adjusted models, NSEC was not associated with retention, though being in the 4th vs. 1st NSEC quartile was associated with lack of viral suppression (RR = 0.88; 95% CI: 0.80-0.97). Residing in the most adverse NSEC was associated with lack of viral suppression. Future studies are needed to confirm this finding

Primary Care Research Priorities in Low-and Middle-Income Countries

PURPOSE To identify and prioritize the needs for new research evidence for primary health care (PHC) in low-and middle-income countries (LMICs) about organization, models of care, and financing of PHC. METHODS Three-round expert panel consultation of LMIC PHC practitioners and academics sampled from global networks, via web-based surveys. Iterative literature review conducted in parallel. Round 1 (pre-Delphi survey) elicited possible research questions to address knowledge gaps about organization and models of care and about financing. Round 2 invited panelists to rate the importance of each question, and in round 3 panelists provided priority ranking. RESULTS One hundred forty-one practitioners and academics from 50 LMICs from all global regions participated and identified 744 knowledge gaps critical to improving PHC organization and 479 for financing. Four priority areas emerged: effective transition of primary and secondary services, horizontal integration within a multidisciplinary team and intersectoral referral, integration of private and public sectors, and ways to support successfully functioning PHC professionals. Financial evidence priorities were mechanisms to drive investment into PHC, redress inequities, increase service quality, and determine the minimum necessary budget for good PHC. CONCLUSIONS This novel approach toward PHC needs in LMICs, informed by local academics and professionals, created an expansive and prioritized list of critical knowledge gaps in PHC organization and financing. It resulted in research questions, offering valuable guidance to global supporters of primary care evaluation and implementation. Its source and context specificity, informed by LMIC practitioners and academics, should increase the likelihood of local relevance and eventual success in implementing research findings.The authors agreed to bid for funding through their shared professional network–the World Organization of Family Doctors (WONCA)–because the aim of the grant aligns with WONCA’s academic mission. Funding came from Ariadne Laboratories through Brigham and Women’s Hospital, which is the recipient of a Bill & Melinda Gates Foundation grant

Disarming Pseudomonas aeruginosa Virulence by the Inhibitory Action of 1,10-Phenanthroline-5,6-Dione- Based Compounds: Elastase B (LasB) as a Chemotherapeutic Target

negative pathogen Pseudomonas aeruginosa, and this enzyme orchestrates several physiopathological events during bacteria-host interplays. LasB is considered to be a potential target for the development of an innovative chemotherapeutic approach, especially against multidrug-resistant strains. Recently, our group showed that 1,10-phenanthroline-5,6-dione (phendione), [Ag(phendione)2]ClO4 (Ag-phendione) and [Cu(phendione)3](ClO4)2.4H2O (Cu-phendione) had anti-P. aeruginosa action against both planktonic- and biofilm-growing cells. In the present work, we have evaluated the effects of these compounds on the (i) interaction with the lasB active site using in silico approaches, (ii) lasB proteolytic activity by using a specific fluorogenic peptide substrate, (iii) lasB gene expression by real time-polymerase chain reaction, (iv) lasB protein secretion by immunoblotting, (v) ability to block the damages induced by lasB on a monolayer of lung epithelial cells, and (vi) survivability of Galleria mellonella larvae after being challenged with purified lasB and lasB-rich bacterial secretions. Molecular docking analyses revealed that phendione and its Ag+ and Cu2+ complexes were able to interact with the amino acids forming the active site of lasB, particularly Cu-phendione which exhibited the most favorable interaction energy parameters. Additionally, the test compounds were effective inhibitors of lasB activity, blocking the in vitro cleavage of the peptide substrate, aminobenzyl-Ala-Gly-Leu-Ala-p-nitrobenzylamide, with Cu- phendione having the best inhibitory action (Ki = 90 nM). Treating living bacteria with a sub-inhibitory concentration (1/2 × MIC value) of the test compounds caused a significant reduction in the expression of the lasB gene as well as its mature protein production/secretion. Further, Ag-phendione and Cu-phendione offered protective rg 1 August 2019 | Volume 10 | Article 1701 1,10-Phenanthroline-5,6-Dione-Based Compounds: As Anti-Virulence Drugs action for lung epithelial cells, reducing the A549 monolayer damage by approximately 32 and 42%, respectively. Interestingly, Cu-phendione mitigated the toxic effect of both purified lasB molecules and lasB-containing bacterial secretions in the in vivo model, increasing the survival time of G. mellonella larvae. Collectively, these data reinforce the concept of lasB being a veritable therapeutic target and phendione-based compounds (mainly Cu-phendione) being prospective anti-virulence drugs against P. aeruginosa