ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria

The stringent response is a survival mechanism used by bacteria to deal with stress. It is coordinated by the nucleotides guanosine tetraphosphate and pentaphosphate [(p)ppGpp], which interact with target proteins to promote bacterial survival. Although this response has been well characterized in proteobacteria, very little is known about the effectors of this signaling system in Gram-positive species. Here, we report on the identification of seven target proteins for the stringent response nucleotides in the Gram-positive bacterium Staphylococcus aureus. We demonstrate that the GTP synthesis enzymes HprT and Gmk bind with a high affinity, leading to an inhibition of GTP production. In addition, we identified five putative GTPases—RsgA, RbgA, Era, HflX, and ObgE—as (p)ppGpp target proteins. We show that RsgA, RbgA, Era, and HflX are functional GTPases and that their activity is promoted in the presence of ribosomes but strongly inhibited by the stringent response nucleotides. By characterizing the function of RsgA in vivo, we ascertain that this protein is involved in ribosome assembly, with an rsgA deletion strain, or a strain inactivated for GTPase activity, displaying decreased growth, a decrease in the amount of mature 70S ribosomes, and an increased level of tolerance to antimicrobials. We additionally demonstrate that the interaction of ppGpp with cellular GTPases is not unique to the staphylococci, as homologs from Bacillus subtilis and Enterococcus faecalis retain this ability. Taken together, this study reveals ribosome inactivation as a previously unidentified mechanism through which the stringent response functions in Gram-positive bacteria

Evolutionary adaptation of the essential tRNA methyltransferase TrmD to the signaling molecule 3,5-cAMP in bacteria.

The nucleotide signaling molecule 3',5'-cyclic adenosine monophosphate (3',5'-cAMP) plays important physiological roles, ranging from carbon catabolite repression in bacteria to mediating the action of hormones in higher eukaryotes, including human. However, it remains unclear whether 3',5'-cAMP is universally present in the Firmicutes group of bacteria. We hypothesized that searching for proteins that bind 3',5'-cAMP might provide new insight into this question. Accordingly, we performed a genome-wide screen, and identified the essential Staphylococcus aureus tRNA m1G37 methyltransferase enzyme TrmD, which is conserved in all three domains of life, as a tight 3',5'-cAMP binding protein. TrmD enzymes are known to use S-adenosyl-L-methionine (AdoMet) as substrate; we shown that 3',5'-cAMP binds competitively with AdoMet to the S. aureus TrmD protein, indicating an overlapping binding site. However, the physiological relevance of this discovery remained unclear, as we were unable to identify a functional adenylate cyclase in S. aureus and only detected 2',3'-cAMP but not 3',5'-cAMP in cellular extracts. Interestingly, TrmD proteins from Escherichia coli and Mycobacterium tuberculosis, organisms known to synthesize 3',5'-cAMP, did not bind this signaling nucleotide. Comparative bioinformatics, mutagenesis and biochemical analyses revealed that the highly conserved Tyr86 residue in E. coli TrmD is essential to discriminate between 3',5'-cAMP and the native substrate AdoMet. Combined with a phylogenetic analysis, these results suggest that amino acids in the substrate binding pocket of TrmD underwent an adaptive evolution to accommodate the emergence of adenylate cyclases and thus the signaling molecule 3',5'-cAMP. Altogether this further indicates that S. aureus does not produce 3',5'-cAMP, which would otherwise competitively inhibit an essential enzyme

Can Reproductive Health Voucher Programs Improve Quality of Postnatal Care? A Quasi-Experimental Evaluation of Kenya’s Safe Motherhood Voucher Scheme

This study tests the group-level causal relationship between the expansion of Kenya’s Safe Motherhood voucher program and changes in quality of postnatal care (PNC) provided at voucher-contracted facilities. We compare facilities accredited since program inception in 2006 (phase I) and facilities accredited since 2010-2011 (phase II) relative to comparable non-voucher facilities. PNC quality is assessed using observed clinical content processes, as well as client-reported outcome measures. Two-tailed unpaired t-tests are used to identify differences in mean process quality scores and client-reported outcome measures, comparing changes between intervention and comparison groups at the 2010 and 2012 data collection periods. Difference-in-differences analysis is used to estimate the reproductive health (RH) voucher program’s causal effect on quality of care by exploiting group-level differences between voucher-accredited and non-accredited facilities in 2010 and 2012. Participation in the voucher scheme since 2006 significantly improves overall quality of postnatal care by 39% (p=0.02), where quality is defined as the observable processes or components of service provision that occur during a PNC consultation. Program participation since phase I is estimated to improve the quality of observed maternal postnatal care by 86% (p=0.02), with the largest quality improvements in counselling on family planning methods (IRR 5.0; p=0.01) and return to fertility (IRR 2.6; p=0.01). Despite improvements in maternal aspects of PNC, we find a high proportion of mothers who seek PNC are not being checked by any provider after delivery. Additional strategies will be necessary to standardize provision of packaged postnatal interventions to both mother and new-born. This study addresses an important gap in the existing RH literature by using a strong evaluation design to assess RH voucher program effectiveness on quality improvement

Does a voucher program improve reproductive health service delivery and access in Kenya?

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.Background - Current assessments on Output-Based Aid (OBA) programs have paid limited attention to the experiences and perceptions of the healthcare providers and facility managers. This study examines the knowledge, attitudes, and experiences of healthcare providers and facility managers in the Kenya reproductive health output-based approach voucher program. Methods - A total of 69 in-depth interviews with healthcare providers and facility managers in 30 voucher accredited facilities were conducted. The study hypothesized that a voucher program would be associated with improvements in reproductive health service provision. Data were transcribed and analysed by adopting a thematic framework analysis approach. A combination of inductive and deductive analysis was conducted based on previous research and project documents. Results - Facility managers and providers viewed the RH-OBA program as a feasible system for increasing service utilization and improving quality of care. Perceived benefits of the program included stimulation of competition between facilities and capital investment in most facilities. Awareness of family planning (FP) and gender-based violence (GBV) recovery services voucher, however, remained lower than the maternal health voucher service. Relations between the voucher management agency and accredited facilities as well as existing health systems challenges affect program functions. Conclusions - Public and private sector healthcare providers and facility managers perceive value in the voucher program as a healthcare financing model. They recognize that it has the potential to significantly increase demand for reproductive health services, improve quality of care and reduce inequities in the use of reproductive health services. To improve program functioning going forward, there is need to ensure the benefit package and criteria for beneficiary identification are well understood and that the public facilities are permitted greater autonomy to utilize revenue generated from the voucher program.This work was supported by the Bill and Melinda Gates Foundation to the Population Council as part of a multi country study evaluation of voucher-andaccreditation interventions. Grant number 51761

Extracellular ATP released by osteoblasts is a key local inhibitor of bone mineralisation

Previous studies have shown that exogenous ATP (>1µM) prevents bone formation in vitro by blocking mineralisation of the collagenous matrix. This effect is thought to be mediated via both P2 receptor-dependent pathways and a receptor-independent mechanism (hydrolysis of ATP to produce the mineralisation inhibitor pyrophosphate, PPi). Osteoblasts are also known to release ATP constitutively. To determine whether this endogenous ATP might exert significant biological effects, bone-forming primary rat osteoblasts were cultured with 0.5-2.5U/ml apyrase (which sequentially hydrolyses ATP to ADP to AMP + 2Pi). Addition of 0.5U/ml apyrase to osteoblast culture medium degraded extracellular ATP to <1% of control levels within 2 minutes; continuous exposure to apyrase maintained this inhibition for up to 14 days. Apyrase treatment for the first 72 hours of culture caused small decreases (≤25%) in osteoblast number, suggesting a role for endogenous ATP in stimulating cell proliferation. Continuous apyrase treatment for 14 days (≥0.5U/ml) increased mineralisation of bone nodules by up to 3-fold. Increases in bone mineralisation were also seen when osteoblasts were cultured with the ATP release inhibitors, NEM and brefeldin A, as well as with P2X1 and P2X7 receptor antagonists. Apyrase decreased alkaline phosphatase (TNAP) activity by up to 60%, whilst increasing the activity of the PPi-generating ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs) up to 2.7-fold. Both collagen production and adipocyte formation were unaffected. These data suggest that nucleotides released by osteoblasts in bone could act locally, via multiple mechanisms, to limit mineralisation

Cluster randomized evaluation of Adolescent Girls Empowerment Programme (AGEP): study protocol.

BACKGROUND: Adolescents in less developed countries such as Zambia often face multi-faceted challenges for achieving successful transitions through adolescence to early adulthood. The literature has noted the need to introduce interventions during this period, particularly for adolescent girls, with the perspective that such investments have significant economic, social and health returns to society. The Adolescent Girls Empowerment Programme (AGEP) was an intervention designed as a catalyst for change for adolescent girls through themselves, to their family and community. METHODS/DESIGN: AGEP was a multi-sectoral intervention targeting over 10,000 vulnerable adolescent girls ages 10-19 in rural and urban areas, in four of the ten provinces of Zambia. At the core of AGEP were mentor-led, weekly girls' group meetings of 20 to 30 adolescent girls participating over two years. Three curricula - sexual and reproductive health and lifeskills, financial literacy, and nutrition - guided the meetings. An engaging and participatory pedagogical approach was used. Two additional program components, a health voucher and a bank account, were offered to some girls to provide direct mechanisms to improve access to health and financial services. Embedded within AGEP was a rigorous multi-arm randomised cluster trial with randomization to different combinations of programme arms. The study was powered to assess the impact across a set of key longer-term outcomes, including early marriage and first birth, contraceptive use, educational attainment and acquisition of HIV and HSV-2. Baseline behavioural surveys and biological specimen collection were initiated in 2013. Impact was evaluated immediately after the program ended in 2015 and will be evaluated again after two additional years of follow-up in 2017. The primary analysis is intent-to-treat. Qualitative data are being collected in 2013, 2015 and 2017 to inform the programme implementation and the quantitative findings. An economic evaluation will evaluate the incremental cost-effectiveness of each component of the intervention. DISCUSSION: The AGEP program and embedded evaluation will provide detailed information regarding interventions for adolescent girls in developing country settings. It will provide a rich information and data source on adolescent girls and its related findings will inform policy-makers, health professionals, donors and other stakeholders. TRIAL REGISTRATION: ISRCTN29322231 . March 04 2016; retrospectively registered

Heat transfer in separated flows on the pressure side of turbine blades

Heat transfer in separated flows on the pressure side of a typical high lift turbine profile is numerically investigated by means of an in-house CFD code. The numerical code was first validated on attached flows in turbine blades. To obtain flow separation cases, the profile is subject to large negative incidences so that a separation bubble is obtained at the pressure side. The numerical results are compared to available experimental data for code validation. It is shown how local minima and maxima values of the heat transfer coefficient are related to the separation and reattachment points, where the velocity component perpendicular to the wall is shown to have a significant effect on the heat transfe

Radical, Reformist, and Garden-Variety Neoliberal: Coming to Terms with Urban Agriculture’s Contradictions

For many activists and scholars, urban agriculture in the Global North has become synonymous with sustainable food systems, standing in opposition to the dominant industrial agri-food system. At the same time, critical social scientists increasingly argue that urban agriculture programmes, by filling the void left by the rolling back of the social safety net, underwrite neoliberalisation. I argue that such contradictions are central to urban agriculture. Drawing on existing literature and fieldwork in Oakland, CA, I explain how urban agriculture arises from a protective counter-movement, while at the same time entrenching the neoliberal organisation of contemporary urban political economies through its entanglement with multiple processes of neoliberalisation. By focusing on one function or the other, however, rather than understanding such contradictions as internal and inherent, we risk undermining urban agriculture\u27s transformative potential. Coming to terms with its internal contradictions can help activists, policy-makers and practitioners better position urban agriculture within coordinated efforts for structural change, one of many means to an end rather than an end unto itself

Influence of crop residue ration supplementation on the attainment of puberty and postpartum reproductive activities of Red Sokoto goats

The general objective of this study was to come up with an appropriate, affordable and locally available crop residue supplementation package that would enhance reproductive performance in small ruminants. Specifically, 28 Red Sokoto weaner does between 3 and 4 months of age weighing between 2 and 3 kg were used in the first experiment to determine the influence of crop residue supplementation on age and weight at puberty as determined by blood progesterone levels. In the second experiment, another 28 adult does (equal to or greater than 2 years old) of the same breed in the same flock with lactation numbers between 1 and 3 were used to determine the length of postpartum acyclic period. In both experiments, a 3 x 2 factorial experimental design comprising three dietary supplements (A, B, C) at two feeding levels (1% and 2% of body weight) fed in addition to a basal diet of Digitaria smutsii hay and natural pasture ad libitum with an unsupplemented negative control group (D) and four goats per treatment was utilized. In ration A, a conventional concentrate supplement consisting of maize, wheat offal, cottonseed cake and bonemeal was utilized; in rations B and C, the supplement consisted of guinea-corn bran, cowpea husk and groundnut haulms; and maize offal, groundnut shells and groundnut haulms respectively. Unsupplemented (ration D) weaner does reached puberty at a later age and had lighter body weights than all the others. Weaner does on ration 2A (concentrate fed at 2% of body weight) attained puberty at the earliest age and heaviest body weight, although the age at puberty was not significantly different from those on rations 1A (concentrate fed at 1% body weight), 1C and 2C. Blood progesterone profiles before and after puberty ranged from 0.05 to 9.0 ng/ml, respectively, and was highest in does fed rations A and C and least in the unsupplemented does. The mean interval between kidding and initiation of ovarian activity was 54.28 plus or minus 17.61 days and the mean interval between kidding and conception was 63.04 plus or minus 25.34 days. Only 25% of the unsupplemented does conceived again during the period under study compared with 100% in rations 1A, 2A, 1C and 2C; 75% in ration 2B and 50% in ration 1B. It was concluded that implementation of supplementary feeding in the dry season improves reproductive performance in the Red Sokoto doe. Furthermore, ration C, a crop residue-based ration, was a suitable dry season supplementation alternative to the expensive conventional concentrate ration for the smallholder goat farmer in the subhumid tropics of Nigeria

Exploring Public Health's roles and limitations in advancing food security in British Columbia

OBJECTIVES: This research analyzes the roles and limitations of Public Health in British Columbia in advancing food security through the integration of food security initiatives into its policies and programs. It asks the question, can Public Health advance food security? If so, how, and what are its limitations? METHODS: This policy analysis merges findings from 38 key informant interviews conducted with government and civil society stakeholders involved in the development of food security initiatives, along with an examination of relevant documents. The Population Health Template is used to delineate and analyze Public Health roles in food security. RESULTS: Public Health was able to advance food security in some ways, such as the adoption of food security as a core public health program. Public Health's leadership role in food security is constrained by a restricted mandate, limited ability to collaborate across a wide range of sectors and levels, as well as internal conflict within Public Health between Food Security and Food Protection programs. CONCLUSIONS: Public Health has a role in advancing food security, but it also faces limitations. As the limitations are primarily systemic and institutional, recommendations to overcome them are not simple but, rather, require movement toward embracing the determinants of health and regulatory pluralism. The results also suggest that the historic role of Public Health in food security remains salient today