A cluster randomised controlled trial of the community effectiveness of two interventions in rural Malawi to improve health care and to reduce maternal, newborn and infant mortality

<p>Abstract</p> <p>Background</p> <p>The UN Millennium Development Goals call for substantial reductions in maternal and child mortality, to be achieved through reductions in morbidity and mortality during pregnancy, delivery, postpartum and early childhood. The MaiMwana Project aims to test community-based interventions that tackle maternal and child health problems through increasing awareness and local action.</p> <p>Methods/Design</p> <p>This study uses a two-by-two factorial cluster-randomised controlled trial design to test the impact of two interventions. The impact of a community mobilisation intervention run through women's groups, on home care, health care-seeking behaviours and maternal and infant mortality, will be tested. The impact of a volunteer-led infant feeding and care support intervention, on rates of exclusive breastfeeding, uptake of HIV-prevention services and infant mortality, will also be tested. The women's group intervention will employ local female facilitators to guide women's groups through a four-phase cycle of problem identification and prioritisation, strategy identification, implementation and evaluation. Meetings will be held monthly at village level. The infant feeding intervention will select local volunteers to provide advice and support for breastfeeding, birth preparedness, newborn care and immunisation. They will visit pregnant and new mothers in their homes five times during and after pregnancy.</p> <p>The unit of intervention allocation will be clusters of rural villages of 2500-4000 population. 48 clusters have been defined and randomly allocated to either women's groups only, infant feeding support only, both interventions, or no intervention. Study villages are surrounded by 'buffer areas' of non-study villages to reduce contamination between intervention and control areas. Outcome indicators will be measured through a demographic surveillance system. Primary outcomes will be maternal, infant, neonatal and perinatal mortality for the women's group intervention, and exclusive breastfeeding rates and infant mortality for the infant feeding intervention.</p> <p>Structured interviews will be conducted with mothers one-month and six-months after birth to collect detailed quantitative data on care practices and health-care-seeking. Further qualitative, quantitative and economic data will be collected for process and economic evaluations.</p> <p>Trial registration</p> <p>ISRCTN06477126</p

Effects of fou8/fry1 mutation on sulfur metabolism: Is decreased internal sulfate the trigger of sulfate starvation response?

The fou8 loss of function allele of adenosine bisphosphate phosphatase FIERY1 results in numerous phenotypes including the increased enzymatic oxygenation of fatty acids and increased jasmonate synthesis. Here we show that the mutation causes also profound alterations of sulfur metabolism. The fou8 mutants possess lower levels of sulfated secondary compounds, glucosinolates, and accumulate the desulfo-precursors similar to previously described mutants in adenosine 5′phosphosulfate kinase. Transcript levels of genes involved in sulfate assimilation differ in fou8 compared to wild type Col-0 plants and are similar to plants subjected to sulfate deficiency. Indeed, independent microarray analyses of various alleles of mutants in FIERY1 showed similar patterns of gene expression as in sulfate deficient plants. This was not caused by alterations in signalling, as the fou8 mutants contained significantly lower levels of sulfate and glutathione and, consequently, of total elemental sulfur. Analysis of mutants with altered levels of sulfate and glutathione confirmed the correlation of sulfate deficiency-like gene expression pattern with low internal sulfate but not low glutathione. The changes in sulfur metabolism in fou8 correlated with massive increases in 3′-phosphoadenosine 5′-phosphate levels. The analysis of fou8 thus revealed that sulfate starvation response is triggered by a decrease in internal sulfate as opposed to external sulfate availability and that the presence of desulfo-glucosinolates does not induce the glucosinolate synthesis network. However, as well as resolving these important questions on the regulation of sulfate assimilation in plants, fou8 has also opened an array of new questions on the links between jasmonate synthesis and sulfur metabolism

Comparison of mRNA levels of key genes of sulfur metabolism.

<p>Col-0, <i>fou8</i>, <i>cad2</i>, <i>rax1</i>, and <i>sultr1;2</i> plants were grown for 2.5 weeks on MS-agarose plates. Relative mRNA levels of <i>ATPS4</i> and <i>APR1</i> in <b>A</b> leaves and <b>B</b> roots and of <i>LS2</i> (At5g48850) and <i>LS5</i> (At5g26220) in <b>C</b> leaves and <b>D</b> roots and genes of glucosinolate synthesis in <b>E</b> leaves were determined. The qRT-PCR reactions were performed in triplicate, the values in Col-0 were set to 1 for all genes. Results are presented as means ± SE from six biological replicates from plants grown in two independent experiments. Different letters mark values significantly different at P<0.05 or in <b>E</b> asterisks show values significantly (P<0.05) different from Col-0.</p

Accumulation of PAP and PAPS in <i>fou8</i> related genotypes.

<p>The various Arabidopsis lines were grown for 5 weeks in controlled environment room. Leaves were harvested and the levels of <b>A</b> PAP and <b>B</b> PAPS were determined by HPLC. Results are presented as means ± SD from four individual plants, grown in two independent experiments. Asterisks represents levels under detection limit, different letters mark values significantly different at P<0.05.</p

Sulfate uptake and flux through sulfate assimilation in <i>fou8</i> and related mutants.

<p>WT Col-0 and mutants <i>fou8</i>, <i>fou2</i>, and <i>aos</i> were grown for 3 weeks on MS-phytagel vertical plates in controlled environment room. The seedlings were incubated for four hours with their roots submerged in nutrient solution adjusted to sulfate concentration of 0.2 mM and supplemented with 6.7 μCi [³⁵S]sulfate. Shoot and root material was harvested separately, and the flux was determined as incorporation of ³⁵S from [³⁵S] sulfate to thiols and proteins. <b>A</b> sulfate uptake, <b>B</b> Percentage of ³⁵S transported to leaves from the [³⁵S]sulfate taken up, <b>C</b> relative flux through the sulfate assimilation in the leaves calculated as % of incorporation in thiols and proteins from total [³⁵S]sulfate taken up. Results are presented as means ± SE from six independent pools of 8 seedlings grown in two independent experiments. Values marked with an asterisk show significant (P≤0.05) difference from Col-0.</p

Expression analysis of Arabidopsis lines.

<p>The various Arabidopsis lines were grown for 5 weeks in controlled environment room. Total RNA was isolated from leaves and the transcript levels of genes involved in sulfur metabolism, glucosinolate synthesis, and jasmonate synthesis were determined by quantitative RT-PCR. The qRT-PCR reactions were performed in triplicate for each of the six independent biological samples from plants grown in two independent experiments. Results are presented as a heat map of relative mRNA levels compared to Col-0. For comparison, sulfate levels are presented in the same way on the far right.</p

<i>fou8</i> is affected in glucosinolate synthesis.

<p>Col-0, <i>fou8</i>, <i>apk1 apk2</i>, and <i>fou8 apk1 apk2</i> plants were grown for 5 weeks in controlled environment room. The total content of <b>A</b> glucosinolates and <b>B</b> desulfo-glucosinolates was measured in leaves. <b>C</b> Total RNA was isolated from leaves and the transcript levels of six genes involved in glucosinolate synthesis was determined by quantitative RT-PCR. The qRT-PCR reactions were performed in triplicate for each biological sample. The values in Col-0 were set to 1 for all genes. Results are presented as means ± SE from six pools of three individual plants grown in two independent experiments. Different letters mark values significantly different at P<0.05; asterisks mark values significantly different from Col-0 at P<0.05.</p