Polygonum minus Huds.is a culinary flavouring that is common in South East Asian cuisine and as a remedy for diverse maladies ranging from indigestion to poor eyesight. The leaves of this herb have been reported to be high in antioxidants. Flavonoids which have been associated with memory, cognition and protection against neurodegeneration were found in P. minus.P. minus aqueous extract (Lineminus™) for its antioxidant activity using the Oxygen Radical Absorbance Capacity (ORAC) assay, the ex vivo Cellular Antioxidant Protection of erythrocytes (CAP-e) assays and for potential anticholinesterase activity in vitro. Cognitive function and learning of Lineminus™ was evaluated using scopolamine induced cognition deficits in a Barnes maze, rodent model of cognition.This study examined a in vitro antioxidant activity with a total ORAC value of 16,964 μmole TE/gram. Cellular antioxidant protection from free radical damage using the CAP-e assay, with an IC50 of 0.58 g/L for inhibition of cellular oxidative damage, was observed. The extract inhibited cholinesterase activity with an IC50 of 0.04 mg/ml with a maximum inhibition of 68%. In a rodent model of cognition using scopolamine induced cognition deficits in the Barnes maze, the extract attenuated scopolamine induced disruptions in learning at the higher dose of 100 mg/kg.The extract displayed P. minus possesses antioxidant and anticholinesterase activity and demonstrated enhanced cognition in vivo. The data suggest neuroprotective properties of the extract.These data shows that  Polygonum minus Huds. synonymous to Persicaria minor is from the family Polygonaceae and is commonly referred to as Kesum or laksa leaf in Malaysia. It is used as a flavouring ingredient in culinary dishes and also consumed as an ulam  for preventive healthcare [P. minus has the ability to increase blood circulation [althcare ,2. Tradialthcare . It is pculation .P. minus are high in antioxidants [P. minus have shown superior antioxidant activity when compared to other popular herbs such as ulam raja, selom, pegaga and curry leaves, where antioxidant activity was similar to the synthetic antioxidant butylhydroxytoluene (BHT) [P. minus had higher antioxidant activity, measured by the total phenolic content (TPC), when comparing to ginger  and turmeric (Curcuma longa) [Polygonum minus was shown to possess 98.3% of lipid peroxidation inhibitory activities and is proposed as a candidate for nutraceutical and cosmeceutical product [Several studies have shown that leaves of oxidants -9. It hane (BHT) . Water ea longa) . Polygon product . It has  product .Free radicals, including reactive oxygen species, have been shown to cause aging and several degenerative diseases such as atherogenesis, cardiovascular and neurodegenerative diseases ,12. AlthP. minus have shown the presence of flavanoids, flavones, cathecin, epicatechingallate and terpenoids [Phytochemical screening of rpenoids . Flavonorpenoids ,14. Studrpenoids .P. minus as a food with other more established high antioxidant foods. The ORAC assay has since been commercialized by Brunswick Labs, Wareham, MA, USA. The DPPH and FRAP method on the other hand is a SET method, which measures the ability of the antioxidant to transfer one electron to reduce a specific oxidant. In the case of DPPH assay, it measures direct reactions with the DPPH radical, which is dependent on the structure of an antioxidant compound therefore only giving a general indication of the radical scavenging abilities of antioxidants.While DPPH method is a rapid and convenient method to measure antioxidant activity it diverges from biological conditions the most, by the use of an artificial DPPH radical and methanol as the solvent [The nutraceutical industry uses a standardized chemical antioxidant method, ie. the Oxygen Radical Absorbance Capacity (ORAC) test to evaluate antioxidant strength. The ORAC is a HAT-based assays measuring the capability of an antioxidant to quench free radicals  by H-atom donation . In addi solvent . This is solvent .in vivo as one of the manifestation of the antioxidant property.However, there is a need to demostrate the antioxidant capacity in serum to show bioavailibity. Since a clinical trial is costly and time consuming, a more intermediary cell-based study was developed to test to what extent a substance protects against oxidative stress in a biologically relevant sytem. The Cellular Antioxidant Protection of erythrocytes (CAP-e) assay is a cell-based antioxidant protection assay using erythrocytes to address the question of whether antioxidants in complex natural products enter the cytosol and contribute to the reduction of oxidative damage within the cell  and was P. minus. Screening for herbal plants with acetylcholinesterase inhibitory activity would open new possibilities for cognition improving herbal products.As improvements in cogniton may be multi-pronged, the herb is also tested for antiacetylcholinesterase activity. Acetylcholine is a neurotransmitter related to learning and memory . It is min vivo models have been used to investigate learning and memory in animal models of which Scopolamine, a muscarinic receptor antagonist, produce deficits in spatial navigation tasks in rodents [Several  rodents . Scopola rodents .The Barnes maze was developed as a sensitive tool for testing hippocampus-dependent spatial memory in rats  and is tP. minus in protecting against oxidative stress in a cell-based study and in memory improvement in vivo. Currently the plant extract most popularly researched for the ability to enhance cognition is Gingko biloba. Extracts of G. biloba were shown to improve memory and normalizing cognitive deficits in animal models [P. minus was tested in vitro and the protection against oxidative damage demonstrated in red blood cells. The paper attempts to draw a parallel between the protective antioxidant affect of the herbal extract to the cognition enhancing effect, in an animal model induced with cognitive deficits by scopolamine, whose activity can also be attenuated by an anti acetylcholinesterase. The activity of the herb was compared to the more traditionally and scientifically documented, G. biloba which has been reported to possess anti-acetylcholinesterase activity and improve cognition in vivo.This study was performed to investigate l models , and in l models . In thisPolygonum minus was procured from Biotropics Malaysia Berhad, Malaysia. The plant material was identified based by a Taxonomist from Institute Bio Science, University Putra Malaysia (UPM) based on their exomorphic characters and literature review of the plant. The voucher specimen of the plant (SK 2077/12) was deposited in the Herbarium Institute Bioscience, UPM Malaysia. The aerial parts of plants including stem and leaves were extracted to produce an aqueous extract for in vitro and in vivo assays.P. minus, standardised to Quercetin-3-glucuronide 0.59% and 0.27% Quercitrin was prepared by Biotropics Malaysia Berhad according to process outlined in Malaysian Patent Pending No. PI2012003882 [P. minus water extract was obtained according to the HPLC method using Kinetex 1.7 μm C18 (2.1 × 150 mm) column. The mobile phase consisted of solvent A-0.10% formic acid in water and B-0.10% formic acid in acetonitrile mixed according to a linear gradient program of between 5-89% of solvent A and 95-11% of solvent B. Two major peaks in the fingerprint profile were isolated and identified to be quercetin-3-glucuronide and quercitrin based on their mass(es) and MS fragmentations. LC-MS-MS was performed using a Shidmadzu UFLC system equipped with a PDA and IT-TOFMS. Peaks at retention times 7.15 and 13.96 min identified as Quercetin glucuronide and Quercitrin respectively were further confirmed by comparing their retention time values and the obtained UV max with those of the standards. .The comparative plant extract of P. minus was shipped to Brunswick Laboratories, Norton, MA, an independent contract laboratory specialising in standardised natural product assays, to test for ORAC values. Data were obtained for ORAC hydrophilic testing using fluorescein as the fluorescent probe and 2,2′-azobis (2-amidinopropane) dihydrochloride as a peroxyl radical generator, ORAC lipophilic testing for lipid antioxidants capable of quenching peroxyl free radicals, HORAC testing for antioxidants capable of quenching hydroxyl free radicals, NORAC testing for antioxidants capable of quenching peroxynitrite, and SORAC testing for superoxide dismutase-like activity  [Extract of the lab) .The CAP-e antioxidant capacity was estimated according to the modified method of Honzel , modifieThe cells were exposed to oxidative damage by addition of the peroxyl free-radical generator 2,2’ azobis (2-amidinopropane hydrochloride) (AAPH). Using the indicator dye Dichlorofluorescein diacetate (DCF-DA), which becomes fluorescent as a result of oxidative damage, the degree of antioxidant damage was recorded by measuring the fluorescence intensity of each sample in a TECAN Spectrafluor plate reader. The inhibition of oxidative damage was calculated as the reduced fluorescence intensity of product-treated cells, compared to cells treated only with the oxidizing agent, in reference to the baseline levels of oxidation in untreated cells. The CAP-e value which is in Gallic Acid Equivalent (GAE) units, reflects the IC50 dose of the test products, i.e. the dose that provided 50% inhibition of oxidative damage. This is then compared to the IC50 dose of the known antioxidant Gallic acid.The extracts was screened for anticholinesterase activity using ProfilingScreen procured from Ricerca Pharmacology Services, Taiwan. The extract was tested in duplicates at a concentration of 10, 30 and 100 μg/ml. Concurrent vehicle 1% DMSO and reference standard Physostigmine were conducted with each assay using Human Recombinant HEK-293 cells.Two to six month old adult male C57BL/6 mice (20–25 g), (n = 12–14) were supplied by BioLASCO (Taiwan). The mice were group-housed under a 12/12-h light/dark (200–300 Lux) cycle (lights on 07:00 h) with free access to food  and water and humidity kept between 50%–70%. The experiment was approved by the Institutional Animal Care and Use Committee of Cerca Insights Sdn Bhd and was conducted in accordance with the Singapore NACLAR Guide (2004) for the care and use of laboratory animals.P. minus, 100 mg/kg P.minus, 50 mg/kg G.biloba or vehicle (water) were given daily via oral gavage for fourteen days prior to Barnes maze testing. This treatment continued during the five days of Barnes maze testing. During the Barnes maze testing these mice received i.p. injections of either scopolamine (0.5 mg/kg) or saline vehicle. A further group received i.p. injections of donepezil (1 mg/kg) and scopolamine (0.5 mg/kg). The dose and time of scopolamine administration has been previously shown to produce deficits in spatial navigation tasks in rodents [Herbal extract treatments of 50 mg/kg  rodents .The Barnes maze (BM) was created to evaluate spatial learning ,26. The Barnes maze testing consisted of three phases, an adaptation period, an acquisition period and a probe trial. A pre-trial (adaptation period) was given prior the start of trial on days one and two. Each subject underwent four trials per day for four days (acquisition period) and then a probe trial was performed twenty-four hours after the final acquisition trial. Thirty minutes prior to the first trial the test subjects received an injection of either scopolamine, saline vehicle, or scopolamine and donepezil.The subject was placed in the center zone of the maze, shrouded in a chamber for 10 seconds. The chamber was removed and the subject was allowed to explore the maze for 30 seconds, then gently guided to the escape hole. If the subject did not then enter the escape hole, it was placed inside. The hole was then covered and the subject remained there for 3 minutes. The subject was then returned to its home cage and the platform cleaned with 70% ethanol.The chamber was removed and the subject was allowed to explore the maze for 5 minutes and then gently guided to the escape hole. If the subject did not then enter the escape hole, it was placed inside. The hole was then covered and the subject remained there for 1 minute. The subject was then returned to its home cage and the platform cleaned with 70% ethanol. The next trial was run after following an inter-trial interval of two minutes.For the probe trial, an identical white disc was placed on the platform that covered all the holes. The subject was placed in the center zone of the maze, shrouded in a chamber for 10 sec. The chamber was removed and the subject was allowed to explore the area for 90 seconds. The subject was then returned to its home cage and the platform cleaned with 70% ethanol.The behavior of the experimental subjects was captured by video camera and recorded on the hard drive of a desktop PC. An analysis of these recordings was performed using EthoVision® XT tracking system for the automatic tracking and analysis of animal movement. For each parameter, the performance in the trials on each day were averaged. The following parameters were measured during the test and processed - (i) Total path length, the distance moved by the subject during the entire session. Total path length is the total distance moved over the whole time of the experimental trial including the distance moved after the first encounter with the escape-hole (when the mouse has encountered the escape hole but failed to enter it). An increase in path length demonstrates a decrease in performance; (ii) Total errors, the count of the number of errors made by the subject throughout the trial. Total errors are the number of approaches to the non-escape hole, when the mouse has interacted with the escape hole but not escaped and further explored the maze. Both total path length and total errors measure learning; (iii) Total latency, the latency for subject to complete the task. Total latency describes the time taken by the mouse to enter the escape hole; (iv) For the probe trial the arena was divided into 8 right equal segments and duration in each segment by the subject was measured. The mice are allowed to explore the maze and the time the mice stay in various areas on the maze is recorded. This ‘probe’ trial is used to assess memory.Data was analyzed using two-way repeated measures ANOVA, with ‘day’ as repeated-measure factor within subjects and ‘treatment group’ as between-subject’s factor. Post-hoc pairwise comparisons between groups using Tukey HSD test were carried out if significant effect was found. Data analysis was performed using Sigma Plot statistical software.FN provides a measure of the total antioxidant power of a food/nutrition product against the five predominant reactive species. The ORACFN values for the extract are shown in Table There are five predominant reactive species found in the body: peroxyl radicals, hydroxyl radicals, peroxynitrite, super oxide anion and singlet oxygen. Total ORACThe extract inhibited cholinesterase activity with an IC50 of 0.0405 mg/ml with maximum inhibition of 68%.P. minus extract was shown to be 55 gallic acid equivalents per gram extract, based on an IC50 value of 0.58 g/L  and day  as shown in Table G. biloba, donepezil or 100 mg/kg P. minus. The scopolamine induced deficit was not reversed by treatment with 50 mg/kg P. minus. These mice showed a significant decrease in performance when compared to vehicle treated mice on day two and three. However, the donepezil, G. biloba and the 100 mg/kg P. minus treated mice did not show a scopolamine induced deficit. There was a significant effect of day  and treatment  on total errors. A significant increase in total errors was seen in the scopolamine treated mice on days one and two (p < 0.05). A significant increase in total errors was also seen in P. minus (50 mg/kg) treated mice on days two and three (p < 0.05). The deficits for total errors were not observed after treatment with P. minus at 100 mg/kg, G. biloba or donepezil for days one, two and three  and treatment  on total latency. A significant increase (p < 0.05) in total latency was see in the scopolamine and P. minus (50 mg/kg) treated mice on days one, two and three ; the scopolamine with donepezil treated ; the G. biloba treated  and the P. minus -100 mg/kg  mice, but not the scopolamine alone nor the scopolamine plus P. minus- 50 mg/kg treated mice  iron to ferrous (II) iron in FRAP reagent, probably as a result of high flavonoid and total phenolic content [P. minus was demonstrated and by evaluating ORAC values, the standardised P. minus extract-Lineminus™ could be compared with other antioxidant foods.The extract was shown to possess strong antioxidant capacity, as measured by the oxygen radical absorbance (ORAC assay). The ORAC value of ectively . In addiectively . Previou content ,9. Usingex-vivo by the use of erythrocyte cells (in the CAP-e bioassay study), where oxidative stress was reduced in a dose dependent manner by the presence of the extract. Natural products including flavonoids that exhibit anti-oxidative effects have been found to attenuate memory impairments [in vitro[in vivo studies showed that flavonoids are able to be absorbed after oral administration, pass the blood–brain barrier and do have various effects on the CNS [P. minus[in vivo, this could help reduce increased oxidative stress such as that reported to occur in the aging brain and so may be therapeutically useful. Hence the possibility of flavonoids of P. minus to cross blood brain barrier thus qualifying the procognitive effect in the animal study conducted here, should be further investigated.In this study, we managed to evaluate antioxidant effect airments . Some isairments . Some fl[in vitro. The in  the CNS . Derivat[P. minus, possiblG. biloba extract.They have both been reported to reverse scopolamine induced deficits in learning [P. minus extract, when administered at 100 mg/kg, attenuated scopolamine induced deficits in the acquisition phase of the Barnes maze task.In the present study, scopolamine treatment induced deficits during the early part of the acquisition (learning) phase of the Barnes maze task. The scopolamine induced deficits in learning were evidenced by an increase in the number of errors, latency, and path length. Considering the learning curve, the impairment of scopolamine treated animals seemed most pronounced early in training, after which some constancy was reached between the treatment groups. This is best illustrated by the increased path length in the scopolamine treated mice on days one, two and three, but not day four. These deficits were attenuated by the positive controls – donepezil and the learning . The P. P. minus extract also reversed scopolamine induced deficits in the retention  aspect of the task. In the probe trial, all the treatments except the lower dose of 50 mg/kg of P. minus attenuated scopolamine induced deficits. These deficits were described by no significant preference for the target segment by the mice. The lack of effect of the lower dose and the significant effect of the higher dose would suggest that there was a dose dependent action of the extract. The results suggest that an extract of P. minus can attenuate scopolamine induced learning and memory deficits in mice.The dose of 100 mg/kg P. minus and G. biloba group where the animals spent a longer duration in the target segment during probe trial as an indicator that the target segment location is remembered. The mice in lower doses of P. minus, the donepezil and scopolamine group demonstrated a shorter period within the target segment suggesting poorer memory for the target segment location that it was exposed to initially during the probe trial. The in vitro data from the present study, demonstrates that the extract has measurable anticholinesterase activity (68%), hence it may be that the extract induced increases in cholinergic tone, additionally providing an explanation for the attenuation of scopolamine induced deficits.Decreases in cholinergic tone are associated with cognitive dysfunction and are reported in neurodegenerative diseases such as Alzheimer’s ,39. IncrP. minus has been reported to possess up to 98.3% of lipid peroxidation inhibitory activities [P. minus[Scopolamine memory impairments have also been associated with brain oxidative stress  and scoptivities . The ant[P. minus. It has P. minus extract used in this study produced neither mortality nor changes in behavior or any other physiological activities [In considering human dosage, safety has been demonstrated in the acute toxicity test where oral administration of 2000 mg/kg of the standardised tivities . The samP. minus water extract as one of its ingredient, natural ingredients in brain health of individuals aged 35–65 years of age was evaluated [P. minus which was one of its major ingredient.In a recent randomized, double-blind, placebo-controlled crossover study of a propriety herbal blend (SuperUlam) containing 150 mg of valuated . There wP. minus has antioxidant activity with a high ORAC value of 16,964 μmole TE/gram and was able to reduce oxidative stress in a dose dependent manner. Higher dose of P minus (100 mg/kg) was able to attenuate scopolamine induced deficit in cognition in vivo by a reduction of total path length travelled and total errors prior to finding escape hole and increased duration in target segment during probe trial, indicating improved memory. These properties suggest that further investigations into the therapeutic potential of this extract for cognition could be a fruitful endeavour.The present study confirms that water extract of The authors declare that Annie George and Wong Hoi Jean are employees of Biotropics Malaysia Bhd. Biotropics Malaysia Bhd funded this study and the article processing fee. Chee Perng Ng, Matthew O’Callaghan and Gitte S. Jensen declare they have no conflict of interest. The findings of the study have been applied for patent by Biotropics Malaysia Bhd.in vivo design and worked on the draft of manuscript. CPN and MOC designed, conducted and interpreted the research outcome of the in vivo study, performed the statistical analysis and worked on the draft of manuscript. GSJ performed the cell based antioxidant assay and made contribution to the revision of the draft manuscript. HJW worked on the standardisation of the herbal extract used in this study. All authors read and approved the final manuscript.AG was responsible for the conception of the study and participated in the The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/14/161/prepub


The Urban Health Initiative (UHI) was initiated in 2009 with the goal of increasing family planning (FP) use among the poor in urban areas of Uttar Pradesh, India. The Measurement, Learning & Evaluation project (MLE) was tasked with rigorous impact evaluation of the UHI. This paper presents the impact evaluation findings of the UHI program.The MLE design includes a longitudinal sample of women and health facilities with baseline (2010) and endline (2014) data collection in six cities in Uttar Pradesh, India. At baseline, samples representative of women in each city were selected with oversampling of the poor. Eighty-four percent of women interviewed at baseline were reinterviewed 4 years later at endline. The longitudinal data support a within/fixed-effects approach to identification of program impact on changes in modern FP use.Impact evaluation results show significant effects of exposure to both demand and supply side program activities. In particular, women exposed to brochures , billboards/posters/wall hangings , and FP on the television  were significantly more likely to be using a modern method at endline. In addition, we found borderline significance for being exposed to a community health worker  and living close to an improved public and private supply environment where UHI undertook activities .UHI program activities were designed to complement the Government of India's strategies aimed at ensuring access to and provision of FP to urban poor populations. The effective demand- and supply-side strategies of the UHI program are therefore likely to be sustainable and scalable to other urban areas in India.Findings from this study are important for designing sustainable and scalable FP strategies for urban India where increases in FP use will be relevant for meeting international FP targets. The Summit sparked a global FP agenda to achieve 120 million new users of modern contraception by 2020 (termed “120 by 20”) Nearly twelve and a half million of the new Indian users are expected to come from the state of Uttar Pradesh (UP) This paper presents recent evidence from the rigorous impact evaluation of the UHI. It is divided into the following sections: 2The UHI, funded by the Bill & Melinda Gates Foundation (BMGF) and launched in 2009 by a consortium of partners led by FHI360, implemented FP programs in urban areas of UP, India. UHI was designed to complement and support national and state urban health strategies. UHI's goals were to increase mCPR, reduce maternal and infant mortality and use evidence-based strategies aligned with GoI programs to ensure sustainability. UHI objectives were to (a) improve the quality of FP services in high-volume facilities, (b) develop cost-effective interventions to integrate FP with postpartum and postabortion services, (c) test innovative private-sector approaches to increase access to and use of FP by the urban poor, (d) develop interventions to sustain demand for quality contraceptive services and supplies and (e) advocate for funding and a supportive policy environment for FP supplies and services. The main activities of UHI included provision of postpartum and postabortion FP, training providers to improve technical competence and client–provider interactions, expanding the role of the private sector in FP service provision, using community health workers (CHWs) for outreach efforts, and using mid- and mass-media to promote demand for FP services. A key UHI strategy was to task CHWs with visiting every home in slums of target cities to offer information about FP methods; counsel on postpartum FP; accompany women to a health facility; refer women to a health facility or fixed service days; and, if requested, provide short-term methods (pills and condoms).3endline) The Measurement, Learning & Evaluation (MLE) project, also funded by BMGF, is led by the Carolina Population Center at the University of North Carolina at Chapel Hill. The MLE project was tasked with developing a rigorous evaluation of the UHI; longitudinal data were collected from women and facilities in six cities in UP. Data presented here come from MLE's baseline (2010) and follow-up survey 4 years later in 2014 . As with UHI radio and television, these variables are coded 0 at baseline. The mid-media and print media variables are not specific to UHI in this analysis because the program intentionally integrated activities into those of the government. Because these were not asked at baseline, it is not possible to examine change in these variables between baseline and endline, and thus, we may be overestimating the effect of these variables on modern method use over time.PSU/cluster. This variable is created as a non-self-weighted cluster average of the number of positive responses to exposure to a CHW in the last 3 months divided by the total number of women in the PSU at baseline or endline.Another key UHI activity involved outreach in slums by CHWs. Notably, other programs, including government initiatives, use CHW, and it was not possible to distinguish UHI CHW and non-UHI CHW. At baseline and endline, women were asked if they were exposed to a CHW in the last 3 months. We use a community-level CHW exposure variable to capture community presence of CHWs at baseline and endline in the community, here defined as the Other general  FP-related variables include whether or not the woman participated in the last 3 months in a community group that discussed FP (measured at endline). Because this was not measured at baseline, all women are coded 0 at baseline; as for the variables above, this variable may overestimate the effects on modern method use. At baseline and endline, women were also asked about exposure to FP on the radio or television in the last 3 months; this is not specific to UHI programming. There are separate general radio and television exposure variables for each time point.UHI supply variables in the model capture whether women live within a specific distance of a facility where UHI operated. Four variables are included: exposure to a public facility within 1 kilometer (km) with UHI full implementation, exposure to a public facility within 1 km with UHI partial implementation, exposure to a private facility within 1¾ km with UHI full implementation and exposure to a private facility within 1¾ km with UHI partial implementation. The distance band for private facilities accounts for greater willingness to travel for private services.Multivariable analyses exploit the longitudinal data with fixed-effects regression models with the pooled samples (baseline and endline) to control for both the possible endogeneity of recall to the exposure variables and program targeting of underserved areas. Fixed-effects methods remove time-invariant unobservables for either the respondent or the community that could bias estimates of program impact. Fixed-effect analyses control for the clustered sampling design. The final model specification includes all of the relevant UHI exposure variables measured in the endline survey. Following initial model runs, a small number of variables were removed or regrouped to address high correlation across program activities; the removed variables were not significant in any of the models. All models control for standard control variables related to socioeconomic status that have been used in prior analyses that examine modern method use in the UP context 4Eighty-four percent of women eligible for endline interview were tracked and interviewed . As expeA number of the program variables were not significant in our models. These included mid-media and community group activities; exposure to UHI radio and UHI television was also insignificant . This latter result is not surprising given that the UHI radio and television activities were halted in the second half of the UHI program.5These results demonstrate impacts of the UHI program on modern method use across the six cities in UP where programs were implemented and evaluated. Our results are the first for urban settings in India to show program impacts and relative contribution of different strategies for both demand-side and supply-side activities on modern contraception use. Previous studies from India have shown some positive associations between modern contraceptive use and either demand-side  or supply-side  program activities It is worth noting that the program activities that were significant in this study are the types of activities typically included in FP programming in India and elsewhere. What is noteworthy is the comprehensive approach taken by the UHI program to implement these activities with a particular focus on meeting the needs of urban women and specifically urban poor women. In particular, the program used field-generated inputs to develop a full set of communication materials for each type of FP method. The set of materials included handouts such as counseling cards, fact sheets and frequently asked questions  as well as posters and wall paintings that included similar information by method. The materials all had similar branding and consistent messages that were meant to complement the GoI (and GoUP) FP strategies. These communication materials were used by CHWs for counseling in health facilities and appeared as posters on walls in facilities and communities. In addition, the program specifically targeted the urban poor by having CHWs map slum areas and undertake intensive outreach efforts within every slum identified and mapped. Future programs in urban India should consider including the significant activities found in this study  and use local-level targeting of poor areas to create comprehensive, multicomponent programs; these programs should also ensure that activities are linked, as done by UHI, to support broader exposure and impact.The findings from this study can be used by the GoI to inform strategies to attain their FP 2020 commitments and to support the new National Urban Health Mission (NUHM) which is a sub-mission under the National Health Mission; the NUHM was launched in 2013 with the goal to provide primary health care services particularly to the urban poor


Anticipatory guidance around the management of sleep and crying problems in early infancy has been shown to improve both infant behaviour and parent symptoms of postnatal depression. Digital technology offers platforms for making such programs widely available in a cost-efficient manner. However, it remains unclear who accesses online parenting advice and in particular, whether the parents who would most benefit are represented amongst users. It is also unknown whether the uptake of online programs can be improved by health professional recommendations, or whether parents require additional prompts and reminders to use the program. In this study we aim to: (1) determine whether weekly email prompts increase engagement with and use of a brief online program about infant sleeping and crying, (2) determine whether encouragement from a maternal and child health nurse promotes greater engagement with and use of the program, (3) examine who uses a brief online program about infant sleeping and crying; and, (4) examine the psychosocial characteristics of participants.This study is a randomised, parallel group, superiority trial, with all participating primary carers of infants aged 2 to 12 weeks, receiving access to the online program. Two modes of recruitment will be compared: recruitment via an online notice published on a non-commercial, highly credible and evidence-based website for parents and carers and via the parent’s Maternal and Child Health nurse. After baseline assessment, parents will be randomised to one of two support conditions: online program alone or online program plus weekly email prompts. Follow up data will be collected at 4 months of infant age.Results from this trial will indicate whether involvement from a health professional, and/or ongoing email contact is necessary to engage parents in a brief online intervention, and promote parental use of strategies suggested within the program. Results of this trial will inform the development of recruitment and engagement strategies for other online interventions.ACTRN12613001098729. Registered 01 October 2013.Australian New Zealand Clinical Trials Registry:  Cry Baby, was developed for online delivery. The aim of this study is to examine the characteristics of parents (and their infants) who seek help for infant behaviour via a parenting website, and identify any under- or over- representation of population subgroups within the sample. We also seek to determine whether encouragement from a health professional to take part in an online program, and/or regular weekly email support, promotes greater engagement with the program and greater use of suggested strategies.Infant sleep and crying are common concerns for parents of infants  with 12 Infant sleep problems are common (38 % of parents of 4 week old infants report infant sleep problems) , and areInfant crying is normal and has a natural peak in frequency of around 2.5 h of crying per day at around 6–8 weeks of age . While iMothers who report infant sleep and crying difficulties are at increased risk for PND , 17–19. Baby Business program, which provided anticipatory guidance on infant sleep and crying with content delivered via booklet, DVD, telephone consultation and a parent group session [Several research trials have established the effectiveness of interventions designed to improve infant sleep or help parents manage infant crying , 21, 22. session . CompareWhile the efficacy of face-to-face interventions has been established, low income parents and those residing outside of major metropolitan areas, face major obstacles to accessing parenting interventions. Key barriers include difficulties with transportation, problems accessing childcare to attend appointments, and inflexible work hours . StudiesOne way this imbalance may be addressed is for infant health care providers to encourage participation. In the Australian state of Victoria all newborn infants are assigned a Maternal and Child Health (MCH) nurse, who provides free health checks at 7 to 10 days, 2 weeks, and then at 1, 2, 4, 8, 12, 18, 24 and 42 months of age. Access is almost universal – 98 % of infants participate in the first (in-home) visit, and 96.6 % attend the MCH clinic for the 2 week (infant age) check up . MCH nurexisting sleep problems in infants/toddlers aged 6 to 36 months, resulted in increased infant and mother sleep duration, and reduced infant sleep onset latency [Research examining the reach and acceptability of online parenting programs is still in its infancy, however, results so far look promising. A trial of an online intervention designed to treat  latency . Partici latency , increas latency  and trea latency , to name latency .preventing infant sleep and crying problems has been consistently supported, to the authors’ knowledge, there have been no trials examining whether the same advice can be effectively delivered via an online program. Such an approach may be particularly appropriate for parents who face complex and competing demands in the first few weeks postpartum. Parents may favour a resource that can be accessed at any time day or night; can be quickly and easily navigated; and that allows access to specific relevant content when the parent needs it. Initial encouragement from a MCH nurse, and/or onging participant support via email, may also be helpful in bolstering parent engagement and rentention to the program. Such strategies would be relatively easily built in to existing postpartum health services or as a feature in an online platform. With this in mind, the Cry Baby online program was developed. The current research aims to examine factors influencing participant engagement and retention to the Cry Baby online program, with the intention that results will inform the design of a larger efficacy trial.While the effectiveness of face-to-face interventions for whether randomisation of participants to a condition that receives additional email prompts that encourage parents to log in to the program, will result in greater use of program strategies (primary outcome) as well as greater retention to the research at follow-up (at 4 months of infant age)whether parents who have the program recommended by their MCH nurse are more likely to engage with the program (as measured by program completion rates), than those who are recruited via an online advertisement for the program; and, whether they are also more likely to use the suggested strategiesthe demographic profile of parents who choose to take part in the online program with a view to identify any over- or under-representation of parents  based on: household income, socioeconomic status, education, language spoken and support available to parents; or child characteristics, including birth order of infant, infant age, gender, birth weight, gestation, and where the baby sleeps at night; and, to see how this differs for participants who are recruited via a popular parenting website compared to those recruited via their MCH nursethe psychosocial well being of participants , with a view to identify any specific concerns that may be addressed in the development of new online programs for parents of infantsOur objectives are to examine:Cry Baby trial is a randomised, controlled, superiority trial with two parallel groups , or recommendation from their MCH nurse. The Raising Children Network website is a non-commercial, evidence-based, quality assured, government-funded online resource that covers a broad range of parenting topics and had over 5.7 million visits in the 2013 calendar year . A notice will be placed on relevant pages on the Raising Children Network, as well as its social media . This notice will contain a link to the study website where parents can gain more information and take part in the program if desired.Participants will be primary carers of infants aged 2 to 12 weeks, recruited by one of two recruitment modalities: advertising on the Raising Children Network  Local Government Area (LGA) of the City of Yarra for MCH nurse recruitment, due to both its high birth rate per annum , and broPower analysis using Stata 13.0  was condCry Baby Only or the Cry Baby with Additional Email Prompts condition, with a 1:1 allocation as per a computer generated randomisation schedule, performed as a simple randomisation. Participants will be randomised using a feature within the online survey program, which offers an online, central randomisation service. Allocation concealment will be ensured, as the service will not release the randomisation code until the participant has been recruited into the trial, and baseline measures have been completed.Participants will be randomly assigned to either the www.raisingchildren.net.au or who are seen by their MCH nurse at their 2 week check in the City of Yarra will be invited to participate in the study. However, parents will be excluded from the study if they are under the age of 18 years, have a child over the age of 12 weeks at the time of enrolment into the study, have insufficient English comprehension to complete the questionnaires, have an infant born before 33 weeks gestation, or if the parent and/or infant suffer from a serious medical condition.Participants will provide informed consent online (by selecting a checkbox that appears after the Information Statement and before the baseline questionnaire), before any study procedures occur. All parents of newborn infants aged less than 12 weeks, who access Cry Baby Only or the Cry Baby with Additional Email Prompts groups. The online Cry Baby program covers evidence-based information and strategies on parent self-care , infant crying  and infant sleep . All content is delivered within the online platform. The program is brief, taking no longer than an hour to complete. The program has inbuilt activities, for example, parents can move objects to make a cot a safe place for a baby to sleep, they can also ‘burst’ bubbles (using the mouse pointer) to bust common myths about baby care. There is a video showing how to wrap (swaddle) a baby for sleep, links to a coping plan for parents who are feeling overwhelmed by persistent infant crying, as well as links to local parenting support services.Eligible participants, who have completed baseline measures, will be randomised to the Cry Baby Only condition will receive log in details to the Cry Baby program. Participants can log in or out of the program whenever they please and can move around different areas of the program according to their interests and requirements. If participants decide to complete the program in one sitting it will take approximately 1 hour to finish. The program is designed to be accessed during the first 6 to 8 weeks post-partum, as this is the time when infant crying is most frequent and the advice offered may be most useful. However, we will allow parents of infants up to 12 weeks of age to take part, as evidence indicates that infants who are very unsettled  are at increased risk for AHT [Participants allocated to the  for AHT . The CryCry Baby with Additional Email Prompts condition will receive access to the Cry Baby program and brief, weekly email prompts from the research team. These messages will contain evidence-based, age appropriate information on infant sleep and crying and parent self care, that is already provided within the online Cry Baby program. The email prompts will also direct participants back to the program for more detailed information. As above, participants will be encouraged to access the program before their infant is 6 to 8 weeks of age (when possible), and are encouraged to revisit the Cry Baby program as often as needed.Participants who are randomly allocated to the Cry Baby with Additional Email Prompts condition may withdraw from receiving the additional email prompts at any time.Participants may withdraw from the study at any time for any reason. Likewise, participants allocated to the Our primary outcome measure will be the frequency with which participants use the strategies recommended in the program (see description of the Follow-Up Questionnaire below). This will be compared across recruitment mode (recruited via the Raising Children Network or via MCH nurse) and trial arms .Secondary outcome measures include: retention to the program at follow up; program completion rates (indicated by every module of the program having been accessed); participant demographic characteristics; infant characteristics; whether the participant would recommend the program to family/friends; report of infant sleeping, crying and feeding problems; average time the infant spends awake during each night waking; and, parent depressive symptoms, fatigue and cognitions surrounding infant sleep.Socio-economic Indexes for Areas [The participant will be asked to complete a baseline questionnaire online. This will collect demographic information about the parent  and infant . Area-level socioeconomic disadvantage will be determined using the or Areas  based onA secure web link to the follow-up questionnaire will be emailed to participants when their infant is 16 weeks of age. This represents an age at which most infants ‘sleep through the night’  .Never to 5 = All the time. Mean scores will be computed for the three types of intervention strategies. Participants will also be asked whether they would recommend the program to friends or family, how much of the program their partner participated in , what was the best part of the program and how could the program be improved.A series of questions were developed specifically for the current study. Participants will be asked how often they use the settling (8 items), safe sleep (3 items), and parent wellbeing (5 items) components of the intervention on a 5-point scale, 1 = Baby Business trial [Participants will rate the level of support or help they received from (1) their partner, (2) family and friends living elsewhere , and 3) how often they feel they need support or help but can’t get it from anyone  . These i how ofteNo; 1 = Yes). If participants indicate that any of these infant behaviours are a problem, they will be asked to rate the extent on an 8-point scale, 1 = Hardly a problem to 7 = Severe problem. These items have been used in prior infant sleep research [Participants will be asked whether infant daytime sleep, night-time sleep, feeding or crying behaviours have been a problem over the last 2 weeks ; at each attendance, the number of minutes the participant spends with their infant (<10mins – 2 h); and the average number of feeds the infant has over a 24 h period (items previously used ).Symptoms of depression will be assessed using the Edinburgh Postnatal Depression Scale , a 10 itNever to 5 = Always), where higher scores indicate higher levels of fatigue. The scale has good reported psychometric properties and has been validated for use with parents [Strongly Disagree to 7 = Strongly Agree), where higher scores reflect higher levels of fatigue.Both the Fatigue Assessment Scale  (FAS) an parents . The 9 iStrongly disagree to 5 = Strongly agree.Four subscales of the Maternal Cognitions about Infant Sleep Questionnaire  will assData will be collected via the programming of the online system, recording whether the program was completed , how many times the participant logged in to the program, and how much time in total they spent within the program.The recruitment phase will run for 12 weeks in each recruitment modality. An advertisement placed on the Raising Children Network will provide interested parents with a link to the program’s online landing page. MCH nurses will provide all parents of newborn infants at the 2 week infant check with an advertising postcard containing the link to the landing page.Cry Baby alone or Cry Baby with Additional Prompts). The final page of the baseline survey will provide participants with a secure link to the Cry Baby website, where they can register their login details and immediately access the Cry Baby program.The landing page will provide more information describing the study, what participation involves, and a secure link to the online baseline survey where participants can read the projects’ plain language statement and complete eligibility criteria and consent. After completion of the baseline survey, participants will receive email notification of their allocation to condition  until their infant reaches 12 weeks of age. Each email will contain a brief summary of information from the online program.All participants will receive a welcome email which contains further information about the program and their group allocation. Participants allocated to the When participants’ infants are approximately 16 weeks of age, they will receive an email link to the follow up survey which has their unique ID code embedded. If participants have not completed their follow-up questionnaire after 2 weeks, a reminder email will be sent a maximum of three times over a 6 week period. At this time point, some parents will only have had access to the program for 4 weeks, and others for 14 weeks, however, we expect that if parents are going to try out strategies suggested in the program, they would do so within 4 weeks of their first access to the program. Setting the follow up time point according to infant age, rather than time since enrolment, controls for the large variability in infant development that might otherwise impact on our results.All data will be collected electronically using an online survey tool which provides a secure survey website (SSL encryption). Password protected data files will be utilised for participant tracking during the study and data analyses. Access to the study data will be restricted to the research team. Data will be securely stored for a period of 7 years after the completion of the study. Backup of electronic data will be performed regularly and stored securely on a password protected internal network.d. All analyses will control for child age given that time of enrolment varied between 2 and 12 weeks postpartum. Finally, intention-to-treat analysis will be conducted using Full Information Maximum Likelihood for missing follow-up data.A range of descriptive and multivariate data analyses will be used to assess the aims of the study. Descriptive statistics  will be presented for the sample demographics, number of times participants access the program, and proportion of participants completing the program. Demographic characteristics of the sample will be compared to country-wide averages obtained from the most recent Australian Census data. Characteristics of infants taking part in the research will be compared to MCH data for babies born within the same geographic area at the time of study recruitment. A mixed-design analysis of variance (split-plot ANOVA) will be conducted to compare the intervention conditions (between-subjects factor) on each of the continuous outcomes at follow-up (within-subjects factor). Effect sizes will be reported where appropriate, with 0.01, 0.06 and 0.14 as small, medium and large effect sizes for multivariate η2, while 0.2, 0.5 and 0.8 are small, medium, and large effect sizes for Cohen’s The current study has received approval from the Parenting Research Centre’s Human Research Ethics Committee . Any modifications to the study protocol which may impact on the conduct of the study, potential benefits of the participants or may affect their safety, including changes of study objectives, study design, participant population, sample sizes, study procedures, or significant administrative aspects will require a formal amendment approved by the principal investigators and HREC.Cry Baby is the first randomised, controlled, superiority trial to examine whether evidence based advice on managing infant sleep and crying can be effectively delivered online. This trial will establish whether involvement from a health professional and/or weekly email prompts are necessary to engage parents of newborns with online programs, and additionally, provide insight in to the demographic characteristics of parents who choose to access advice online. Our findings will inform the development, design and retention strategies of other trials of online programs.


During the late 19th century, their behavior in filtration tests applied to the agent causing the 'plant mosaic disease' eventually led to the discrimination of viruses from bacteria. Thereafter, they promoted the development of biophysical cornerstone techniques such as electron microscopy and ultracentrifugation. Since the 1950s, the robust, helically arranged nucleoprotein complexes consisting of a single RNA and more than 2100 identical coat protein subunits have enabled molecular studies which have pioneered the understanding of viral replication and self-assembly, and elucidated major aspects of virus–host interplay, which can lead to agronomically relevant diseases. However, during the last decades, TMV has acquired a new reputation as a well-defined high-yield nanotemplate with multivalent protein surfaces, allowing for an ordered high-density presentation of multiple active molecules or synthetic compounds. Amino acid side chains exposed on the viral coat may be tailored genetically or biochemically to meet the demands for selective conjugation reactions, or to directly engineer novel functionality on TMV-derived nanosticks. The natural TMV size (length: 300 nm) in combination with functional ligands such as peptides, enzymes, dyes, drugs or inorganic materials is advantageous for applications ranging from biomedical imaging and therapy approaches over surface enlargement of battery electrodes to the immobilization of enzymes. TMV building blocks are also amenable to external control of in vitro assembly and re-organization into technically expedient new shapes or arrays, which bears a unique potential for the development of 'smart' functional 3D structures. Among those, materials designed for enzyme-based biodetection layouts, which are routinely applied, e.g., for monitoring blood sugar concentrations, might profit particularly from the presence of TMV rods: Their surfaces were recently shown to stabilize enzymatic activities upon repeated consecutive uses and over several weeks. This review gives the reader a ride through strikingly diverse achievements obtained with TMV-based particles, compares them to the progress with related viruses, and focuses on latest results revealing special advantages for enzyme-based biosensing formats, which might be of high interest for diagnostics employing 'systems-on-a-chip'.The rod-shaped nanoparticles of the widespread plant pathogen  As seAround the turn of the 19th century, a new type of infectious agent, the 'virus', was discovered . During Tobacco mosaic virus . In 188aviridae . Martinuaviridae –22.From 1935 on, Wendell Stanley examined the tobacco and a similar tomato disease and attributed them to an infectious crystallizable protein –24. Soon, as they may undergo a conformational change into a short helical “lockwasher” -enzymes, and a spatial decoupling of the enzymes and the TMV scaffold.To circumvent the need for extensive changes to the natural TMV structure, the enzymes were immobilized on the surface of moderately engineered TMV sticks by a two-step procedure . This coterminus  served atin; see  for detaCys subunits could be decorated with biotin linkers, and virtually all thereof with enzymes  released lower-order CPCys/Bio aggregates, (iii) or stoichiometric amounts of maleimide-PEG11-biotin linkers, respectively, or into (iv) uncoated plates resulting in mere enzyme adsorption  (PDMS), a standard polymeric material used for the fabrication of microfluidic devices by master-replication techniques, was already shown to allow a site-specific bottom-up integration of TMV carrier sticks . This waSo far, however, it remains to be tested whether TMV particles immobilized via protruding RNA ends or through non-covalent attachment would retain their arrangement inside the flow channels of microfluidic devices in operation . To ensFast, sensitive and cost-saving biosensors often employ label-free read-out, in which signal transduction occurs without a need of reporter molecules, e.g., by optical, electrochemical, mass- or surface plasmon resonance-sensitive techniques –172. TheTMV has accompanied human history for more than a century, threatening farmers and the tobacco industry as well as supporting and inspiring scientists. Once a key player in the origins of the virology discipline, enabling breakthrough findings on virus structure, properties and interplay with the host, concomitantly advancing the development of analytical techniques, the virus currently adapts novel roles: As a richly available, particularly robust biomolecular complex, TMV allows for extensive exploration studies on prospects and definite limitations of novel synthetic, nanostructured protein-containing material combinations. With respect to the general knowledge that a lot of frogs need to be kissed  before meeting a handsome prince , researcIt is only within the past few years that plant-made effector proteins, pharmaceuticals and virus-like particles especially for vaccination purposes are regarded economically valuable, attractive alternatives to conventionally produced compounds. This is attested by a rapidly growing number of present reports and companies specializing in this field –184. Pla


The obesity epidemic is well established, particularly in rural settings. Programs promoting healthy lifestyles for rural women are urgently needed; however, participant engagement is challenging. In the context of a large randomized controlled trial targeting the prevention of weight gain in rural women, we explored successful recruitment strategies and aimed to understand participants’ barriers, enablers and reasons for program participation.n = 649) and qualitative semi-structured interviews conducted for a subgroup of participants (n = 45). Data were collected at three time points: baseline, 6 and 12 months post program commencement.We recruited women (aged 18–55 years) from the general rural Australian population. A mixed-methods approach was applied to explore factors that influenced program participation, including quantitative questionnaires for all participants  and social (peer persuasion and support). Identified enablers included convenience of the program location, perceived program utility, such as weight management and optimization of lifestyle choices, as well as attending the program with peer support. Barriers to engagement, which are likely exacerbated in rural communities included lack of anonymity, self-consciousness and segregated social networks in rural settings. Participants reported that eliciting local support and maximizing publicity is fundamental to improving future program engagement.Multiple program promotion strategies including communication, marketing and partnering, as well as mobilization of social networks and peer persuasion, enabled engagement of rural women into a healthy lifestyle program. These recruitment strategies are consistent with successful strategies utilized previously to recruit urban-dwelling women into lifestyle programs. Future engagement efforts in rural settings could be enhanced by hosting multiple sessions within existing socio-cultural networks and assuring participants that they will not need to share their personal health information with others in their community.ACTRN12612000115831. Date of registration 24 January 2012.Australia & New Zealand Clinical Trial Registry. Trial number The online version of this article (doi:10.1186/s13063-015-0860-5) contains supplementary material, which is available to authorized users. The obesity epidemic is increasing globally and is a serious threat to public health. Socially disadvantaged populations such as those living in rural areas are at an increased risk of being overweight or obese compared with the general population. Higher rates of obesity in rural settings are associated with lower income and education levels, higher rates of unemployment and living in disadvantaged neighborhoods , 2. MoreThe efficacy of healthy lifestyle weight gain prevention programs has been established , 9. The Comprehensive program evaluation is required to inform scale-up through the exploration of implementation strategies and sharing of learning . AssessmOf the limited literature available, motivators for program engagement of disadvantaged populations include perceived personal benefits, minimal time commitments and program flexibility . Social n = 21) or control (n = 20) groups.The Healthy Lifestyle Program (HeLP-her) is an evidence-based healthy lifestyle weight gain prevention program for women , 31. TheCommunities selected had a population size between 1500–10,000 people and were located 100–400 km from the capital city, Melbourne. Women aged 18–55 years of any BMI and residing in or close to participating communities were invited to participate. Exclusion criteria included pregnancy, or serious medical condition impacting program participation. The detailed study design methodology is comprehensively reported elsewhere . This stParticipant recruitment commenced in September 2012 and was completed in April 2013. A comprehensive communication and engagement protocol was developed to promote effective recruitment . The recDevelopment of a multilevel partnering strategy across all local communities;Flyers distributed to all families though partnership with the local primary schools, pre-schools and kindergartens within each town;Staff presence and visits within the local towns promoting the program;Media releases ;Flyers distributed at additional locations ;Mobilization of social networks through (a) encouraging women who had expressed interest and registered for the program to bring friends and family along to the program and (b) identifying community champions to promote the program to their community, workplace and social networks and accessing partners databases for email contacts.Specific recruitment strategies incorporated:Women expressed interest in study involvement by sending SMS text messages, phone, email or return of an expression of interest form.The program was delivered predominately within each community’s local primary schools, and session times were scheduled to align with school drop off and pick up. In addition community locations such as local town halls, community centers and sports clubs were utilized for evening sessions to ensure multiple opportunities to participate. Sessions were able to accommodate young children.We applied a mixed-methods approach to explore factors that influenced healthy lifestyle program recruitment and engagement. Here, we have defined engagement as factors that motivated, encouraged and enabled program participation, extending our definition beyond recruitment to include completion of the early phase of the HeLP-her Rural program (0–6 months). This phase included attending a single group education session, receiving SMS text messages, completing phone coaching and utilizing the program manual, described in detail in the study protocol . We alsoBaselineThe baseline questionnaire included demographic, nutrition, physical activity information and a brief devised questionnaire exploring factors influencing healthy lifestyle program participation. The enablers and barriers investigated included questions related to social factors , program location , program costs and program facilitator .Six months post program commencementQualitative methods (in-depth semi-structured interviews) were conducted with HeLP-her Rural participants. Purposeful sampling techniques were applied to obtain a representative sub-sample from the larger RCT. Women from 10 randomly selected communities (6 interventions and 4 control communities) were invited to participate in the semi-structured interviews and 65 women volunteered. Interviews were conducted until a point of data saturation, determined when no new ideas emerged from the interviews, as per standard methods . One staTwelve-months post program commencementThe results of our semi-structured interviews informed the questions included in the 12-month questionnaire relating to potential community barriers to program engagement such as competing commitments, disinterest in health, self-consciousness and inconvenience of the program location and time. This enabled an opportunity to conduct data triangulation.Data were collected at three time points.Statistical analysis was performed using STATA version 12 for Windows  and descriptive statistics used to explore participant demographic and anthropometric data at baseline (recruitment data). Continuous variables are presented as mean (SD) and categorical data as relative frequencies. Program retention data were assessed via Student’s t-test to determine whether there were differences in characteristics between participants that remained in the study (completers) compared to those that did not remain in the study (non-completers) at 12 months. However, categorical data  were compared between completers and non-completers using Fisher’s exact test because of the reduced numbers of participants within each category. Therefore, the categorical tests need to be interpreted with caution, as applying more robust statistical measures was not possible.Qualitative transcripts were analyzed independently by two investigators via thematic analysis and key themes and patterns in the data identified. Thematic coding of data and development of models were assisted by the NVivo software program .n = 301) and intervention (n = 348) participants were 39.6 ± 6.7 years and 28.8 ± 6.9 kg/m2, respectively, with no significant difference between groups  with a mean BMI of 30.25 ± 8.1 kg/mn = 95) were excluded post screening. Reasons for exclusion included pregnancy, breastfeeding, taking weight control medication, diagnosis of a serious physical or psychological condition or not contactable . This represented approximately 7-10 % of the eligible target population. Based on predefined and limited exclusion criteria, less than 12 % of women , this suggested that the study cohort was representative of the wider Australian regional population, according to household income  and education (tertiary education reported in 30 %) .As there was no difference in demographic characteristics between those in the controls and interventions at baseline, results were pooled for all participants. Baseline results revealed factors influencing program engagement were related to the timing, venue and cost of the program, social influences as well as the mode of delivering the program . Program location (within women’s township) had the greatest impact on increasing the likelihood of participation and engagement (79 % agreeing with this statement), followed by the cost (69 % agreeing) and ability to attend the program with peer support (54 % agreeing). Conversely, few participants indicated that they would attend a healthy lifestyle program if it were held in another township (6 % agreeing), if the program was computer based (10 % agreeing) and if the program cost more than 50 dollars (13 % agreeing) Fig. .Fig. 2FaOur thematic analysis of interviews revealed that the major reasons for participation in the HeLP-her Rural lifestyle program could be grouped into two broad categories: personal and social factors. Personal factors were divided into two sub-themes (1) perceived program benefits and (2) logistic factors associated with attendance. Social factors were also divided into sub-two themes: (1) peer support and persuasion and (2) psychosocial influences. Figure Perceived program benefits“I actually needed to lose some weight and I thought something like that might motivate me to actually do it” (Control participant).Perceived personal program benefits were the strongest driver for program enrollment. Broadly participants described being motivated to participate for reasons related to weight management and to optimizing their health by improving their lifestyle choices. Participants frequently reported attending the HeLP-her Rural program as they were seeking external motivation to modify their lifestyle.“I was sort of on watch to manage my weight and change some habits to keep my blood sugar under control” (Intervention participant).Others reported attending the program to receive advice for weight-related conditions such as high-cholesterol, impaired glucose tolerance and polycystic ovary syndrome.There's nothing here [healthy lifestyle program] that I've heard about…there are a lot of myths, there’s a lot of this…but I really wanted to know how my body works” (Control participant).““The research I found very interesting, yeah, because, I mean, it’s going to tackle obesity big time, hopefully, and improve things” (Control participant).(b)Logistical factorsHowever, participants also described enrolling in the HeLP-her Rural program for reasons unrelated to weight management. Women described joining the program because it was a unique opportunity and they were interested in women’s research.“ease” and “convenience” of the program location, flexibility of session times and minimal participant burden. Hosting the program sessions at the local primary school was described as ideal as this venue was familiar to participants and enabled them to feel comfortable.“I think it was very appropriate, having it at the school because the school is a big part of our community” (Intervention participant).Participants reported that their program involvement was aided by the “didn’t have to make an extra effort” to attend the program.“The program meetings were the right time of the day for me particularly but I imagine it’d probably be the right time of the day for most of the mums with children that are school aged” (Intervention participant).Participants also explained that as the sessions were run at school drop off and pick up times, this assisted program attendance as women “access to everybody.”As the program was free this was also described as a motivating factor for program recruitment, allowing “was well advertised, we had it almost every week in the school newsletter… and it was advertised in the [local] paper.” On the other hand a participant from the same town reported, “I think it was just possibly that there weren’t enough people that sort of knew it was actually happening.” Participants described a range of other potential personal logistical barriers that may have prevented other women participating such as work, farming and family commitments, disinterest and limited access to childcare.Interestingly, while program awareness was described as high among program participants, lack of program awareness was speculated as a key barrier to greater program engagement. One woman explained that the program Social support and peer persuasion“[I went] because there were a couple of my friends going” and “word of mouth is always the best way in small towns” (Intervention participant).A strong relationship emerged between program engagement and the social dynamics and influences within rural settings. Members of participants’ social networks played a pivotal role in persuading and encouraging program involvement. The importance of program promotion through community members and social networks also resonated strongly.“confidence” and provided “moral support.”“A lot of people don’t want to go to these things  by themselves; they might be uncomfortable and embarrassed” (Intervention participant).Moreover, participants described that attending the program with peer support boosted “safety in numbers.” Indeed, almost all participants irrespective of their weight expressed preferring to attend a healthy lifestyle program with peer support rather than alone.“Amongst friends, amongst family,” you don’t feel out of place when you’re in a group” (Control participant).(b)Psychosocial factorsThe need to attend a healthy lifestyle program with peer support was described as particularly important among obese women. Attending the program with friends reduced anxiety for program enrollment as there is “know everyone” can result in women feeling “self-conscious” and “embarrassed” and can be a “hurdle.”“This is a small community—they’re nervous about doing something that everybody is going to be watching” (Control participant).Lack of anonymity in rural settings emerged as a prominent barrier for greater community program engagement and uptake. Participants speculated that attending a healthy lifestyle program in a rural setting where you “People are always self-conscious…do you want to admit that you want to lose weight in front of a whole heap of other mums at the school?” (Control participant).The prospect of being judged by peers was exacerbated in obese women compared to lean women.“fear of the unknown.” Women revealing that there is a “stigma” attached with attending an urban, externally led community program.“People [are] apprehensive of what they are going to get into” and “what is going to be asked of them” (Intervention participant).Others speculated potential psychosocial obstacles to program recruitment were distrust of a university-based program and off-putting” and “hindered” and led to mothers of children from other schools feeling “uncomfortable," discouraging program participation.“The schools are very segregated” and mothers of children from other schools may “have thought it  was just for our primary school” (Intervention participant).Furthermore, a heightened sense of identity as well as place associated with community structures in rural settings was emphasized by participants. Participants revealed that while hosting a lifestyle program at the local government primary school was an ideal location for mothers of children at this school, this venue may have been “The participants described various strategies they believed would assist with improving future healthy lifestyle program engagement. These strategies were grouped broadly into four themes: (1) obtain community support via involving the community in the promotion of the program, 2) utilize multiple channels  to advertise the program in order to ensure maximum program awareness, (3) host multiple sessions within a range of rural community socio-cultural networks and (4) provide healthy lifestyle-related incentives  and work commitments (64 %). Social factors such as feelings of being embarrassed to attend the program were also reported by participants (40 %) Table .Table 3P2 versus 28.4 kg/m2) and a greater age (46.8 years versus 35.9 years). Other potential contributors including, household income, education and employment status were not associated with attrition. An additional file shows this analysis in more detail . Reported reasons for missed data were censoring due to pregnancy, onset of a serious illness, relocation interstate, withdrawal and loss to contact. Overall, program attrition rates were higher among women with a heavier baseline BMI (BMI 31.7 kg/mOur 12-month retention strategy prior to review included: (1) sending invitational letters to all participants 2 weeks prior, (2) reminder phone calls made the week prior and (3) a text message sent the day prior. The invitation letter emphasized the low participant burden of the 12-month review with the invitation letter stating, “This review takes just a few minutes.” The review was scheduled at the same location as the baseline group session  during school drop off and pick up to further minimize the participant burden. Additionally, successful retention strategies included flexible review schedules , frequent communication with local partners, rigorous tracking of participants, a skilled team and persistence. Gift packs were also provided to encourage women to return for the 12-month follow-up  and social factors were associated with peer support and persuasion. Additional social factors included the lack of anonymity in small rural communities, which may result in some women feeling apprehensive and too self-conscious to join a weight-related programs.While the motivators for healthy lifestyle program enrollment have previously been established in obese women, which include improvements in physical appearance, self-esteem and confidence , there aLikely exacerbated in rural settings, our results indicated that social influences have an important role in both promoting and discouraging program participation in rural settings. Consistent with previous literature, we report that group-based programs promote opportunities for social contact and interactions  and thatInterestingly, both our quantitative and qualitative results revealed that the greatest barrier to healthy lifestyle engagement was a lack of program awareness, despite the utilization of a multi-strategy communication and engagement approach. We report that participants required multiple personal verbal (peer persuasion) and non-personal visual  prompts prior to engaging with this program. Participants frequently reported that personal peer persuasion and word of mouth comprise the most valuable avenue to engaging rural women. Our results add to the findings of a recent systematic review indicating that increased awareness of a healthy lifestyle program among potential participants improves the likelihood of program engagement . FurtherAnother important consideration when implementing healthy lifestyle programs in rural communities is the selection of an appropriate venue. As noted in other studies, the importance of ensuring cultural community sensitivity when attempting to gain entry for healthy lifestyle programs is vital . In ruraDespite young women being notoriously difficult to retain in healthy lifestyle programs , we retaWe also report lower retention rates in women with heavier baseline BMIs, and this has been previously attributed to unrealistic weight loss expectations, leading to early disappointment and subsequent program drop out . Our finStrengths of the current study included the application of a mixed-methods approach to a rigorously designed healthy lifestyle prevention RCT. We also applied robust qualitative data analysis using a theoretical framework and utilized two independent staff for data analysis. Moreover, the purposeful sample utilized increases the generalisability of the qualitative results to the wider RCT cohort. In addition, it is unlikely that our results were influenced by differential recall bias at 12 months between intervention and controls as no statistical difference was reported between groups. Limitations include a lack of specific information regarding the impact and effectiveness of each individual recruitment strategy; therefore, we are unable to identify which engagement strategy was most effective in recruiting women from diverse socioeconomic backgrounds. Additionally, the barriers to engagement identified were speculative based on enrolled program participant’s views as we were unable to interview women who did not participate in this program.The HeLP-her Rural program provided a unique opportunity to explore participant engagement including recruitment and participation in a healthy lifestyle program specifically targeting rural-dwelling women. We engaged communities and rural women through multiple channels of communication  in order to reach program recruitment targets. Our findings suggest that factors that need to be considered when engaging rural women into a healthy lifestyle program include using existing community social networks to deliver programs, promoting the program multiple times through local community networks, maximizing program awareness and understanding community social dynamics. Future program engagement would be enhanced by hosting multiple programs within existing socio-cultural networks and assuring participants that they will not be required to share personal health information with other women living in their community. Ultimately, the learning and practical implications reported in this study can be applied to future prevention programs more broadly, enhancing program engagement.