Cash transfer programs (CTPs) aim to strengthen financial security for vulnerable households. This potentially enables improvements in diet, hygiene, health service access and investment in food production or income generation. The effect of CTPs on the outcome of children already severely malnourished is not well delineated. The objective of this study was to test whether CTPs will improve the outcome of children treated for severe acute malnutrition (SAM) in the Democratic Republic of the Congo over 6 months.We conducted a cluster-randomised controlled trial in children with uncomplicated SAM who received treatment according to the national protocol and counselling with or without a cash supplement of US$40 monthly for 6 months. Analyses were by intention to treat.P = 0.007). The adjusted hazard ratios in the intervention group for relapse to moderate acute malnutrition (MAM) and SAM were 0.21  and 0.30  respectively. Non-response and defaulting were lower when the households received cash. All the nutritional outcomes in the intervention group were significantly better than those in the control group. After 6 months, 80% of cash-intervened children had re-gained their mid-upper arm circumference measurements and weight-for-height/length Z-scores and showed evidence of catch-up. Less than 40% of the control group had a fully successful outcome, with many deteriorating after discharge. There was a significant increase in diet diversity and food consumption scores for both groups from baseline; the increase was significantly greater in the intervention group than the control group.The hazard ratio of reaching full recovery from SAM was 35% higher in the intervention group than the control group  = 1.10 to 1.69, CTPs can increase recovery from SAM and decrease default, non-response and relapse rates during and following treatment. Household developmental support is critical in food insecure areas to maximise the efficiency of SAM treatment programs.NCT02460848. Registered on 27 May 2015.ClinicalTrials.gov, The online version of this article (doi:10.1186/s12916-017-0848-y) contains supplementary material, which is available to authorized users. Childhood malnutrition is a significant cause of ill health and poor development worldwide. High-quality nutrition is essential in early childhood to ensure healthy growth, proper organ formation and function, a strong immune system and neurological and cognitive development. Children with severe acute malnutrition (SAM) are at high risk of morbidity and death . There aAlthough considerable progress has been made in treating SAM –4, one wThere is a particular need to determine the effect of CTP strategies and their impact on vulnerable households with malnourished members among different target groups and contexts. Most underlying causes of malnutrition are a function of people’s resources and social context. What households produce as well as the time they have to care for dependent members are determined by a range of social, economic and political factors; these are thought to include the division of labour, gender inequality, educational opportunities and property and power relations. When households have more money, they can diversify their diets by buying or growing food of a higher quality, being able to afford to attend the health centre and investing capital to create ongoing income-generating opportunities. Here, we hypothesised that additional cash will have a direct effect to improve the final outcome of children enrolled in a community-based management of acute malnutrition (CMAM) program. Specifically, within the household the cash would decrease intra-household sharing of the RUTF and improve the food diversity and consumption. The cash would improve the outcome of the malnourished child by reducing death, morbidity, transfer to hospital, defaulting, relapse or other causes of failure. The child would have higher rates of mid-upper arm circumference (MUAC) and weight gain and derived anthropometric indices.This paper presents the findings from a cluster-randomised trial comparing the outcome of a standard Outpatient Therapeutic Program (OTP) for SAM and infant and young child feeding (IYCF) counselling with and without a monthly cash transfer over a 6-month period in the Democratic Republic of the Congo (DRC).2 in the city of Mbuji-Mayi; GPS 6°11'S 23°54'E) in the Kasaï-Oriental province. There are 52 functional health centres where management of SAM was established using the OTP module of the National Integrated Management of Acute Malnutrition protocol. This setting was selected for the following reasons. First, the socioeconomic homogeneity of the whole livelihood zone was confirmed by three baseline surveys: (1) a market chain analysis [The present study took place in the DRC, where around two million children younger than 5 years old are affected by SAM every year , 27. Thianalysis , (2) a Hanalysis  and (3) analysis . Second,analysis . Third, analysis .A cluster was defined by a health centre and its catchment area. There were 52 eligible health centres, of which 20 were selected. The health centres were selected at random by sequentially drawing, in public, sealed, numbered papers from a basket in the presence of all 52 health centre representatives. A priori, in order to minimise contamination bias between clusters if a subsequent health centre was drawn but was found to have a catchment area adjoining a health centre that had already been selected, that centre was eliminated from the draw and a further sealed paper drawn; the health centre representatives were aware of this procedure before the draw took place. When the 20 health centres that were to be included in the trial were chosen, a second selection round was conducted with each selected centre drawing a sealed paper with either a 1 or 2 written on it to indicate to which arm of the trial that centre would be entered. A third selection was made in private when one of the arms was assigned at random to receive standard SAM management plus counselling and the other arm standard SAM management plus counselling plus cash transfer. In order to detect an expected recovery rate of 70% after 8 weeks and an expected difference between groups of 10%, with an α-error = 5%, a β-error = 20% and an intra-class correlation of 0.001, a sample size of 1392 children was required. Assuming a study dropout of up to 15%, a total sample size of 1600 children was projected with an average of 80 participants per cluster: 800 per arm. Because we wished to avoid any confounding from temporal variation in the centres’ recruitment rate, we determined the potential case load from the admissions during the previous year when there had not been any active screening of the population. We then conducted an exhaustive, active screening of the whole catchment area of each health centre in order to recruit the required number of children (about 4 per centre per day) over as short a time as possible. The staff in each health centre comprised the nurse in charge and two Save the Children staff dedicated to check and collect the data. The study’s staff were supervised by Pronanut (the DRC Government nutrition agency) and UNICEF. The study teams were trained intensively for 3 weeks prior to the start of the study by the Principal Investigator, and the details of the national protocol were revised with the nurses in charge of the centres.Inclusion criteria for the trial required participants to be eligible for outpatient SAM treatment according to the integrated management of acute malnutrition (IMAM) national protocol , have noThe objectives and procedures of the study were explained to heads of households or principal child caregivers before inclusion. An informed consent statement was read aloud in the local dialect before consenting adults signed or gave their fingerprint. It was emphasised that participation in the study was not a pre-condition for obtaining nutritional treatment and free medical services. It was clearly stated that participants were free to withdraw from the study at any time. The protocol was approved by both the National Ethical Committee of the School of Public Health from the Faculty of Medicine of University of Kinshasa and the Ministry of Public Health. The study was registered on ClinicalTrials.gov as NCT02460848 and was performed in accordance with Good Clinical Practice (GCP) guidelines for clinical trials and according to the tenets of the Declaration of Helsinki.In both study arms, children with SAM received treatment according to the IMAM national protocol for OTP and counselling on IYCF . At admiAfter discharge, children were followed up monthly: medical history, physical examination, and anthropometry were repeated at each visit until the end of the study. Observations were concluded 6 months after recruitment. Thus, those who were under treatment for longer had a shorter post-discharge follow-up; as failure to respond was defined as still being malnourished after 12 weeks of treatment, the minimum follow-up period was 14 weeks, so that individual children had either three, four or five monthly follow-up assessments. Children were defined as having relapsed to SAM if they again reached any of the three criteria defining SAM at least once during the monthly follow-up visits after being discharged as recovered. Children’s relapse to moderate acute malnutrition (MAM) was defined as the development of WHZ < –2.0 and ≥ –3.0 (WHO Growth Standards 2006) or MUAC <125 mm and ≥115 mm  at least once during the monthly follow-up visits, without the child developing SAM criteria during any other follow-up visit. ‘Unknown’ was defined as a defaulter not confirmed by a home visit or with no information for the child at the end of the trial. Withdrawal from the study was defined as participants who elected to stop the study for personal reasons.At enrolment, trained health workers recorded the socioeconomic characteristics of the household and categorised it into wealth groups according to local definitions of wealth and assets from the HEA assessment. The Household Dietary Diversity Scores (HDDSs)  and the All participating caretakers from the intervention group with one or more children with SAM received an unconditional cash transfer of US$40 value each month during treatment and follow-up for a total of 6 months . We emphasised to the participants that they could use the funds in any way they saw fit in a completely unrestricted way without any conditions being imposed on how the cash was used. The cash was distributed directly to the child’s caretaker for each household in the intervention group without informing or involving the health centre staff. Each month the cash was given from 10 separate administrative offices  by two Save the Children financial staff and two food security supervisors attached to the study and completely independent of the health centre staff. The dispensing of the money was spread throughout the month according to the patient's admission date, so that daily attendance was minimised. This mechanism was considered the most secure to preserve confidentiality and to avoid contamination bias; electronic and other forms of cash transfer were not available in the area at the time of this study.The amount given to each household was fixed and not adjusted by the size of the household or the number of malnourished children. This amount was calculated to provide 70% of the monthly household income for a household of seven persons characterised as very poor using HEA criteria . SeventyThe cash transfer for a family of seven persons amounts to 18 cents US per person per day. As the area is isolated without reliable ground transportation of goods, it tends to be more expensive than less isolated areas. The cash given in other CTPs in the DRC varies from US$110 to US$135 monthly (except for one pilot project which dispensed US$20.5 per month). The amount given in this study was judged by the Emergency Department of UNICEF to be a sustainable amount that could be supported by donors and other stakeholders if the program were to be scaled up; the higher amounts given in other programs were judged to be unsustainable operationally .All data were collected on standardised paper forms and double-entered into EpiData version 3.1 (EpiData Association) by staff unaware of the arm to which each health centre belonged. Any anthropometric data which fell outside the limits of biological plausibility, using WHO criteria, were eliminated from the database . Changest test for continuous variables and the chi-square test for categorical variables.Significance testing for differences between intervention and control groups at baseline was performed using the independent sample Student's P values. We checked for possible deviation from the proportional hazards assumption of the Cox regression model by using the non-proportionality test on the basis of the Schoenfeld residuals.Differences in child recovery between trial arms of the primary outcomes and relapse rate after discharge from therapeutic home treatment were tested by using a mixed-effects Poisson regression model, with health centre as random effect to estimate the incidence rate ratio (IRR). Next, we estimated hazard ratios (HRs) and 95% confidence intervals (CIs) using marginal Cox proportional hazards models adjusted for baseline values, where the outcome variable is time from recruitment to the event (recovery) and the time scale is calendar week. All 95% CIs used robust estimates of the variance to account for clustering at the health centre level as well as a shared-frailty model as developed by Andersen . PotentiComparisons between arms for the secondary outcomes , time to recovery, length/height change, IDDS, HDDS, FCS and daily, weekly and monthly anthropometrics changes were made by using linear mixed-effects models for continuous outcome variables, whereas mixed-effects logistic regression models were used for proportions, with health centre as random effect and models adjusted for baseline values. Analyses of anthropometric data which depended upon body weight excluded children with oedema; oedematous children were included in all other analyses.P values were computed with the robust score test.We produced and used Kaplan-Meier plots to estimate the probability of failure to achieve and maintain nutritional recovery up to 6 months from enrolment. Survival curves of two groups were compared using the Cox regression analyses with robust estimates of the variance to account for clustering at the health centre level, and Between 16 July 2015 and 31 July 2015, 1600 children were admitted to the centres with a diagnosis of SAM; 119 (7.4%) children did not meet the inclusion criteria and were excluded. Among those, some were admitted using the IMAM unisex weight-for-height table but were ineligible using sex-specific assessment, a few lived outside the catchment area and others were referred directly to the hospital. Figure P > 0.05 . The mothers had a higher school achievement in the intervention group, but were otherwise not different. The HEA assessment showed that 75% of the households were classified as poor or very poor in both arms. The household size and number of children younger than 5 years were both greater in the intervention group. However, there were fewer household members younger than 5 years in the intervention group compared to the control group (5.3 vs 5.5), so that the increased number of young children, and hence the dependency ratio, in the intervention group would tend to make this group more vulnerable to nutritional deficits. In both groups, less than 20% of the households had an acceptable diet diversity score with 43% being in the lowest category; 53% were considered to have poor or borderline food consumption. These results are consistent with previous assessments conducted in Kasaï-Oriental [The control and intervention (cash) group’s baseline characteristics are shown and compared in Table Oriental  and showThe results of the treatment are given in Table During treatment, changes in weight, WHZ, weight-for-age Z-score (WAZ), body mass index Z-score (BMIZ) and IDDS were all significantly greater in the intervention group compared to the control group; however, there was not a difference in the rate of increase in MUAC or height. Both groups gained less height than expected when compared with the standards for age of height-for-age Z-score (HAZ); the changes were not significant. The mean length of stay among children in the therapeutic program was 6.9 weeks (±2.5). It did not differ significantly between the groups, although the median was 1 week shorter in the intervention group.th week. Adjustment of the analysis for the significant baseline household and maternal characteristics increased the difference between the groups by a trivial amount. The mixed-effects Poisson regression analysis gave similar results.The Cox regression analyses Table  showed tTable This deterioration in the control children’s status post-discharge is confirmed by the proportion of children who again developed MAM or SAM. Altogether 44% of the control children deteriorated after treatment, 11% to a level where they would be readmitted for SAM. Most of the children who relapsed to MAM did so with both WHZ and MUAC. Furthermore, as there was no significant gain in height, the deterioration in WHZ for those who relapsed to MAM or to SAM could not be ascribed to a disproportionate gain in height relative to weight.χ2 = 2.7, P = 0.10). In contrast, 47 (7.1%) of the control children re-developed anthropometric SAM having been discharged as cured.For those who relapsed to SAM, only 8 of the 707 (1.1%) intervened children relapsed anthropometrically by the WHZ criterion and none by the MUAC criterion. The main type of relapse in the intervened children was re-development of oedema. There were slightly more children who relapsed with oedema in the control group (26 vs 16), but the difference was not significant , water (2.8%), bill payment (2.3%), tuition fees (1.8%), health costs (1.5%) and the remaining 5.3% on other activities and basic needs. The extra expenditure on food should translate to an improvement of the quality of the diet for the index child and the whole family.P < 0.001). However, the increment in the cash-intervention group was very much greater than that in the control group; the increment amounted to between 2.6 times for the index child’s dietary diversity to 5.3 times the control group value for the household diet diversity score. By the end of the study, 60% of the intervention group’s households had achieved a high dietary diversity, whereas only 17% of the non-intervened group had an acceptable dietary diversity at this time. Their food consumption scores mirrored these data with only 3.8% of the group receiving cash having a poor or borderline score compared to about 25% of the control group.Table P < 0.001).The comparison of the children who relapsed after recovery with those who maintained their recovery is given in Additional files To our knowledge, this is the only study to assess the direct effect of supporting households with cash during the course of treatment and follow-up of children with SAM. The analysis by intention to treat showed that the cash supplement significantly improved all aspects of treatment. Six months after admission, 80% of the children whose families were given additional support remained within the normal range of WHZ and MUAC. In contrast, less than 40% of those whose families did not receive this additional support had a good outcome; this is not only statistically significant but also biologically highly significant.For a child to develop severe acute malnutrition shows the child, and presumably the siblings and whole household, to be at particular risk of death and the other serious consequences of being severely malnourished. Follow-up of young children in a DRC community with good medical facilities, but without specific management of SAM, shows that about 5% with a WHZ < –3Z are dead within 3 months; this increases to 15 to 20% at < –4Z and 30% for those approaching –5Z –46.The parents of malnourished children need to choose between attending the health centre and all their other competing activities essential to the integrity of the household. The poorer the household, the more important each individual economic activity is, because the survival of the household is fragile and continuously at risk from even minor additional stress. If the parents consider that the treatment at the health centre is not helping, competing activities critical or the costs of attending excessive, attending will not be a priority and they will default. Thus, the defaulting rate is one measure of the quality of the service provided. Children are much less likely to recover if they default from treatment . The casDuring treatment the intervention group gained weight faster than the control group, more children recovered and fewer failed to respond to treatment. This occurred despite the same amount of RUTF being dispensed to the families and all other aspects of treatment being the same apart from the cash delivery. The question arises of how the cash delivered to the family effected this improvement in recovery from wasting. We hypothesise that it was due either to the children receiving a higher proportion of the dispensed therapeutic food or to the child getting a higher quality diet from the family pot or a combination of these factors. As most of the funds were spent on food, it is unlikely that the immediate environment (water and sanitation for example) improved over a short time or that there was significantly more health-seeking behaviour.maximum average intake of the RUTF is around 70% of the amount dispensed; the actual intake is likely to be much lower because some of the 120 kcal/kg taken by the child will come from the family food and not from the RUTF. Furthermore, if the RUTF were being taken exclusively, there would have been no other foods taken and the IDDS would have shown no diversity at all. As this was not the case, it confirms that the children were indeed consuming less than the computed 70% of the dispensed RUTF. Unrealised low compliance can be a major reason why randomised controlled trials report false negative results [We collected data on the sharing of the RUTF. The data is not presented because we judged it to be very inaccurate. The respondents from both groups reported very low levels of sharing within the family. In fact, compliance is usually poor in resource-poor settings with extensive sharing and there is an incentive for the families to exaggerate the amount taken by the child in order to ensure continued enrolment in the program , 49. The results . It is pDietary diversity is associated with a child’s nutritional status –54. GlobIn agreement with our results, CTPs in African countries and programs have reported an increase in household consumption with the majority of the additional income from the cash intervention being spent on a variety of foods with a resulting improvement of diet diversity –21. CashThe most dramatic finding of our study was the difference in the relapse rate between the children of households who received ongoing support to the end of the 6-month period and those who were simply returned ‛cured’ to their households. Relapse after discharge was the main reason for failure of the program without the cash transfer; of those admitted initially less than 40% were deemed a success after 6 months. These are the most vulnerable children in the community; after treatment they are returned to exactly the same environment and circumstances that they endured whilst becoming malnourished. Although after treatment they are older and healthier and thus more likely to demand and receive their share of the household food, they are still at high risk of relapse without continued support.The relapse rate without family support after discharge varies greatly from study to study , but is The state of food security at discharge has a decisive effect in countries with seasonality. This is elegantly shown in a study from India. Discharge when food security is low was followed by a relapse rate of 35% to MAM and 6.5% SAM; with moderate food security this fell to 29% and 3.8% and with high food security to 8.7% and 0.7% respectively . These msecure households in the same area at the same time as the control group households remained food-insecure. The difference in outcome for the two groups is clear. The intervention children continued to catch up from –1.5Z weight-for-height and MUAC for age towards the median; in contrast, the control group without this household support significantly deteriorated with a high proportion of the children relapsing.Thus, the very large differences between the relapse rates during food-insecure and food-secure times is artificially mirrored in the DRC with our study. The cash converted the food-insecure households with restricted diets and high levels of malnutrition to relatively food-inter alia differences in the types of food available, cultural practices, taboos, woman’s roles, seasonality and climate between the DRC and Niger. This raises the question of the external validity of such studies. However, it is noteworthy that IYCF was not included in the Niger study, and it is possible that such counselling affected the choices, behaviour and disbursements of the recipient households in our study to the benefit of the children.Other forms of post-discharge support may also have a beneficial impact on the further fate of the children; for example, a quasi-controlled analysis showed a better outcome of MAM with prolonged supplementary feeding . Cash maThere was no difference by group in the proportion of children developing oedema after discharge. We do not have an explanation for this observation, but it may depend upon individual nutrients, such as sulphur or particular antioxidants, being generally deficient from the foods available in this area .Our study emphasises that the protocols specifically developed for short-term relief in emergency situations may not be sufficient for use in impoverished communities in a developmental context. Having identified households with a malnourished child in the poorer sections of the society, giving short-term treatment to increase the weight of the child is appropriate to prevent imminent death, but is insufficient when the ‛cured’ children are simply returned to their original poverty-stricken households without other interventions. In this context the ‛emergency’ home treatment should be combined with ‛developmental’ support to the family that can be sustained in the longer term and lead to an improvement in their circumstances. It is unknown what happened to the children in this study when the cash transfer program ceased, because the children were not followed for logistical reasons. They may have deteriorated subsequently in the same way that the control group deteriorated without family support. Given the dramatic findings of this study, in terms of relapse, longer term follow-up should be investigated in further studies.Although the increases in food diversity and food consumption scores were much greater in the intervention group, there was also an improvement in the control group which was significantly greater than their baseline assessments. This could be due to temporal changes in the whole community; alternatively, it could be ascribed to the IYCF counselling. Whatever the cause, it is clear that IYCF counselling did not have a dramatic effect on preventing deterioration or relapse in the control group. Knowledge about IYCF by the household could not compensate for the effect of poverty on their ability to purchase higher quality foods and presumably to follow the advice given about young child feeding. However, IYCF was probably critical for the changes in the intervention group, who now had the resources to implement the advice given during counselling.It is important that distribution of cash respects the autonomy of households to decide how to best meet their own requirements. However, we expect that any significant effect on child nutritional status depends on the duration of exposure to the intervention and the amount of cash transfer received per household, per month, relative to the local costs and the local availability of high-quality food. Information on program costs, although a key indicator for public health decision-makers and program managers, was not an objective of this study. Nevertheless, it usually costs less to get cash transfers to people than in-kind assistance because aid agencies do not need to purchase, transport, store and then pay to distribute relief goods. A four-country study comparing cash transfers and food aid found that 18% more people could be assisted at no extra cost if everyone received cash instead of basic food (i.e. not including fresh foods) . In EthiIn the DRC, and other large countries with a relatively scattered population and poor transport, preventive programs like general food distribution or blanket feeding are logistically too difficult and expensive and so have never been implemented country-wide. Supplementary feeding programs for treating MAM are sporadic and not functional in most of the DRC. In this context, CTPs should be considered as an alternative to in-kind assistance and services or as a complement to more traditional interventions. Normally national protocols for treating SAM state that children who have recovered from SAM should be enrolled in a supplementary feeding program to be followed and receive a fortified food ration for at least 3 months after discharge. This provision is frequently unrealistic. The results of the present study, which shows that a cash supplement effectively prevents relapse and allows for continued catch-up, demonstrate CTP to be a viable and more easily implemented alternative to a supplementary feeding program , 69. FurMost programs for managing severely malnourished children do not automatically include a follow-up program and only report the  excellent ‛recovery’ rates. Such would have been the results of both arms of our study. By including the follow-up in the assessment of our program, we have shown that many reports can be misleading in terms of the overall success of a program. We strongly recommend that such continued support and its evaluation should be routine for all CMAM/IMAM programs in relatively stable countries.This study showed that there was no catch-up in height-for-age in either group. Thus, the program had no effect on stunting. It may be that increased rates of normal growth, indicated by a height increase, are delayed beyond the study period, as observed by Heikens et al. . NeverthIt was not possible, of course, to blind the participants to the transfer of cash into their hands. Indeed, this intervention might have been the incentive to continue participation in the study and may have affected the results by preventing defaulting which was then analysed by intention to treat so that all defaulting children were included in the analyses and, where possible, followed up with home visits. Although the health centre staff were not involved in any way with the cash transfer, it is difficult on a practical level to prevent the service personnel from becoming aware of who received the intervention and who did not.As the participants were not blinded, it is possible that the intervention group’s respondents were more disposed towards the study than those of the control group, which could have affected the answers they gave to the questionnaires. We do not think that this is likely to have biased the results, as they both reported clearly erroneous reports of RUTF sharing to the same extent, and there were similarities of the two groups’ responses at baseline. Nevertheless, it was not possible to verify the accuracy of the other questionnaire data with direct home observations. Clearly, this potential bias will not affect the anthropometric data.There is likely to have been an ascertainment bias so that the patients were not properly representative of severe malnutrition in the community, because those who were recruited by MUAC criteria were selected from community screening and those who were selected by WHZ criteria were taken from attendees at the health centre. Survey data from the DRC indicate that 32% of the severely malnourished children by WHZ have a MUAC above the cut-off point for SAM . They wiAs there is no real seasonality in this location and as the two arms were conducted concurrently, we do not consider the time of discharge as a cause of bias. The data for the non-intervened group are consistent with the reports of other programs of outpatient treatment in many other locations, and their rates of relapse are similar to those of many other reports. We therefore consider the study to have reasonable external validity for the control group. The effect of the intervention, however, is dependent upon variables such as the availability of varied nutrient-dense products on the market, reasonable market access for the participants and reasonable price stability; therefore, the external validity of the intervention arm needs to be confirmed in other contexts with differing potential effectors.This research demonstrates the benefits of cash assistance; any potential negative impacts were not considered or examined (such as the extra cash in the society increasing market prices to the detriment of the control group). However, one possible negative effect of a therapeutic treatment program is that the child will be purposefully kept malnourished in order to receive the benefit. This is not thought to be a problem in the present study, as it was made clear to the beneficiaries of the intervention arm that they would receive the cash transfer monthly for 6 months independently of the recovery rate of the child, provided that they did not default from the program. Thus, the cash is likely to have deterred defaulting (a good outcome), but there was no incentive to maintain the child in a malnourished state to continue to benefit from the program. It is possible that such an effect was present for some children in the control arm of the study; however, the mean length of stay under treatment was not different between the groups. If this did occur, it is a further benefit of the cash transfer for the intervention group.The study does not provide evidence of a greater positive effect of providing cash assistance rather than in-kind or other forms of assistance. The study estimates the impacts of cash when US$240 was delivered per household over the course of 6 months to children with SAM. The findings should not be extrapolated to different amounts or time frames. Moreover, this trial was conducted in a semi-urban area of the DRC without marked seasonal variation that had during the study a relatively poor food insecurity and a high prevalence of wasting. Therefore, our results need to be extrapolated with care and interpreted within the given context.This study shows that giving cash in impoverished communities can be effective in improving the outcome of children treated for SAM and provides a safety net that prevents relapse and allows for continued catch-up in weight and MUAC up to 6 months from admission. This very positive impact over a relatively short time on the children’s nutritional status is most easily explained by the improved access to high-quality food, enabling households not only to meet minimal needs for survival but also to diversify their diet within a society characterised by a high level of endemic malnutrition. In the DRC, where supplementary feeding interventions are logistically difficult and expensive to implement, to reduce acute malnutrition and improve its coverage, carefully designed cash transfer is shown to be a viable and highly effective intervention. Such innovative programs merit further investigation in different contexts to assess their cost-effectiveness compared to other interventions. Nutritional rehabilitation programs, often using the procedures derived directly from emergency relief operations, should always consider the feasibility of incorporating developmental programs such as micro-credit, home gardening, etc. into their procedures to enable the gains from acute nutritional intervention to be sustained.Additional file 1: Table S1.Changes in diet diversity and food consumption score between children who relapsed and recovered. (DOC 35 kb)Additional file 2: Table S2.Changes in anthropometric indicators between children who relapsed and those who did not relapse after discharge from therapeutic home treatment for severe acute malnutrition. (DOC 51 kb)


Here we report on the observation of this so-called topological magnetoelectric effect. We use monochromatic terahertz (THz) spectroscopy of TI structures equipped with a semitransparent gate to selectively address surface states. In high external magnetic fields, we observe a universal Faraday rotation angle equal to the fine structure constant α=e2/2hc (in SI units) when a linearly polarized THz radiation of a certain frequency passes through the two surfaces of a strained HgTe 3D TI. These experiments give insight into axion electrodynamics of TIs and may potentially be used for a metrological definition of the three basic physical constants.The electrodynamics of topological insulators (TIs) is described by modified Maxwell's equations, which contain additional terms that couple an electric field to a magnetization and a magnetic field to a polarization of the medium, such that the coupling coefficient is quantized in odd multiples of  et al. observe a universal Faraday rotation angle equal to the fine structure constant, evidencing the so-called topological magnetoelectric effect.The electrodynamics of topological insulators has been predicted to show a new magnetoelectric term, but this hasn't been observed. Here, Dziom  Pc(E) and magnetization Mc(B) as a function of the applied electric and magnetic fields, respectively. Soon after the theoretical prediction124Pt(B) and Mt(E) when time-reversal symmetry is weakly brokenMaxwell's equations are in the foundation of modern optical and electrical technologies. To apply Maxwell's equations in conventional matter, it is necessary to specify constituent relations, describing the polarization N is an integer and α≈1/137 is the fine structure constant. The derivation of α, when a linearly polarized electromagnetic radiation passes through the top and bottom topological surfaces691011Here θF=−4α/(1+nsub), that is, it depends on the refractive index of the substrate nsub and hence is not fundamental.In real samples, the TME may be screened by nontopological contributions1314α. Strained HgTe layers grown on CdTe, that are investigated in the present work, are shown to be a 3D TI911Here we report on the observation of the universal Faraday rotation angle equal to the fine structure constant α (for N=0) comes from two spatially separated topological surfaces in a 3D TI. This corresponds to the half-quantized Hall conductivity e2/(2h) per surface or, equivalently, to the TME occurring at each surface separately. Therefore, the observed Faraday effect 2(N+1/2)α is intimately related to the TME, which distinguishes qualitatively our 3D TI from 2D or quasi-2D materials. There is also a quantitative difference. Even without the substrate (nsub=1), the Faraday rotation in graphene would be quantized as 4(N+1/2)α, including the spin and valley degeneracies. The minimum Faraday rotation angle is then α (for N=0)Nα, where the factor of 2 comes from the equal contributions of the up- and down-spin subsystems, which independently exhibit the integer QHE. This is because in GaAs/AlGaAs heterostructures, the Zeeman splitting for magnetic fields below α (for N=1)The observed Faraday rotation angle 0.7Hg0.3Te layers (0.7Hg0.3Te layers have a thickness of 51 nm (lower layer) and 11 nm (top/cap layer), respectively. The purpose of these layers is to provide the identical crystalline interface for top and bottom surface of the HgTe films as well as to protect the HgTe from oxidization and adsorption. This leads to an increase in carrier mobility with a simultaneous decrease in carrier density compared to uncaped samples11 cm−2 and a carrier mobility of 2.2 × 105 cm2 V−1 s−1. The optical measurements are carried out on a sample fitted with a 110 nm thick multilayer insulator of SiO2/Si3N4 and a 4 nm thick Ru film. The Ru film (oxidized in the air) is used as a semitransparent top-gate electrodeThe strained HgTe film is a 58 nm thick HgTe layer embedded between two Cde layers . The Cd0v<1 THz) are carried out in a Mach–Zehnder interferometer arrangement232. Using wire grid polarizers, the complex transmission coefficient t=iφ is obtained both in parallel tp In general case, the light propagating along the iωt− time dependence is assumed for all fields. As the wavelength of 856 μm for v=0.35 THz is much larger than the HgTe layer thickness, we use the limit of thin film, and the corresponding transfer matrix σxx) and Hall (σxy) components of the conductivity tensor For normal incidence, the fields across the conducting interface are connected by the Maxwell equation c is the cyclotron resonance (CR) frequency, σ0 is the dc conductivity, and τ is the scattering time. For classical conductors, the CR frequency is written as Ωc=eB/me, where me is the effective electron cyclotron mass.Here ΩV on both sides of the sample and hence contains full information about the transmission and reflection coefficients. Thus, when ac transport properties of the HgTe layer, in accord with tp and tc based on the transfer matrix formalism as well as the exact form of the transfer matrices are presented in Methods section.The total transfer matrix e) Ωce at tp and tc indicates a high purity of our HgTe layer. The scattering time is significantly longer than the inverse THz frequency ac conductivity reveals a resonance-like behaviour σxx, σxy∝1/(ω2).Magnetic field dependence of the THz transmission is dominated by a sharp CR of surface electrons  is the energy dispersion, B is the magnetic field and A is the area enclosed by the wave vector k, we calculate for the topological surface state To understand the origin of the experimentally observed resonances, we analyse the band structure of tensile strained Cdtp and tc allows the extraction of all transport characteristics, that is, conductivity, charge carrier density, scattering time and CR frequencyExperimentally, simultaneous fit of the real and imaginary parts of μ=τΩc/B. The surface states demonstrate high mobility dc transport data. Since the e-CR and s1,s2 resonances occur at different magnetic fields, their contributions to the ac transport can be clearly separated, as presented in ac conductivity of the surface states dominates at large gate voltages. In what follows, we concentrate therefore on From the obtained scattering time and the CR positions in the magnetooptical spectra of σxy. The overall behaviour is provided by the high-field tail of the classical Drude model, that is, σxy with growing magnetic field. In addition to the classical behaviour, regular oscillations in ∂σxy/∂B can be recognized, which are linear in inverse magnetic field e2/h per surface  is provided by the classical curve of σxy can be approximated as σxy(ω)≈σ0Ωce/[(ω2)τ], which in the limit σxy=ne/B, being a multiple of e2/h. In low magnetic fields, the CR frequency Ωce becomes comparable to the THz frequency ω, destroying the regularities in σxy(ω).In magnetic fields above 5 T, the Hall conductivity clearly shows a plateau close to  surface . Anotherac quantum Hall conductivity σxy(ω) calculated from the Kubo formula for both top and bottom surface states within the Dirac model13Since in strained HgTe, the Fermi level lies in the bulk band gap see , we attr&∂;σxy/∂B. However, the model predicts much sharper transitions between the QHE plateaus, as observed in the experiment. One of two possible explanations is the heating of the surface carriers by the THz field, resulting in a higher effective temperature compared to that of the lattice. Such a heating can occur due to inefficient energy relaxation in the electronic system through the emission of LO phonons at low temperaturesT=25 K  with density fluctuations within 10% relative to their nominal values. As the fits are nearly indistinguishable, we cannot quantitatively determine the contributions of both mechanisms leading to the smearing of the THz QHE plateaus.Our two-surface Dirac model describes well the surface carrier CR  surface conductivity σa=σb≈50e2/h. Its large value indicates high-surface carrier mobility, insuring that the condition for the quantum Hall regime, B>1 T. Here τa,b are the scattering times of the top and bottom carriers, R0=h/(2e2) is the resistance quantum, and vF is the Fermi velocity of the Dirac surface states.where B>5 T is determined by the conductivity quantum G0=e2/h (Na=Nb=0), we turn to the central result of this work, the THz Faraday effect. Owing to the TME of Exeiωt− (magnetic Hyeiωt−) field of the linearly polarized THz radiation induces in a 3D TI an oscillating magnetic αHxeiωt− (electric αEyeiωt−) field. The generated, in such a way, secondary THz radiation is polarized perpendicular to the primary polarization and its amplitude is α times smaller. This can be viewed as a rotation of the initial polarization by an angle Having established that the THz response of the topological surface states in high magnetic fields ηF is shown in α for We rigorously characterize the THz Faraday effect, and the Faraday ellipticity α=e2/2hc=e2μ0c/2h is a direct consequence of the TME, confirming axion electrodynamics of 3D topological insulators. We use monochromatic terahertz spectroscopy, providing complete amplitude and phase reconstruction, which can be applied to investigate topological phenomena in various systems, including graphene, 2D electron gas, layered superconductors and recently experimentally discovered Weyl semimetals0=h/G0=e2/h are suggestede, h and c  have been carried out in a Mach–Zehnder interferometer arrangementMagnetooptical experiments in the THz frequency range  components of electric  and magnetic  fields, which may be combined in form of a four-component vector V=. The propagation of light between two points in space separated by a distance V1 and V2 can be described via 4 × 4 transfer matrix To analyse the experimental transmission spectra, we follow the formalism described by Berreman24Z=μ=1. The Berreman procedure is in general not limited to the case of normal incidence. However, in such geometry the choice of tangential field components simplifies the treatment. Electric and magnetic fields across the interfaces are connected by the Maxwell equation iωt− is assumed.Here V consists of (i) the amplitude of the linearly polarized wave (Ex) propagating in the positive direction, (ii) the amplitude of the wave with the same polarization propagating in the negative direction, and (iii) and (iv) of two waves with perpendicular polarization (Ey). The propagation matrix in the new basis is The full transfer matrix t) and reflected (r) waves, respectively. The equation connecting all waves is given by:The present experiment is described by a linearly polarized incident wave and by two components of the transmitted  and crossed (tc) polarizers are given by:To interpret the experimental data, we use the whereZ0≈377 Ohm is the impedance of free space, Z=k=σxx(ω), and σxy(ω), are given by Here As has been discussed previouslyand the influence of the substrate is minimized.The data that support the findings of this study are available from the corresponding authors upon reasonable request.How to cite this article: Dziom, V. et al. Observation of the universal magnetoelectric effect in a 3D topological insulator. Nat. Commun.8, 15197 doi: 10.1038/ncomms15197 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figure 1


We present a novel finite-time average consensus protocol based on event-triggered control strategy for multiagent systems. The system stability is proved. The lower bound of the interevent time is obtained to guarantee that there is no Zeno behavior. Moreover, the upper bound of the convergence time is obtained. The relationship between the convergence time and protocol parameter with initial state is analyzed. Lastly, simulations are conducted to verify the effectiveness of the results. In recent years, many applications required a lot of vehicles or robots to work cooperatively and accomplish a complicated task. Given this, many researchers have devoted themselves to the studies of coordination control of multiagent systems , 2. The In practical multiagent systems, each agent is usually equipped with a small embedded microprocessor and has limited energy, which usually has only limited computing power and working time. These disadvantages drive researchers to develop event-triggered control schemes, and some important achievements have been made recently –11. For Moreover, the convergence time is a significant performance indicator for a consensus protocol in the study of the consensus problem. In most works the protocols only achieve state consensus in infinite time interval, that is, the consensus is only achieved asymptotically. However, the stability or performance of multiagent systems in a finite time interval needs to be considered in several cases. The finite-time stability focuses on the behavior of system responses over a finite time interval , 18. TheRecently, there are few results reported in the literature to address finite-time event-triggered control consensus protocols for multiagent systems. To the best of our knowledge, in  the authThe main contributions of this paper can be summarized as follows: (1) a new finite-time consensus protocol based on the event-triggered control strategy for multiagent systems is presented, and the system stability is proved. The protocol is simpler in formulation and computation. (2) The lower bound of the inter-event time is gotten to guarantee there is no Zeno behavior. (3) The upper bound of convergence time is obtained. The relationship between the convergence time and protocol parameter, the initial state, is analyzed.The rest of this paper is organized as follows. In Section x, E is the Euler number (approximately 2.71828).In this subsection, we introduce some basic definitions and results of algebraic graph theory. Comprehensive conclusions on algebraic graph theory are found in . Moreoven vertices, L has always a zero eigenvalue, and 1 is the associated eigenvector. We denote the eigenvalues of L by For an undirected graph Suppose that a functionis differentiable and satisfies the conditionwhereandThenreaches zero atandfor allwhereIfandthenn agents, and the state of agent i is denoted by The multiagent system investigated in this study consists of equality . We suppWith the given protocol s solved . In addiIn the event design, we suppose that each agent can measure its own state u is held constant and can be formalized as Between events are triggered, the value of the input It is well known that the control algorithm is a piecewise constant function, and the value of the input is equal to the last control update.The protocol based on event-triggered control utilized to solve the finite-time average consensus problem is In this subsection, we study protocol . Now, weSuppose that the communication topology of a multiagent system is undirected and connected and that the triggered function is given bywhereandThen, protocol (solves the finite-time average consensus problem for any initial state. Moreover, the settling timeTsatisfiesprotocol  solves tGiven that the topology is undirected and connected, Let κ is time invariant.Therefore, Consequently, Define the measurement error as follows: Then we get Let Here we have taken the Lyapunov function By differentiating Equation  results Suppose that entclass1pt{minimaThen we get Then Combining the above formulas results in According to the triggered function of Theorem Then we get T satisfies Therefore, according to Lemma If For each agent, an event is triggered as long as the triggered function satisfies μ in the triggered function (μ is large, the allowable error is large. This means that when μ is large, the trigger frequency is low. From equation (μ is large, the convergence time is long.The role of the parameter function  is adjusfunction  we know equation  we know Note that if we set udied in . Howeverudied in  does notτ. This is proven in the following theorem.In the event-triggered control conditions, the agent cannot exhibit Zeno behavior. Namely, for any initial, the interevent times equation  are loweConsider the multiagent system (with consensus protocol (Suppose that the communication topology of the multiagent system is undirected and connected. The trigger function is given by equation (Then the agent cannot exhibit Zeno behavior. Moreover, for any initial state, the interevent timesare lower bounded bygiven bywheret system  with conprotocol . Supposeequation . Then thSimilarly to the main result in , define By differentiating Then Then we can get that the interevent times are bounded by the time as follows: From equation  we know Combining the above formulas results in μ.From equation  it is eaNote that if we set α is studied.In this subsection, the relationship between convergence time and other factors, including initial state and parameter Firstly, we study the relationship between convergence time and initial states. In the consensus problem, rather than the size of the initial states, the disagreement between states is more concerned. By definition, equation  we easilα. Supposing that Then we study the relationship between convergence time and parameter equation  we obtaiThen α becomes large, and when Letting Convergence time is defined as the amount of time the system consumes to reach a consensus. The precise convergence time of the studied nonlinear protocol is difficult to obtain. The above conclusions were obtained based on the upper bound of convergence time in equation .In this section, the simulations are conducted to verify the efficiency of the conclusions. Consider the multiagent system with mentclass2pt{minimThe trajectories of agents under protocol  when \doentclass1pt{minimaWhen entclass1pt{minimaα, α when α becomes large, and when Moreover, the relationship between the convergence time and parameter The proposed protocol can solve the finite-time average consensus problem.The lower bound of the interevent time was obtained to guarantee that there is no Zeno behavior.α becomes large, and when The larger the difference in the initial state, the longer the convergence time. Moreover, if We presented a novel finite-time average consensus protocol based on event-triggered control strategy for multiagent systems, which guarantees the system stability. The upper bound of convergence time was obtained. The relationship between convergence time and protocol parameter with initial state was analyzed. The following conclusions were obtained from simulations. In this paper, the authors only considered first-order multiagent systems. Our future works will focus on extending the conclusions to second-order or higher-order multiagent systems with switching topologies, measurement noise, time delays, and so on.


A 40-year-old male suffering from hallucinations and bizarre behavior was brought to our emergency room (ER) by the police. His drug and alcohol screens were positive for amphetamines and a blood alcohol content of 0.029 mg/dL. His past medical history was significant for alcohol use disorder, end-stage liver disease, ascites, esophageal varices, portal hypertension, and hepatic encephalopathy. He was admitted in an encephalopathic state and developed worsening hematochezia and hemodynamic instability over the course of days. Multiple investigations including contrast enhanced computed tomography (CT), upper and lower endoscopy, and mesenteric angiography did not identify a clear cause of the bleeding. Eventually, his source of bleeding was found to be from cecal varices. A transjugular intrahepatic portosystemic shunt procedure and coil embolization of the right colic and ileocolic veins stabilized the patient and he was discharged home a few days later. Cecal varices are a poorly characterized cause of gastrointestinal bleeding and are rare compared to varices in other locations in the gastrointestinal tract . They usA 40-year-old male suffering from hallucinations and bizarre behavior was brought by police to our emergency room (ER). His vitals on arrival were: temperature 36.9°C, pulse 124 BPM, respiration 20 per minute, blood pressure 104/57, and pulse oximetry 95% on room air. A urine drug screen was positive for amphetamines and his blood alcohol level was 0.029 mg/dL. His past medical history was significant for alcohol use disorder, end-stage liver disease, portal hypertension, ascites, esophageal varices, and hepatic encephalopathy. On examination, the patient was lethargic and difficult to arouse with an ammonia level of 109.5 umol/L. He was admitted for acute treatment of hepatic encephalopathy but developed hematochezia within 24 h of admission. An esophagogastroduodenoscopy (EGD) demonstrated grade II esophageal varices, which were banded, and portal hypertensive gastropathy. This seemed to resolve the hematochezia; however, two days later he had another episode of bright red blood per rectum. Sigmoidoscopy was performed, which demonstrated nonbleeding internal hemorrhoids. Over the next 36 h the patient complained of increasing lower abdominal pain and had intermittently bloody stools; however, a computed tomography (CT) scan of the abdomen and pelvis was negative for any acute changes. He then had two large, bloody stools and developed hypotension overnight; additionally his creatinine increased from 0.6 to 1.2 within 12 h. Given the intermittent nature of his gastrointestinal bleeding, a Model for End-Stage Liver Disease (MELD) score of 20 and concerns that he may have been developing hepatorenal syndrome, the gastroenterologist determined colonoscopy too risky. Instead, a tagged red blood cell scan was ordered as a less invasive modality to seek out intermittent bleeding. It showed abnormalities in the duodenum and stomach as well as bleeding from the right colon. The patient was taken to interventional radiology for a mesenteric angiogram. No active bleeding was identified; however, the portal venous phase of the superior mesenteric arteriogram did show dilated varices within the mesentery of the right colon.Given the grave prognosis, the patient decided to transition to palliative care and became no code status for four days. He continued to worsen during this time period, though he later decided he would like to transition off palliative care and after much discussion, he elected to proceed with transjugular intrahepatic portosystemic shunt (TIPS) procedure in an effort to reduce his portal hypertension in hopes of reducing his bleeding risk. Interventional radiology first recommended a triphasic CT scan to better evaluate arterial/venous anatomy relative to cross-sectional anatomy. Triphasic CT scan was performed and demonstrated varicosities throughout the abdomen with a focus of varicosities in the right lower quadrant, likely the right colon Figure . TIPS waDuring the first nine months of follow-up, the patient has had a complicated course related primarily to his chronic liver disease. He has suffered from intermittent abdominal pain and has been hospitalized or seen in clinic for lactic acidosis, bouts of abdominal pain, an incarcerated right inguinal hernia, significant scrotal edema, and methicillin-resistant Staphylococcus aureus bacteremia. He has, however, attempted positive lifestyle changes, including abstaining from alcohol and illicit drugs and improving his social support. He has had neither recurrent episodes of hematochezia nor has he suffered additional bouts of hepatic encephalopathy. The patient continues to be followed closely as an outpatient.Ectopic varices are defined as varices outside the cardio-esophageal junction . ApproxiDue to the paucity of data surrounding cecal varices, as well as the various underlying causes, diagnosis and best treatment strategies remain equivocal. Most literature currently points to selective mesenteric angiography as the ideal diagnostic approach as it may allow for precise localization of hemorrhage as well as immediate therapeutic options , 8. HoweNonbleeding cecal varices have even less available treatment data. One case reported successfully reduced cecal variceal size with the use of propranolol, but the patient could not tolerate the side effects of the medication . UltimatIn this case, the decision to embolize the right colic and ileocolic veins post-TIPS procedure was made in an urgent setting to stabilize a hemodynamically unstable patient despite the known risk of potential bowel ischemia. Post-embolization, the patient showed no evidence for bowel ischemia. We postulate that upon embolization of the right and ileocolic veins, the presence of a mesocaval shunt provided venous return from the right colon, which the pressure gradient now favored. Some authors have previously attempted mesocaval shunt procedures as therapy for small intestinal bleeding secondary to portal hypertension . This prAs demonstrated in this case, colonic varices can be a life-threatening and somewhat illusory cause of gastrointestinal bleeding. The presence of a spontaneous mesocaval shunt may allow safe embolization of the colonic varices when concerned about ischemic bowel. As demonstrated in this case, cecal varices can be a life-threatening and illusory cause of gastrointestinal bleeding. The presence of a spontaneous mesocaval shunt may allow safe embolization of cecal varices when concerned about ischemic bowel.


Disturbance is known to affect the ecosystem structure, but predicting its outcomes remains elusive. Similarly, community diversity is believed to relate to ecosystem functions, yet the underlying mechanisms are poorly understood. Here, we tested the effect of disturbance on the structure, assembly, and ecosystem function of complex microbial communities within an engineered system. We carried out a microcosm experiment where activated sludge bioreactors operated in daily cycles were subjected to eight different frequency levels of augmentation with a toxic pollutant, from never (undisturbed) to every day (press-disturbed), for 35 days. Microbial communities were assessed by combining distance-based methods, general linear multivariate models, α-diversity indices, and null model analyses on metagenomics and 16S rRNA gene amplicon data. A stronger temporal decrease in α-diversity at the extreme, undisturbed and press-disturbed, ends of the disturbance range led to a hump-backed pattern, with the highest diversity found at intermediate levels of disturbance. Undisturbed and press-disturbed levels displayed the highest community and functional similarity across replicates, suggesting deterministic processes were dominating. The opposite was observed amongst intermediately disturbed levels, indicating stronger stochastic assembly mechanisms. Trade-offs were observed in the ecosystem function between organic carbon removal and both nitrification and biomass productivity, as well as between diversity and these functions. Hence, not every ecosystem function was favoured by higher community diversity. Our results show that the assessment of changes in diversity, along with the underlying stochastic–deterministic assembly processes, is essential to understanding the impact of disturbance in complex microbial communities. Complex microbial communities and ecosystems are highly sensitive to disturbance, which can affect community diversity and structure, but to date the impact of disturbance remains difficult to predict. Here, Stefan Wuertz and colleagues from the Nanyang Technological University in Singapore show how different disturbance frequencies affect microbial population dynamics. Analyses of microbial communities in sludge bioreactors exposed to a toxic pollutant at different rates revealed that populations at the extremes (not exposed and most exposed) showed the lowest α-diversity, whereas populations exposed at intermediate levels were most diverse. Notably, ecosystem function trade-offs were observed between organic carbon removal and nitrification and biomass productivity, with diversity also affecting these functions. These observations highlight the importance of evaluating diversity when determining the effects of disturbance on microbial communities. The IDH has been influential in ecological theory, as well as in management and conservation,32 but its predictions do not always hold true.33 For example, in soil and freshwater bacterial communities, different patterns of diversity were observed with increasing disturbance frequency with biomass destruction34 and removal35 as disturbance type, respectively. Meanwhile, the effect of varying frequencies of non-destructive disturbances on bacterial diversity remains unknown. Furthermore, the IDH predicts a pattern but it is not a coexistence mechanism as it was originally purported to be.36 Hence, its relevance is being debated38 with multiple interpretations and simplicity as the main points of critique. To date, the mechanisms behind the observed patterns of diversity under disturbance remain to be elucidated.40Disturbance is defined in ecology as an event that physically inhibits, injures, or kills some individuals in a community, creating opportunities for other individuals to grow or reproduce.41 which is also known to inhibit both organic carbon removal and nitrification in sludge reactors.42 Microcosm studies are useful models of natural systems,43 can be coupled with theory development to stimulate further research,44 and by permitting easier manipulation and replication can allow inference of causal relationships45 and statistically significant results.46The objective of this work was to test the effect of disturbance on the bacterial community structure, diversity, and ecosystem function of a complex bacterial system, with emphasis on the underlying assembly mechanisms. We employed sequencing batch bioreactors inoculated with activated sludge from an urban wastewater treatment plant, in a laboratory microcosm setup with eight different frequency levels of augmentation with toxic 3-chloroaniline (3-CA) as disturbance. Triplicate reactors received 3-CA either never , every 7, 6, 5, 4, 3, and 2 days , or every day  for 35 days. Chloroanilines are toxic and carcinogenic compounds and few bacteria encode the pathways to degrade 3-CA,We analysed changes in the ecosystem function over time by measuring the removal of organic carbon, ammonia, and 3-CA, as well as biomass. Changes in community structure were examined at different levels of resolution using a combination of metagenomics sequencing and 16S rRNA gene fingerprinting techniques. Such changes were assessed by employing a combination of ordination tools, diversity indices, cluster analysis, univariate and multivariate statistical analyses. We also explored how diversity was related to function, focusing on trade-offs. Furthermore, the role of stochasticity in community assembly was investigated by employing null model techniques from ecology. We hypothesized that time would lead to a decrease in α-diversity at the extreme sides of the disturbance range due to deterministic adaptation to the environment, while less predictable conditions at intermediate disturbance levels would lead to higher α-diversity and stochastic assembly. Consequently, replicates at intermediately disturbed levels should display higher variability in terms of both community structure and function, compared with the ones at the extreme sides of the disturbance range where the opposite  should occur.P = 0.003, Supplementary Table P = 0.15). Disturbance was the factor responsible for the observed clustering .Bacterial community structure displayed temporal changes and varied between disturbance levels, as assessed by 16S rRNA gene terminal restriction fragment length polymorphism (T-RFLP) Fig. . Overalling Fig.  and not The undisturbed community (L0) was the only one with complete dissolved organic carbon (COD) removal and nitrate generation without nitrite residuals, while the press-disturbed community (L7) was the only one where nitrification products were never detected and also had the lowest biomass Fig. . InitialX component was nitrite, but some nitrate was also produced  and nitrate production (ρ = −0.697). Biomass values on day 35 differed significantly among levels with the highest value at L1 and the lowest at L7  and nitrate production (ρ = 0.656)  yielded significant similarity . Procrustes tests of comparisons within ordination methods of PCO .β-Diversity patterns observed from 16S rRNA gene amplicon T-RFLP data on day 35 were significantly similar to those from shotgun metagenomics data. A Mantel test on Bray–Curtis distance matrixes for both datasets  which give higher weight to less abundant operational taxonomic units (OTUs). They displayed the same parabolic pattern . Additionally, there were strong significant correlations between α-diversity and ecosystem function  in terms of stochastic intensity (SI) and standard effect size (SES) values, which corresponded to communities less deviant from the null expectation  and press-disturbed (L7) levels were distinct from each other as well as from the remaining intermediate levels, as supported by multivariate tests (both distance-based and GLMMs). The ordination plots and cluster analyses showed a clear separate clustering for the independent replicates of these two disturbance levels along the experiment, and particularly the constrained ordination plots displayed this with 0% misclassification error. Furthermore, the ecosystem function was clearly differentiated between L0 and L7, as well as being consistent across replicates at each level. We contend that the observed clustering is an indication that both the undisturbed and press-disturbed levels favoured deterministic assembly mechanisms, where the selective pressure due to unaltered succession (L0) or sustained toxic-stress (L7) promoted species sorting, resulting in similar community structuring among biological replicates over the course of the experiment.X products, which was initially hampered when communities were still adapting to degrade 3-CA, was not the same across all equally handled independent replicates. The observed divergence across independent replicates is considered here as a strong indicator of stochasticity in community assembly. Additionally, the lower deviation for L2–L5 from expected β-diversity values estimated via null model analysis indicates a higher role of stochasticity at intermediate disturbance levels. Several processes might be promoting stochastic assembly, like strong feedback processes51 that are linked to density dependence and species interactions,52 priority effects,53 and ecological drift.54 Reactors within this study were designed as closed systems, hence stochastic dispersal processes55 could not affect community assembly.Conversely, the communities from intermediately disturbed levels (L1–6) did not form distinct clusters for any particular level through the experiment. Within-treatment dissimilarity among replicates increased over time, with some replicates being more similar to those of other intermediate levels. Concurrently, ecosystem function parameters also displayed within-treatment variability for L1–6. For example, the conversion of ammonia to NO18 found through null model analysis that both deterministic and stochastic processes played important roles in controlling community assembly and succession, but their relative importance was time-dependent. The greater role of stochasticity we found on day 35 concurred with higher observed variability in the ecosystem function and structure among replicates for intermediately-disturbed levels. Likewise, previous work on freshwater ponds tracking changes in producers and animals49 found β-diversity (in terms of dissimilarity) increasing with stochastic processes. These observed patterns are also in accordance with ecological studies proposing deterministic and stochastic processes balancing each other to allow coexistence,10 with communities exhibiting variations in the strength of stabilization mechanisms and the degree of fitness equivalence among species.9 Thus, it is not sufficient to ask whether communities mirror either stochastic or deterministic processes,8 but also necessary to investigate the combination of such mechanisms that in turn explain the observed community structures along a continuum.9We argue that there were different underlying stochastic–deterministic mechanisms operating in the resulting community assembly along the disturbance range of our study. Similarly, a study on groundwater microbial communities31 both in terms of composition (0D) and abundances . This finding is non-trivial in two aspects. First, Svensson et al.32 have shown that most studies find support for the IDH by using species richness (0D) rather than evenness or other abundance-related indices (like 1D and 2D). They suggested that low evenness at high disturbance levels could be caused by the dominance of a few disturbance specialists. Second, the use of richness for microbial communities is not reliable48 since it is heavily constrained by the method of measurement,56 which makes it hard to compare results from different studies using this metric. Additionally, for complex communities there is often a huge difference between the abundance of rare and abundant taxa. Hence, for microbial systems, it is reasonable to assess diversity in terms of more robust compound indices rather than richness, the reason why we focused on 1D and 2D for diversity-function analyses.We observed the highest α-diversity at intermediate levels as predicted by the IDH,2D decreased over time in agreement with deterministically-dominated processes, probably because such levels represented the most predictable environments within our disturbance range. In contrast, intermediate levels either increased or maintained the same 2D over time , seemingly a case where niche overlap promoted stochastic assembly.8 The emergence of an IDH pattern after time is coherent with findings in previous microcosm studies using synthetic communities of protists57 and freshwater enrichment microbial communities.35 Yet, none of these studies evaluated the relative importance of the underlying assembly mechanisms for the observed diversity dynamics.Importantly, the observed pattern in α-diversity was time-dependent and resulted in an IDH pattern after 35 days. Temporal dynamics were expected since the sludge community experienced an initial perturbation in all reactors after transfer from a wastewater treatment plant to our microcosm arrangement. For the sludge inoculum, this implied changes in reactor volume, frequency of feeding (continuous to batch), type of feeding (sewage to complex synthetic media), immigration rates (open to closed system), and mean cell residence time (low to high). This was a succession scenario in which communities had to adapt to such changes along with the designed disturbance array. For L0 and L7, 1D and 2D were positively correlated with nitrification and productivity, suggesting that higher community evenness favours functionality under selective pressure,58 but were negatively correlated with organic carbon removal. Thus, we cannot affirm that more diverse communities have better functionality without considering trade-offs. This supports the notion that higher α-diversity does not necessarily imply a “better” or “healthier” system.56 In addition to the observed changes in OTU diversity, there was an evident variation in ecosystem function along the disturbance range studied , which are stronger at extreme ends of the disturbance range. Stochastic mechanisms will produce even assemblages (higher α-diversity) at intermediately disturbed levels, whilst infrequent or too-frequent disturbances will favour some species over others (lower α-diversity). We propose this idea as the intermediate stochasticity hypothesis  might display similar responses to disturbance. We argue that changes not only in diversity but also in the underlying deterministic–stochastic assembly mechanisms should be evaluated in studies of the effects of disturbance on such systems. For such an assessment, both replication and wide-enough disturbance ranges are key. Additionally, the ISH could be evaluated within open systems to include the effect of dispersal processes. This calls for more studies in microcosm69) included toxic 3-CA at varying frequencies. Eight levels of disturbance were set in triplicate independent reactors (n = 24), which received 3-CA every day (press-disturbed), every 2, 3, 4, 5, 6, or 7 days (intermediately-disturbed), or never (undisturbed). Level numbers were assigned from 0 to 7 , nitrogen species  and 3-CA, and volatile suspended solids (VSS). On the initial day and from the second week onwards, sludge samples (2 mL) were collected weekly for DNA extraction.We employed sequencing batch microcosm bioreactors (20-mL working volume) inoculated with activated sludge from a full-scale plant and operated for 35 days. The daily complex synthetic feed  was analysed by T-RFLP of the 16S rRNA gene using the 530F–1050R primer set targeting V4–V5 regions. The PCR program included initial denaturation at 95 °C for 10 min, followed by 30 cycles of denaturation , annealing  and extension , and final extension at 72 °C for 7 min. Purified DNA products were digested using the BsuRI (HaeIII) enzyme through incubating at 37 °C for 16 h. Enzyme inactivation was performed at 80 °C for 20 min. Digested DNA was subjected to T-RFLP on an ABI 3730XL DNA analyser. Sequence alignment files from T-RFLP runs were assessed for quality control and pre-processed using the software GeneMapper v.5 (Applied Biosystems).71 Peak areas were normalized to the total area per sample72 and de-noised using a conservative fluorescence threshold of 200 units.73DNA extracted from all sludge samples (n = 24) were subjected to metagenomics sequencing at the SCELSE sequencing facility (Singapore). Library preparation was performed according to Illumina’s TruSeq Nano DNA Sample Preparation protocol. Libraries were sequenced in one lane on an Illumina HiSeq 2500 sequencer in rapid mode at a final concentration of 11 pM and a read-length of 250 bp paired-end. Around 173 million paired-end reads were generated in total and 7.2 ± 0.7 million paired-end reads on an average per sample. Illumina adaptors, short reads, low quality reads or reads containing any ambiguous base were removed using cutadapt .74 Taxonomic assignment of metagenomics reads was done following the method described by Ilott et al.75 High quality reads (99.2 ± 0.09% of the raw reads) were randomly subsampled to an even depth of 12,395,400 for each sample prior to further analysis. They were aligned against the NCBI non-redundant (NR) protein database (March 2016) using DIAMOND (v.0.7.10.59) with default parameters.76 The lowest common ancestor approach implemented in MEGAN Community Edition v.6.5.577 was used to assign taxonomy to the NCBI-NR aligned reads with the following parameters: maxMatches = 25, minScore = 50, min Support = 20, paired = true. On average, 48.2% of the high-quality reads were assigned to cellular organisms, from which in turn 98% were assigned to the bacterial domain. Adequacy of sequencing depth was corroborated with rarefaction curves at the genus taxonomy level . We did not include genotypic information as it was outside the scope of this study, but will do so in future investigations arising from this work.Purified genomic DNA from sludge samples on d0 (inoculum) and d35  of 10%.78 Community structure was assessed by a combination of ordination methods  and multivariate tests 79 on Bray–Curtis dissimilarity matrixes constructed from square-root transformed normalized abundance data using PRIMER (v.7). Additionally, GLMMs, which deal with mean–variance relationships,80 were employed using the mvabund R-package81 fitting the metagenomics dataset to a negative binomial distribution, to ensure that the observed differences among groups were due to disturbance levels and not heteroscedasticity. The 500 most abundant genera  were employed to ensure random distribution of residuals fitted in the model. Significance was tested using the anova function in R with PIT-trap bootstrap resampling (n = 999).82 Hill diversity indices83 were employed to measure α-diversity as described elsewhere,84 and calculated for normalized non-transformed relative abundance data.All reported 85 were applied to compare metagenomics and T-RFLP datasets from all bioreactors on day 35 . Such an approach is valid for the questions asked in this study, since comparisons between NGS and fingerprinting techniques support the use of T-RFLP to detect meaningful community assembly patterns and correlations with environmental variables,61 and such patterns can be validated by NGS on a subset of the fingerprinting dataset.2Mantel and Procrustes tests86 All these statistical tests were performed using the vegan R-package .Bray–Curtis dissimilarity matrixes were computed using square root transformed T-RFLP data and bacterial genus-level taxa tables generated using a metagenomics approach. Mantel tests were then used to determine the strength and significance of the Pearson product–moment correlation between complete dissimilarity matrices. Procrustes tests (PROTEST) were also employed as an alternative approach to Mantel tests in order to compare and visualize both matrices on PCO and NMDS ordinations. The resultant m2-value is a statistic that describes the degree of concordance between the two matrices evaluated.87 which assumes that species interactions are not important for community assembly.88 We employed a null model approach originally applied to woody plants50 and more recently to microbial communities.18 The model defines β-diversity as the β-partition 89 and takes into account both composition and relative abundances. To adapt it to handle microbial community data, we considered species as OTUs (genus taxonomic level) and each individual count as one read within the metagenomics dataset. The model randomizes the location of each individual within the three independent reactors for each of the eight disturbance treatment levels, while maintaining the total quantity of individuals per reactor, the relative abundance of each OTU, and the γ-diversity. We applied it to the metagenomics datasets from d0 and d35.To disentangle the roles of stochastic and deterministic processes as drivers of change in β-diversity it is necessary to incorporate a statistical null model in the analysis,βobs) minus the mean of the null distribution of β-diversity values σexp), SES = Each step of the null model calculates expected mean α-diversities for each disturbance level and then estimates an expected β-partition. After 10,000 repetitions, the mean and standard deviation of the distribution of random β-partitions for each disturbance level are calculated. The output of this model is a β-deviation or SES, which is the observed β-diversity (Further information on experimental design is available in the Supplementary informationReporting Summary


Radiodermatitis is a painful side effect for cancer patients undergoing radiotherapy. Irradiation of the skin causes inflammation and breakdown of the epidermis and can lead to significant morbidity and mortality in severe cases, as seen in exposure from accidents or weapons such as “dirty bombs” and ultimately leads to tissue fibrosis. However, the pathogenesis of radiodermatitis is not fully understood. Using a mouse model of radiodermatitis, we showed that the Transient Receptor Potential Melastatin 2 (TRPM2) ion channel plays a significant role in the development of dermatitis following exposure to ionizing radiation. Irradiated TRPM2-deficient mice developed less inflammation, fewer severe skin lesions and decreased fibrosis when compared to wild type mice. The TRPM2-deficient mice also showed a faster recovery period as seen by their increased weight gain post irradiation. Finally, TRPM2-deficient mice exhibited lower systemic inflammation with a reduction in inflammatory cytokines present in the serum. These findings suggest that TRPM2 may be a potential therapeutic target for reducing the severity of radiodermatitis. Radiation therapy is commonly used to treat several types of cancer  , placed in an X-ray irradiator box and shielded with lead so that only the lower pelvic region was exposed to radiation for five consecutive days at a dose of 8 Gy/day. This dose regimen was chosen because it mimics the radiation therapy regimen of a patient being treated for pelvic cancers  was purchased from Sigma-Aldrich (cat # C6019). CTZ was first solubilized in ethanol (50 mg/ml), further diluted in corn oil to reach a 1% CTZ containing solution, and filter sterilized using membranes of 0.22 µm nominal pore size. Wild-type C57BL/6 male mice were treated topically with this solution. CTZ is clinically very well characterized as it is used as an anti-fungal agent, and its topical application is not toxic. The solution (or ethanol/corn oil as a vehicle control) was applied twice a week on affected skin areas starting as soon as lesions would appear (4 weeks after irradiation).The skin lesions were recorded and scored 12 weeks after irradiation for quality as described in (E.J. 2012) (0) normal, (1) erythema, (2) dry desquamation, (3) wet desquamation, and (4) ulceration  for 5 min and rinsed before differentiating 15 min in 2.5% phosphomolybdic-2.5% phosphotungstic acid. Finally, collagen was stained for 8 min in 2.5% aniline blue, rinsed, and differentiated for 1 min in 1% acetic acid. Sections were dehydrated through graded alcohols, cleared in xylenes, and mounted with Permount .Trichrome stained sections were imaged in brightfield mode, with a 20× objective, on a Leica DM4000 B LED microscope . To measure the collagen density in the skin, each section was imaged over the length of the section requiring ten evenly spaced fields of view. Using ImageJ software, the region of interest  was selected so that only the area containing collagen was included in the analysis. Next, thresholding was used to select only blue pixels (collagen) and excluded purple/red pixels (immune cells and keratin); white hues were excluded to eliminate holes in the tissue. The collagen density was calculated as the number of pixels representing collagen divided by the total number of pixels in the region of interest (ROI). The percent area of tissue comprised of collagen was averaged for each animal and the mean per group reported.The epidermal layer thickness was quantified using the trichrome staining images. For each image, approximately 20 equally spaced measurements were made along the length of the tissue by drawing a line from the junction of the dermis and epidermis to the edge of the epithelial layer. The pixel value was converted to microns using a factor of 3.84 pixels/micron. A mean epidermal thickness was calculated for each animal using all images containing epithelium.Serum was separated using Z-Gel microtubes (Sarstedt), clotting blood for 30 min at room temperature, and serum removed by centrifugation (8000 rpm for 5 min). Meso Scale Discovery V-Plex Proinflammatory Panel 1 (mouse) plates were used according to the manufacturer’s instructions.2O2 in PBS, followed by a 5-min wash. Next, slides were blocked in 10% goat serum for 30 min followed immediately by 1 h incubation in primary antibody. Primary antibodies included CD68 , CD3 , and TRPM2 . Negative stain controls were incubated in blocking buffer without primary antibody for 1 h. Following several washes in PBS, the sections were next incubated in biotinylated goat anti-rabbit secondary antibody  then washed in PBS again. Peroxidase activity was associated to the biotinylated secondary antibody using the Vector Labs ABC Kit (cat. PK-4000) by incubation for 30 min in ABC buffer. Finally, DAB substrate was applied to detect the proteins of interest  for 5–7 min until the brown color was visible under a microscope. Slides were counterstained by briefly dipping in Harris hematoxylin , then dehydrated through graded alcohols, cleared in xylenes, and mounted with Permount solution.Tissue sections were deparaffinized in xylenes and rehydrated in graded alcohols, then rinsed in running deionized water. Antigen retrieval was performed by boiling slides in 10 mM sodium citrate buffer, pH 6.0 for 20 min, followed by a 20-min cool down, and a 10-min PBS wash. Endogenous peroxidases were quenched for 5 min in 3% HFor both CD3 and CD68 in skin, six random fields of view were captured with a 20× objective, on a Leica DM4000 B LED microscope. Cells staining a deep brown color were manually counted using the “multi-point” function in ImageJ software. The average cells per field were reported and used for statistical analysis. For TRPM2, serial sections were stained for TRPM2, CD68 and CD3 to determine if TRPM2 expression co-localized in lymphocytes and macrophages.t test was used when only two groups were compared. Differences were considered statistically significant when p < 0.05.Data are expressed as mean ± SEM. One-way analysis of variance was used for multiple comparisons, and Tukey’s post hoc test was applied where appropriate. Student’s −/− male mice were irradiated for five sequential days with 8 Gy/day in the pelvic region. This radiation scheme was used previously in rats  and TRPM2−/− mice up to 5 weeks post-irradiation, TRPM2−/− mice recovered and gained weight steadily from 6 to 12 weeks post radiation whereas the WT mice never recovered from their initial weight loss  WT or TRPM2nce Fig. b. We alsnce Fig. c.−/− mice 4 weeks post irradiation. In accordance with our previous observations, TRPM2−/− mice had reduced inflammatory cytokines, including IL-1β, IL-6 and KC as compared to WT mice  following irradiation of WT mice. As illustrated in Fig. −/− mice are protected from skin damage and overall weight loss associated with lower abdominal radiation exposure. Furthermore, histological analysis of skin lesions showed that TRPM2-deficiency protected the tissue from irradiation-induced damage by limiting the inflammation and the development of fibrosis in irradiated skin. Finally, we showed that TRPM2−/− mice had significantly lower circulating inflammatory cytokines and lower leukocyte recruitment, but apical inhibition of TRPM2 had no effect on radiation-induced dermatitis. Taken together, these data suggest that TRPM2 deficiency is protective against radiation-induced skin damage and helps preserve the function of this organ.In this study, we have demonstrated that TRPM2-deficiency decreases the severity of various side effects associated with radiation exposure. Specifically, we have shown that TRPM2−/− skin lesions showed less infiltration of inflammatory cells as well as decreased levels of systemic inflammatory cytokines, specifically IL-1β, IL-6 and KC. TRPM2 is known to promote inflammation and cytokine production in various situations anthranilic acid inhibits TRPM2 (Kraft et al. 2 inhibitor (Chen et al. Several compounds have been shown to inhibit TRPM2 currents. For instance, as stated previously, we used clotrimazole to see if we could prevent radiation-induced skin injury by apically blocking TRPM2. Other compounds such as 2-aminoethoxydiphenyl borate (Togashi et al. Radiodermatitis is a serious side effect due to radiotherapy to treat many types of tumors found throughout the body, which can lead to the delay of therapeutic treatments. Furthermore, the skin is the first organ that would be affected in a nuclear accident or “dirty bomb” detonation and as such exposed to whole body irradiation. However, given that our understanding of the inflammatory pathways involved in radiodermatitis is still limited, we currently do not have an effective treatment for controlling damage to the skin. Our results emphasize the importance of TRPM2 in mediating radiation-induced inflammatory responses and suggest TRPM2 as a potential target when considering therapeutic interventions for radiodermatitis.