Antibiotic overuse is the main modifiable driver of antibiotic resistance. Factors associated with overuse have been inconsistently reported and vary across populations. Given the burgeoning occurrence of infectious diseases around the world, there remains a great need to identify barriers and solutions to the control of infections. We examined whether knowledge about infections and antibiotic resistance is associated with antibiotic use in a northern European population sample.> 16 years. Participants were asked whether they had taken an antibiotic in the past 12 months; and six questions were asked concerning knowledge about infections and antibiotic resistance. Correct answers to the six knowledge questions defined a knowledge score (score range 0–6 correct answers). We used multivariable logistic regression to estimate odds of self-reported antibiotic use during the last 12 months in association with knowledge score , and response to each knowledge question. Covariates included sex, age group, smoking, alcohol drinking, deprivation index, self-rated health, and satisfaction with life. Results were outputted as Odds Ratios (OR) and 95% Confidence Intervals (CI).The Health Survey Northern Ireland 2014/15 was completed by a cross-sectional sample of 4135 participants aged p < 0.001), with a P-value < 0.001 for trend with increasing knowledge score. Female sex, age, high deprivation, and poor general health, were independently associated with higher odds of antibiotic use. Stratified analyses showed sex and age group differences.Antibiotic use in the past 12 months was reported by 39.0% (1614/4135); and 84.2% (3482/4135) scored < 6/6 correct on knowledge statements. Compared to the lowest knowledge score (0/6 correct), the highest knowledge score (6/6 correct) was associated with higher odds of antibiotic use (adjusted OR 2.03, 95% CI , Knowledge, and other modifiable and non-modifiable risk factors, were positively associated with antibiotic use in the past 12 months. While the causal direction of these associations could not be determined, given the high prevalence of lesser knowledge, as well as independent contributions of other factors including socioeconomic characteristics, health literacy campaigns to raise awareness of antibiotic resistance should take a multi-pronged approach.The online version contains supplementary material available at 10.1186/s12889-021-11018-x. Antimicrobial resistance (AMR) is a global crisis with serious consequences for the individual, public health, and the economy . It has Global commitment and multi-sector partnerships are critical to implement effective interventions to reduce unnecessary use of antibiotics and control AMR , 6. GlobThe World Health Organization’s (WHO) global action plan on AMR has five objectives, the first of which is to improve knowledge and understanding of AMR through communication, education and training . NationaOne model targeting behavioural change related to AMR suggests that knowledge, environmental change, social/professional roles, social influences, and public beliefs and priorities, among others, are important factors. Thus, multipronged public health interventions are necessary to address AMR and to foster behaviour change . QualitaGiven this backdrop, we took advantage of a big data resource – the Health Survey Northern Ireland 2014/15 (HSNI14/15) – to examine the association between knowledge regarding infections and AMR and antibiotic use. Since multi-prong interventions are necessary to decrease AMR, HSNI data allowed us to consider sex, age group, sociodemographic and behavioural factors, deprivation, and self-rated general health and satisfaction with life. These factors have been previously associated with knowledge and antibiotic use –27. GiveThe HSNI is a series of independent, cross-sectional surveys conducted annually since 2010 by Northern Ireland Statistics Research on behalf of the Northern Ireland Department of Health, Social Services and Public Safety. The HSNI examines a wide range of public health issues that are important for the people of Northern Ireland. In this analysis, we utilised data from HSNI14/15, conducted between April 1, 2014 and March 31, 2015. HSNI14/15 consisted of a random sample of 5850 street addresses that were representative of the general population in Northern Ireland . A vacanEligible households were sent a letter of invitation via post. The letter stated that the survey was being conducted on behalf of the Department of Health, Social Services and Public Safety, Central Survey Unit, which is the leading social survey research organisation in Northern Ireland. The letter also stated that an official interviewer would visit the household to explain more about the study and that the survey could be rescheduled if necessary. Non-response occurred when an eligible address was not reached by an interviewer or when respondents refused to participate. To minimise non-response, an interviewer attempted up to four calls to each eligible household unless the respondents had previously refused to participate.HSNI14/15 surveyed knowledge of infections, antibiotics and antibiotic resistance, and use of antibiotics over the past 12 months. Additionally, the survey collected sociodemographic, behavioural , and general health factors information .The study protocol of HSNI14/15 was approved by Northern Ireland Department of Health. Informed consent was provided when the interviewer visited the household. Participants were able to refuse to participate at any stage of the survey . No inceSurvey data from HSNI14/15 were collected using Computer-Assisted Personal Interviewing (CAPI). CAPI is a face-to-face interviewing method. HSNI14/15 interviewers visited the respondents with a computer, read the questions to the respondents, and entered the responses immediately on the computer during the course of the interview. If a participant did not want to engage in a CAPI interview, they were offered a self-administered, closed booklet version of the HSNI14/15 that contained the questions and response options. Responses were manually entered later .Our primary exposure and outcome variables were located in the ‘Medication’ section of the HSNI14/15. Within this section, there was a subsection dedicated to antibiotics. This section was comprised of seven questions.The answer to the first question in the ‘Medications’ section, subsection ‘Antibiotics’, was the primary outcome used in our analysis. The first question was ‘In the past 12 months, have you taken an antibiotic?’ Response options included: ‘Yes’, ‘No’ or ‘Don’t know’ .The following six questions in the section on ‘Medications’, subsection ‘Antibiotics’, were used as the primary exposure. These six questions assessed knowledge about infections, antibiotics and antibiotic resistance Table . Each quTo examine how knowledge about infections, antibiotics and antibiotic resistance was associated with antibiotic use in the past 12 months, the knowledge variable was considered as a correct answer to each individual statement , as well as a composite knowledge score. The knowledge score was the sum of answers to all six statements. The score range was 0–6, where a score of 0 corresponded to incorrect answers on all 6 statements and a score of 6 was a correct answer to all statements.We selected potential covariates and confounders based on published associations with knowledge, behaviour, and awareness and antibiotic use , as wellDemographic characteristics: included sex  and age group  [Health behaviours: included current cigarette smoking and alcohol drinking [drinking . CurrentSociodemographic factors: included education attained and income [d income . A deprid income  was usedGeneral health status: Self-rated general health and life satisfaction were components of the HSNI [the HSNI . Self-raParticipant characteristics and knowledge about infections and antibiotic resistance were described using frequencies and percentages. The Pearson’s chi-squared test was used to test for associations between categorical variables.To examine the association between the exposure, knowledge about infections and antibiotic resistance and the outcome, self-reported antibiotic use over the past 12 months, weighted logistic regression analyses were used with ‘no reported use’ as the referent. Sampling weights were used to estimate robust standard errors in both unadjusted and adjusted models. The HSNI14/15 sample was weighted by sex and age using the Northern Ireland 2014 mid-year population estimates. Odds ratios (OR) and 95% confidence intervals (CI) were estimated.The primary exposures were: 1) a correct answer to each of six knowledge statements, and 2) a composite knowledge score derived as the sum of correct answers to the six knowledge statements about infections and antibiotic resistance. Knowledge scores range from 0 to 6 with a score of 0 representing 0 correct answers to all 6 statements, and a score of 6 representing 6 correct answers to all 6 statements. First, we ran six models estimating whether the correct answer to each individual statement was associated with antibiotic use in the past 12 months. Second, using the sum of correct answers to the six statements regarding knowledge about infections and antibiotic resistance, we estimated whether knowledge score (maximum score 6/6 correct answers) was associated with antibiotic use in the past 12 months when compared to a lower knowledge score (score < 6/6 correct answers).p < 0.10 in univariate logistic analyses were included in multivariable models. Multivariable models were later stratified by sex and age group . Results were considered statistically significant at p < 0.05 using two-sided tests. All statistical analyses were performed using Stata 16.0.Univariate logistic regression analyses were followed by multivariable analyses. Sex, age group, smoking and alcohol drinking, deprivation index, self-rated general health, and satisfaction with life, were evaluated a priori as potential covariates in the multivariable models. Potential covariates with a n = 72) had missing data for questions comprising knowledge about infections and antibiotic resistance, self-reported antibiotic use, current cigarette smoking status, drinking status, self-rated general health, satisfaction with life or sample weight, and were excluded. This left 4135 participants included in our analysis. The majority of these HSNI14/15 participants were women (58.9%), older than 25 years (94.0%), current non-smokers (79.2%), and current alcohol drinkers (75.3%). There was a relatively equal distribution of participants across deprivation quintiles.Of 5850 randomly selected street addresses, 981 were ineligible since the address was vacant, leaving an effective sample of 4869 households. Among these households, 28.6% (1394 of 4869 households) refused to participate and 8.1% (392 of 4869 households) were not reached. Thus, 63.3% (3083 of 4869) of households completed the survey, with some households providing data from more than one resident. As a result, HSNI14/15 data provided by the UK Data Service included 4207 individuals aged 16 years and older . Of these 4207 participants, 1.8% (n = 1614). Women were more likely to have used an antibiotic compared to men . Participants who provided correct answers to the six statements regarding knowledge about infections and antibiotic resistance, which comprised our primary exposure, are presented in Table n = 334) of participants responded ‘don’t know’ to all six questions.Among all 4135 participants, use of an antibiotic in the past 12 months was self-reported among 39.0% (p’s < 0.10), except alcohol drinking (p = 0.321). Therefore, alcohol drinking was not included in multivariable-adjusted models.HSNI14/15 participant characteristics by self-reported antibiotic use in the past 12 months are presented in Table p < 0.001) of antibiotic use associated with a correct answer to the statement ‘Antibiotics are used to treat bacteria’ and 19% higher odds  of antibiotic use with a correct answer to the statement ‘Chances of resistant bacteria increase if you don’t finish the course’. A breakdown of knowledge score (the sum of correct answers to six statements about infections and antibiotic resistance (range 0–6)) by number correct answers showed that 8.5% (N = 353) of participants had 0/6 correct answers, 2.8% (N = 115) had 1/6 correct answers, 3.8% (N = 160) had 2/6 correct answers, 21.6% (N = 891) had 3/6 correct answers, 24.6% (N = 1011) had 4/6 correct answers, 23.0% (N = 952) had 5/6 correct answers and 15.8% (N = 653) had 6/6 correct answers.The association between knowledge of infections and antibiotic resistance and antibiotic use was estimated using weighted odds of antibiotic use in the past 12 months predicted by knowledge defined by a correct answer to each individual statement, as well as knowledge score Table . Evaluatp for trend < 0.001), with adjustment for sex, age, current smoking, deprivation quintiles, and self-rated general health and satisfaction with life. However, knowledge was not the only characteristic associated with higher odds of antibiotic use. Women compared to men  and those reporting poor versus good general health  were more likely to report using an antibiotic in the past 12 months. Those aged 45–64 years compared to those age 16–24 years  and those comprising the 5th deprivation quintile (the least deprived) compared to the referent 3rd deprivation quintile  were less likely to report antibiotic use.Estimates of the odds of antibiotic use in the past 12 months associated with our primary exposure, knowledge score and each characteristic, were accomplished using weighted multivariable-adjusted models and are presented in Table Sex and age group stratification illustrated some nuances to these data. Among both women and men, the highest knowledge score (6/6 vs. 0/6 correct answers) was associated with 2 times higher odds of antibiotic use in the past 12 months; and poor self-rated health was linked to higher odds of antibiotic use. However, deprivation index was a characteristic associated with antibiotic use among men only, and when compared to the middle deprivation quintile, men comprising the least deprivation quintiles exhibited 42% lower odds of antibiotic use  HSNI14/15 participants comprised the largest proportion  with lesser knowledge about infections and antibiotic resistance, as has been previously reported  years HS. YoungerIn HSNI14/15, there is some evidence of a gradient of association between deprivation and antibiotic use. Compared with the 3rd deprivation quintile, the odds of antibiotic use in the past 12 months were not different among the most deprived (3rd and 2nd deprivation quintiles), but were lower among the least deprived (4th and 5th deprivation quintiles). NIMDM deprivation index is a composite of income, employment, health deprivation and disability, education, skills and training, proximity to services, living environment, and crime and disorder, with consideration for home address. Many studies have shown that individuals comprising lower socioeconomic classes tend to be less knowledgeable about infections and antibiotic resistance compared with individuals in higher socioeconomic classes . While wPoorer self-rated general health was also associated with higher odds of antibiotic use in HSNI14/15. This may have been observed for several reasons, including: 1) co-occurring physical disease or co-morbidities, some of which may increase the risk of a bacterial infection ; 2) psycOur analysis has several strengths. The HSNI14/15 includes a representative, population-based sample of more than 4000 participants, across a wide age range. Self-reported data were collected using well-validated scales and state-of-the-art CAPI methods without reliance on population-based prescribing statistics; and the calculated deprivation index is comprehensive and informative. In addition, multivariable analyses allow evaluation of the association between knowledge and antibiotic use after controlling for potentially relevant confounders, lending to the robustness of our result. Given the relatively few studies in this field, these data are highly valuable and fill a gap in the literature. Despite these strengths, as with many large-scale population-based surveys, there are limitations. First, the HSNI uses a serial cross-sectional design comprised of independent samples. Despite the agreement of our results with other published studies, we cannot determine causality, i.e., whether knowledge influences behaviour (antibiotic use) or vice versa. Second, HSNI data are self-reported, thus, there is inherent recall bias. Regarding the face-to-face efficiency of CAPI, underreporting is more frequent in face-to-face interviewing of sensitive, stigmatized or unpopular behaviours . This riHSNI14/15 data illustrate that there was a positive cross-sectional association between knowledge about infections and antibiotic resistance and antibiotic use. However, we also observed a number of common modifiable and non-modifiable risk factors associated with both self-reported antibiotic use and basic knowledge of treatment of common infectious diseases and antibiotic resistance. These factors represent potential ‘at risk’ groups and thus, allow the creation of targeted campaigns that are critical for increasing knowledge in general, supporting informed choices, and raising the public’s health literacy. In addition, future research should prospectively examine whether knowledge about infections, antibiotics and antibiotic resistance predicts quantity or frequency of antibiotic use; as well as identify optimal types of information and methods of information dissemination that reduce antibiotic overuse. It is of critical importance that future prospective studies include measures of knowledge and attitudes, as well as dedicated measures of health literacy.Additional file 1.Additional file 2.


The Saving Lives at Birth (SL@B) funding partners joined in 2011 to source, support, and scale maternal and newborn health (MNH) innovations to improve maternal and newborn survival by focusing on the 24 hours around the time of birth. A multi-methods, retrospective portfolio evaluation was conducted to determine SL@B’s impact. Forty semi-structured, key informant interviews (KIIs) were conducted with experts in global MNH based in low- and middle-income and in high-income countries to assess the SL@B program. KIIs were conducted with global MNH technical experts, innovators, government officials in low- and middle-income countries, donors, private investors, and implementing partners to include the full spectrum of voices involved in identifying and scaling innovations. Data were analyzed using thematic analysis. Stakeholders believe the SL@B program has been successful in changing the way maternal and newborn health programs are delivered with a focus on doing things differently through innovation. The open approach to sourcing innovation was seen as positive to the extent that it brought more interdisciplinary stakeholders to think about the problem of maternal and newborn survival. However, a demand-driven approach that aims to source innovations that address MNH priority needs and takes into account the needs of end users  was suggested as a strategy for ensuring that more innovations go to scale. Progress on reducing maternal and newborn mortality has been inadequate. Far too many women and newborns die in the 24 hours around birth, the majority in low- and middle-income country (LMIC) settings –3. ThereThe Saving Lives at Birth (SL@B) program launched in 2011 as a Grand Challenge for Development recognizing that making progress on the Millennium Development Goals (MDGs) related to maternal and newborn survival would require going beyond traditional approaches and that innovation could help leapfrog toward progress . The SL@The SL@B Grand Challenge was designed to crowd-in innovations from a broad array of innovators and support them to reach integrated impact and scale through funding for science and technology, service delivery, and demand generation at the seed, validation, and transition to scale levels. One unique aspect of the SL@B program has been the creation of a “community of innovators” who can rely on each other for support in developing global MNH innovations. The cornerstone of this effort is the DevelopmentXChange (DevX), an annual event traditionally held at USAID headquarters in Washington, D.C. that attracts SL@B innovators, SL@B finalists, and potential scaling partners for SL@B innovations. Innovators compete for SL@B funding at the DevX event and are evaluated by a diverse review panel of MNH experts and investors in order to screen in the most innovative and investable ideas. Each year, SL@B attracted 500+ applications and selected about 60 finalists who competed for between 10–20 awards. Since 2011, the SL@B program has funded 116 unique innovations in eight rounds including 25 innovations that have received multiple rounds of SL@B funding . All participants provided written informed consent.Key informants who were familiar with the SL@B program (n = 30 out of 40) were asked about the pros and cons of an open versus targeted approach to identifying and funding innovation, which is a hallmark of the SL@B program. KIs were asked to think about the relative merits of an open versus targeted approach to identifying innovative ideas and funding different types of organizations  and innovations.Roughly, two-thirds of KIs preferred a targeted approach to soliciting key areas for innovation, where SL@B would drive the innovation agenda on maternal and newborn survival by setting priorities together with LMIC stakeholders. The rest preferred an open approach or had no opinion either way. KIs who were in favor of an open approach thought that this design could bring innovative ideas to the field that would have been impossible to target in advance. KIs who were in favor of a more targeted approach thought that the SL@B platform could be used to focus innovation on priority areas of identified need in maternal and newborn survival. KIs suggested that the needs could be identified in collaboration with LMIC stakeholders: governments, clinicians, civil society organizations, etc. to ensure innovations tackle priority areas and have a higher chance of going to scale.I think we should first have an assessment, then identify special critical areas, and then focus on [those]. Otherwise, even if we have innovations, we will end up without a significant impact after spending all that time and resources. There are areas which we really, really need immediate solutions for in newborn and maternal issues. So, in conclusion, I am for a focused approach personally.”“–LMIC-based MNH expertOverall, most KIs were agnostic about the best type of organization for SL@B to fund . Many commented that no one institution on its own is going to be able to drive change and thus a combination of organizations is needed. Most KIs thought that rather than focusing on the type of organization funded, the innovation should be the focus. If an innovation addresses a critical problem on the ground, is affordable, easy to use, and culturally responsive, then KIs thought that the type of innovator who generated the idea is irrelevant.I think the idea is that innovations should really be recognized that they can come [from anywhere]. No one institution or individual has a monopoly on that, but it’s just that once you find something that works, then how do you make sure the most number of people can benefit from it? At that stage is where you really need to make sure you are building on existing systems, making sure the right people are involved, making sure that people are mobilized around this. It basically reaches that critical mass and it’s very difficult to do when it’s just a single initiative driving that forward.”“–HIC-based MNH expertA few KIs noted that although universities might have advantages in terms of conducting research and development, they may require more support and guidance when it comes to testing and scaling innovations on the ground.There’s credit in working with universities, but again I think it’s facilitating those on the ground connections that are absolutely critical for their successes [which] is sometimes lacking.”“–HIC-based MNH expertAcross the SL@B portfolio, product-based innovations  represent 61 percent of all innovations (n = 88). Innovations classified as drugs/ vaccines (n = 16), mHealth (n = 16), and practice and approach (n = 15), are evenly represented, each making up about ten percent of the portfolio overall. When KIs were asked about where SL@B fits in the innovation-funding ecosystem, the majority were keen to point out that SL@B seems to focus on funding devices. However, some KIs expressed concern that devices alone would not be sufficient to improve maternal and newborn survival and that SL@B should be intentional about sourcing integrated solutions that include a wide range of innovations.One MNH expert observed:But again, it depends on the strength of the systems in that case–you could have the best innovation the world, but if you don’t have a strong [health] system to implement it—then, that’s just not going to work.”“–HIC-based MNH expertThe majority (83%) of awards in SL@B’s portfolio went to organizations based in HICs. KIs were asked to react to this composition and to suggest what could be done to increase LMIC representation, if it were desirable. Almost all KIs (n = 34) weighed in on this question. Two-thirds of KIs emphasized the need to have a more balanced innovation portfolio from the Global South and North. KIs perceived that increased LMIC representation might lead to a more sustainable, cost effective, user friendly, and, most importantly, problem solving innovations in the MNH space.I think there should be a better balance for sure. You know, people from high-income countries are trying to develop innovations that they think would be appropriate for low-income countries and sometimes they are successful and sometimes they are not. People from low-income countries know exactly what the situation is, and they know what the possibilities are, and what the constraints are; so, they might design with those constraints in mind to come up with solutions to work with those constraints. So I definitely think that there should be a better balance.”“–LMIC-based MNH expertOn the other hand, almost a quarter of KIs thought that as long as the innovations that are developed align with needs on the ground, the composition from LMICs is irrelevant.There’s no problem if most of the innovators are from the high-income countries, there is no problem, but the problem will be—do they really understand the real problem in the low resource setting; that will be a challenge. … So to get a solution, in general, be it from HICs or from LMICs, as long as it solves the problem of maternal and child health care, there’s no problem.”“–LMIC-based MNH expertMany KIs hypothesized that LMIC representation in the SL@B portfolio is low due to lack of awareness of the call for proposals and the competition against HIC innovators who may be more skilled at writing grants, especially in English. To address this gap, several KIs suggested that SL@B should work closely with local ministries, USAID Missions, and LMIC-based NGOs to announce the call. A few KIs suggested that SL@B could source ideas through pre-proposal accelerator workshops or regional events that identify local problems and local innovators, which might include partnerships with HIC innovators and institutions.Most KIs thought that the DevX and community of innovators that it has created is one of the major contributions of SL@B. KIs noted that DevX provides a platform like no other that enables innovators to cultivate partnerships that may facilitate market entry and scale, and critical peer-to-peer learning from innovators facing similar challenges (e.g. regulatory hurdles). Most KIs from LMICs thought that SL@B should hold a regional DevX event to enable broader participation from innovators in LMICs who may have trouble obtaining a visa for travel to Washington, D.C., where DevX has traditionally been held.I was amazed to hear the number of innovations going on. I even thought, before even going to DC [for DevX], we could be having a platform or a forum in Africa where the partners, the grantees like us, in Africa, we just meet in the region to share with one another about what we are doing.”“–LMIC- based InnovatorKey informants were asked their opinion on the major achievements of the SL@B program. Twenty KIs out of the total forty KIs (largely representing the MNH experts group) felt they had enough information to respond to this question. The most frequently mentioned achievement was raising awareness for innovation in maternal and newborn survival.Without SL@B, we may not be talking about maternal and newborn survival and innovations; [I think SL@B] tapped attention that would not otherwise have happened.”“- HIC-based InvestorSL@B was also commended by half of the KIs for encouraging and enabling more ideas, new approaches, and unlike minds to work on maternal and newborn survival, including funders from the public and private sector.I think the fact that anyone is putting innovation up the flagpole and getting some public-private partnerships under way, and getting players that typically wouldn’t be at the table to the table to innovate and to join hands in the world of maternal and newborn health is hugely important and a great value add.”“–HIC-based MNH expertFinally, a third of KIs specifically mentioned that SL@B has generated a pipeline of innovative ideas that can be taken to scale, which can lead to a broader impact.What SL@B is doing is unique, especially by identifying areas that should be taken as priority for MNCH work and then focus on those priority areas, and just build a robust pipeline of innovations and innovators that can actually be taken to scale. I think where the issue is, is about choosing well, choosing these interventions well by focusing on those ones that have got the highest propensity to succeed.”“–LMIC-based MNH expertKIs were asked to consider their own opinion and describe specific innovations they saw as success stories. Their responses reflected their own interpretation of SL@B’s success which may reflect how much public attention an innovation has received rather than widespread scale. Specific innovations most frequently cited as success stories by KIs included the rapid scale-up of the use of chlorhexidine for umbilical cord care in Nepal , which SWhile the vast majority of KIs indicated that SL@B has been successful in raising awareness for innovation in maternal and newborn survival, two areas emerged as unrealized potential of the SL@B program. First, many KIs mentioned that, while there have been a few success stories, innovations funded by SL@B often lack scaling plans and have not been integrated into health systems at scale.KIs hypothesized that lack of scaling success may be due to unclear exit strategies for SL@B-funded innovations, an issue that emerges early in the problem identification and sourcing stage. For example, KIs mentioned the following areas as possible explanations for lack of scale: lack of partnerships with bilateral cooperative agreements for validation and scale, lack of funding partners at later stages identified from the early stages, and a feeling that the current MNH ecosystem is not supportive of innovation, and that the work needed to be done may be beyond the grant-making process and should take a more ecosystem building approach.A number of KIs expressed concern that SL@B innovations have not reached widespread scale from when the program began (2011) until the time of this study (2019). One participant reflected on the pace of change in MNH through innovation in general:[That innovative new drug] was another one of the very first round awardees, wasn’t it? So, I think it’s just sobering to see how long it takes [to get an innovation to scale].”“–HIC-based MNH expertSecond, KIs perceived a mismatch between the areas SL@B innovators address and the areas of priority need for MNH innovation in LMICs. KIs expressed their opinion that the innovations funded within the SL@B portfolio do not always address the areas of most need and that the role of SL@B should be, as expressed by one KI, to future-cast the key areas where innovation is needed, by taking a proactive rather than passive approach to the call for innovations so that problems drive the areas for innovation. Several KIs suggested that SL@B could define areas for MNH innovation in collaboration with LMIC partners and design their call for application accordingly.I think the extent of the innovation conversation needs to go further to the frontline… at the end of the day, the people who are living with the problem, I would say are the best litmus test for the solution. I think engaging the global health experts is important, but this is not always about expertise, it is also about understanding the problem in the context. … just creating that ecosystem requires the community from the very beginning, not only at the recipient end, once you have everything signed off.”“–LMIC-based MNH expertAccording to the KIs interviewed, the SL@B program has successfully generated a pipeline of new, early stage ideas that have the potential to save and improve maternal and neonatal lives . StakehoThe findings in this study echo the growing literature on the factors that contribute to successfully scaling innovations in LMIC settings –21. InnoThe qualitative insights from this evaluation of the SL@B program have implications for future MNH-focused innovation programs. Engaging stakeholders in LMICs before issuing a call for innovative ideas could result in a higher probability that MNH innovations, once tested and proven effective, will be more readily adopted by LMIC decision-makers/end users, integrated into health systems, and reach widespread scale. Innovation funding programs should continue to strive for a balanced portfolio of innovators from LMIC and HIC settings, but the primary foci should still be on whether the innovation itself has the potential for health impact in priority areas, whether it resonates with local end users, and if it is scalable and sustainable within resource-limited LMIC settings. To ensure that the portfolio is balanced, SL@B could consider holding a DevX-like event in LMIC settings (e.g. East Africa) and connect LMIC innovators with pre-application support. Other funders could also adopt a similar strategy to engage local stakeholders and provide additional support to make LMIC innovators more competitive in an application process. One unexpected finding from this evaluation was how little disagreement among stakeholders from different perspectives there was on the key questions of how to balance LMIC and HIC voices in sourcing innovations and on the key role of SL@B in shedding light on innovation as a way to improve MNH.This qualitative study of the SL@B program has several limitations. The MNH innovation space has grown considerably in the last decade, but we only included SL@B-funded innovators as KIs, and not non-SL@B innovators in the space. Also, this study includes perspectives from government and implementation partners across Kenya, Ethiopia, Uganda, Nepal, and global, high-level experts from HICs, but does not include individuals from every country where SL@B innovations have been deployed. However, within this small sample from areas where SL@B has a large presence, convergence of themes was apparent. This study included 40 interviews which is well over typical benchmarks for qualitative data saturation . One samEight years of funding innovation through the SL@B program has raised awareness of innovation in MNH and brought new players from the public and private sector to the MNH ecosystem. The open process of sourcing innovative ideas has been driven by innovators rather than identified needs and priorities based on local demand in LMICs. The study highlights the need to make innovation-funding programs more demand-driven so that they provide the most benefit for end users and have a higher likelihood of adoption and wide scale.S1 File(DOCX)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(PDF)Click here for additional data file.S4 File(PDF)Click here for additional data file.


Methionine (Met) deficiency in the diet can limit milk protein production and lead to excessive nitrogen (N) excretions to the environment by dairy cows. We demonstrated that the supplementation of a new rumen-protected Met product to a Met deficient diet increased milk protein yield and decreased manure N excretions of high producing dairy cows. Increased blood flow to the mammary glands and increased apparent total tract digestibility of dietary crude protein seem to be the underlying mechanisms for those improvements in production and the environmental sustainability.p < 0.040) without affecting DMI. Milk protein yield increased by 50 g/d for the 19.3 vs. 0 g/d dose (p = 0.006) but the rate of increment decreased for 27.5 g/d dose. Plasma Met, and MPF increased linearly with RPM dose (p < 0.050). Apparent total tract digestibility of crude protein (p = 0.020) and FN (p = 0.081) decreased linearly with RPM. The UN did not change but total manure N decreased linearly with RPM (p = 0.054). The RPM (19.3 g/d) seemed to help cows overcome the metabolizable Met deficiency while mitigating manure N excretions to the environment.The study objective was to determine the effects of rumen-protected methionine (Met) by microencapsulation (RPM) on amino acid (AA) supply to the udder, milk production, and manure nitrogen (N) losses of dairy cows. A corn and soybean-based diet deficient in metabolizable Met (~10 g/d) was supplemented with RPM providing 0, 11.0, 19.3, and 27.5 g/d of Met. Dry matter intake (DMI), milk production, plasma essential AA (EAA), mammary plasma flow (MPF), and fecal (FN) and urinary N (UN) outputs (g/d) were determined. The RPM increased linearly milk yield, milk protein yield, and energy corrected milk yield ( Methionine (Met) is one of the most limiting amino acids for lactating dairy cows in the US . When su®, Vetagro S.p.A, Reggio Emilia, Italy) on AA supply to the udder, milk production, and manure N losses of high producing dairy cows fed a corn and soybean-based diet.A typical lactating cow diet in the US is based on corn and soybean and supplies 25% less metabolizable Met than the requirement predicted by current nutrient requirement models . Therefoad libitum amount of TMR (110% of previous day intake), which was delivered once daily (0700 h). Each RPM dose mixed in 100 g of ground corn was top-dressed separately on the basal diet of individual cows immediately after the diet was delivered in the morning. Feed allowance and refusals were recorded daily. Cows were milked three times per day at 0400, 1200, and 2000 h.All procedures were approved by the Institutional Animal Care and Use Committee IACUC: 18-091) of Iowa State University . Forty-eight multiparous Holstein cows  were used in replicated 4 × 4 Latin square design including four treatments and four 28 d periods. In each period, cows within a square were assigned randomly to one of four dietary treatments including a total mixed ration (TMR) serving as the control diet (CON), and CON supplemented with 20 (LM), 35 (MM), or 50 (HM) g/cow per day of microencapsulated DL-Met  providing 11.0, 19.3, and 27.5 g of Met to the diet, respectively. The CON was related to a predicted . In conjunction with above model, the linear, quadratic, and cubic contrasts for the relationships of RPM dose with each response variable were analyzed. The contrast coefficients were obtained using the IML procedure of SAS. Statistical significance of the treatment effects and the contrasts was declared at p ≤ 0.05. The tendencies were declared at 0.05 < p ≤ 0.10.Data were analyzed with the MIXED procedure of SAS  using the following model.In this study, we supplemented graded levels of rumen-protected Met by encapsulation (RPM) to a corn and soybean meal-based diet of high producing cows. The objective was to determine the impact on milk production performance, AA supply to and uptake by the mammary glands, and manure N excretions. The RPM contains DL-Met (55%) and lipids (40%). Therefore, 20.0 (LM), 35.0 (MM), or 50.0 (HM) g/d doses provided 11.0, 19.3, and 27.5 g of Met, and 8.0, 14.0, and 20.0 g of lipids to the basal diet (CON), respectively. The predicted  MP suppln = 48) are presented in p = 0.388). Milk yield had a linear relationship with RPM dose (p = 0.026) and increased from 38.1 kg/d for CON to 39.7 kg/d for HM (p = 0.019). Mordenti et al. [p = 0.113). Nonetheless, milk protein yield responded strongly to RPM dose  for MM providing 19.3 g/d of Met to the diet. The current National Research Council model [p = 0.541) suggests that MM might have corrected the deficiency and helped cows meet the Met requirement. Milk fat and lactose percentages did not change with RPM supplementation (p > 0.370). Fat yield did not also have a relationship with the RPM dose (p ≥ 0.193). On the other hand, lactose yield tended to increase linearly with it . The ECM increased by 1.57 kg/d from CON to HM representing a 4% increase (p = 0.037). Milk urea nitrogen concentration remained unchanged at 13.54 mg/dL highlighting that whole-body N catabolism and urinary N output [p ≥ 0.404) indicating no effect of metabolizable Met supply on the mobilization of fat , the mobilization of protein , or the net effect of those two processes on BW and BCS.The means and the relationships of DMI, milk yield, and milk components with RPM dose . We assumed the coccygeal blood would be a reasonable alternative to external pubic arterial blood for studying the arterial EAA concentrations, AVd, and mammary EAA uptake of lactating dairy cows as demonstrated by Zhang et al. [p < 0.001). The LM and HM providing 11.0 and 27.5 g/d of Met to the diet was associated with a 14 and 34% increase in arterial Met relative to CON, respectively (p ≤ 0.019). Because the availability of free AA in the small intestine can positively influence the blood concentrations [p < 0.050), whereas branched-chain amino acid concentration remained unchanged with increasing RPM dose. Although the reason is obscure, Zhou et al. [Arterial plasma concentrations of EAA presented in g et al. . The arttrations ,38, artetrations . Therefou et al.  also obsp > 0.230) indicating an increased utilization of arterial Met by the mammary glands  in response to the RPM supplementation. However, net mammary uptake of Met increased linearly with RPM dose (p = 0.028) likely because of mammary plasma flow that linearly increased with RPM dose (p = 0.043). The literature does not provide much information on relationships between blood flow and graded Met supplementations. Guinard and Rulquin [p = 0.035) and tryptophan (p = 0.027) that decreased linearly with the RPM dose. However, net mammary uptake of valine or tryptophan was not affected by RPM dose possibly because the uptake was also a function of mammary plasma flow that increased with RPM dose. Signifying further the impact of plasma flow, net mammary uptake of lysine, phenylalanine, and arginine increased linearly (p ≤ 0.045) even though AVd did not change with the RPM dose (p > 0.155). Net mammary uptake of Lys and Arg were not correlated with arterial concentrations  but the plasma flow  highlighting the significance of blood flow in supplying precursors for the synthesis of milk. Although a linear decrease in metabolizable lysine: Met could be anticipated with increasing RPM supplementation, the ratio between the mammary uptake of lysine and Met remained similar at 3.9 across the treatments  suggesting a marginal impact of the differences in the ratio between absorbed Met and Lys at mammary gland level. The net mammary uptake of EAA increased for LM compared to CON (p = 0.012), decreased for MM compared to LM (p = 0.051), and increased again for HM compared to CON (p = 0.051) exhibiting a cubic relationship with RPM dose. The net mammary uptake represents the utilization of EAA for milk protein synthesis, tissue protein accretion, and catabolism [p = 0.029) with RPM dose indicating a decreased AA utilization efficiency for milk protein synthesis when AA supply  increased. Similarly, Nichols et al. [The arterio-venous differences (AVd), net mammary uptake, and mammary plasma flow are presented in  Rulquin  report a Rulquin  did not e p = 0.0. The littabolism . The EAAs et al.  observedy p ≤ 0.0 even thon = 8) providing AA data discussed above. Similar to DMI (p > 0.310). Fecal N output (g/d) tended to decrease linearly with the RPM dose (p = 0.081). The linear decrease in fecal N became more significant when it was expressed as a percentage of N intake (p = 0.020) highlighting an improvement in apparent total tract digestibility of dietary CP for the RPM supplementation. The literature provides limited data for relationships between the CP digestibility of dairy cows and graded levels of dietary RPM. In previous studies, the CP digestibility has been assessed very often for a single dose of RPM and shown to be unaffected. Nonetheless, the data on other animal species, such as rats, pigs, poultry, and fish, indicate Met supplementation can enhance the digestion and absorption of proteins by improving small intestinal morphology including the villus height [p > 0.235). It is fair to acknowledge the lack of representativeness of urine volumes (L/d) and N outputs (g/d) determined with creatinine concentrations of spot urine samples as highlighted in Lee et al. [p = 0.054). The linear decrease in manure N output became further prominent (p = 0.006) when it was expressed as a percentage of N intake. Manure N decreasing relative to N intake represents an increased efficiency of the utilization of dietary protein, which is an expensive nutrient accounting for 42% of the total dietary nutrient cost of dairy cows [p = 0.015). Milk protein efficiency (MPE) expressing milk protein yield as a percentage of dietary crude protein intake had a positive but quadratic relationship with RPM dose  while MPE of MM also remained numerically greater than that of CON . According to White [p = 0.009). Cows without RPM were related to a negative N balance of 71.8 g/d that decreased to 4.6 g/d for MM (p = 0.011). Considering together the milk protein yield  to a diet deficient in metabolizable Met (~10 g/d) increased linearly milk yield, milk protein yield, and energy corrected milk yield without affecting DMI. Milk protein yield increased by 50 g/d for the dose providing 19.3 g/d of Met to the diet but the rate of the increments declined for further supplementation of RPM. Arterial Met, arginine, lysine, tryptophan, and plasma flow to the udder increased linearly with RPM dose. Net mammary uptake of EAA increased linearly with RPM dose possibly due to the increased mammary plasma flow because the AVd did not respond significantly to RPM dose. The RPM dose decreased linearly the percentage of dietary N excreted in feces indicating an improved apparent total tract digestibility of dietary crude protein. Despite the unaffected urinary N output, manure (feces + urine) N output decreased linearly while milk protein efficiency increased quadratically with RPM dose. Cows without RPM had a negative N balance, which started to become positive at RPM dose providing 19.3 g/d of Met to the diet. Overall, the present rumen-protected Met product providing 19.3 g/d of Met to the diet seems to help cows overcome the metabolizable Met deficiency of about 10 g/d.


MET assessment for patient stratification. In this study, we aimed to determine the molecular context in which MET is deregulated in CC using a series of genomic and proteomic tests to define MET expression and identify patient subgroups that should be considered in future studies with MET‐targeted agents. To this aim, orthogonal expression analysis of MET was conducted in a population‐representative cohort of stage II/III CC patients (n = 240) diagnosed in Northern Ireland from 2004 to 2008. Targeted sequencing was used to determine the relative incidence of MET R970C and MET T992I mutations within the cohort. MET amplification was assessed using dual‐colour dual‐hapten brightfield in situ hybridisation (DDISH). Expression of transcribed MET and c‐MET protein within the cohort was assessed using digital image analysis on MET RNA in situ hybridisation (ISH) and c‐MET immunohistochemistry (IHC) stained slides. We found that less than 2% of the stage II/III CC patient population assessed demonstrated a genetic MET aberration. Determination of a high MET RNA‐ISH/low c‐MET IHC protein subgroup was found to be associated with poor 5‐year cancer‐specific outcomes compared to patients with concordant MET RNA‐ISH and c‐MET IHC protein expression (HR 2.12 [95%CI: 1.27–3.68]). The MET RNA‐ISH/c‐MET IHC protein biomarker paradigm identified in this study demonstrates that subtyping of MET expression may be required to identify MET‐addicted malignancies in CC patients who will truly benefit from MET inhibition.Clinical trials for MET inhibitors have demonstrated limited success for their use in colon cancer (CC). However, clinical efficacy may be obscured by a lack of standardisation in  MET is deregulated in colon cancer using a series of genomic and proteomic tests. This study demonstrates the limited number of genetic MET aberrations present in a Northern Irish colon cancer cohort and identifies a cancer‐specific poor prognosis MET RNA/c‐MET protein subgroup that may be amenable to targeted therapy.A population representative study looking at the molecular context in which  CCcolon cancerCIconfidence intervalCRCcolorectal cancerCSScancer‐specific survivalDDISHin situ hybridisationdual‐colour dual‐hapten brightfield Epi700a population‐representative Northern Irish colon cancer cohortFFPEformalin‐fixed paraffin embeddedHRhazard ratioIHCimmunohistochemistryISHin situ hybridisationMSImicrosatellite instability1Globally, colorectal cancer (CRC) is the third most common cancer contributing to nearly 10% of all new instances of cancer and cancer‐related deaths . The relMET, a proto‐onocogene located on chromosome 7q31.2, has been linked with both lack of phase III clinical trial efficiency and treatment refractory disease following EGFR tyrosine kinase inhibition  and visualised with chromogenic detection using DAB . RNA‐ISH stained slides were digitised and regions of interest in the tumour epithelium digitally evaluated using the assisted scoring software halo . Digital image analysis enabled precise quantitation of RNA‐ISH tissue data which included the total tumour area and the total probe area within the core. Nuclear segmentation and cell detection were not conducted in this study as sample pre‐treatment required by the automated RNAscope assay led to loss of distinct cellular morphology in the tissue. Hence, we calculated an additional value for analysis representing the number of probe signals per micrometre squared of tumour tissue. This score was created by dividing the values for the total positive probe area by the total tumour area. Similar to c‐MET IHC protein expression analysis, the median probe area across the three TMA cores was taken to generate a MET RNA‐ISH score per patient.The Leica Bond RX automated immunostainer (Leica Biosystems) was used to carry out RNA‐ISH using the RNAscope assay to produce robust staining . RNA‐ISH® Benchmark XT automated immunostainer (Roche Diagnostics). DNA‐ISH staining was conducted using the dual‐colour dual‐hapten brightfield ISH method (DDISH) for evaluation of MET amplification using the MET DNP Probe  and Chromosome 7 DIG Probe  as per manufacturer's instructions. Chromogenic detection of the MET probe was visualised with silver ISH DNP detection  while CHR7 probe was visualised with red ISH DIG detection . The ratio between the black MET and red CHR7 probe signals was determined by manual light microscopy at 400× magnification. Twenty tumour cells were evaluated, and an average score was determined in potentially amplified cores. The amplification cut‐off was set at a MET/CHR7 ratio of three. Each patient was designated as MET amplified or nonamplified via DDISH, if one of the three TMA cores was determined as amplified during DDISH evaluation.DNA‐ISH was carried out on the Ventana2.3r version 3.6.1 . Kaplan–Meier plots and log‐rank P values were used to illustrate the 5‐year cancer‐specific survival (CSS) between dichotomised low and high MET patient groups. CSS was the time between diagnosis and death specifically caused by CRC as determined by ICD cause of death codes C18, C19, C20 and/or C26. Data were right‐censored for patients with incomplete survival information and in patients with greater than 5‐year survival. Univariate and multivariate analyses were conducted using the Cox proportional hazard method to generate hazard ratios (HR) with 95% confidence intervals (CI). Multivariable models were adjusted for age, sex, UICC TNM stage, MSI status and whether adjuvant chemotherapy was received. Sensitivity analysis of multivariable models was conducted on this dataset by left‐censoring patients with a follow‐up of 6 months or less. Association of continuous and categorical variables between groups was assessed using either ANOVA or Pearson's chi‐square test for independence when appropriate. Scatter plots were used to visualise the relationship between orthogonal MET biomarker assays and correlations reported using Spearman's Rho.All statistical analysis was performed in MET in relation to survival within a retrospective, population‐representative cohort of CC. The reporting standards of the current study fulfil these recommendations.This study was conducted in accordance to REporting recommendations for tumour MARKer prognostic studies (REMARK) , 20. The33.1MET expression analysis was carried out in 240 of the original cohort (32.43%)  was therefore assumed to be representative of the retrieved population‐representative, Northern Irish, Stage II/III CC cohort (n = 661) in terms of age, sex, stage, MSI status and chemotherapy administration in the current study.Orthogonal 3%) Fig. . Full cl3.2MET amplification, MET mutation, MET RNA‐ISH and c‐MET IHC protein expression levels were assessed across the patient cohort as described. Variable MET RNA‐ISH and c‐MET IHC protein expression levels were observed within the cohort  of the study cohort with a c‐MET and CHR7 ratio of 4.03 . The patient with MET amplification were found to cluster near patients with increased levels of both MET RNA‐ISH and c‐MET IHC protein expression, while no clustering was observed in patients with MET mutations  patients while nonconcordance was observed in 32 (13.33%) and 35 (14.58%) of the study cohort in patients with low MET RNA‐ISH, high c‐MET IHC protein expressing tumours and in high MET RNA‐ISH and low c‐MET IHC protein expressing tumour, respectively . Whereas no significant difference in survival was seen in patients with nonconcordant low MET RNA‐ISH, high c‐MET IHC protein expression compared to patients with concordant MET RNA‐ISH and c‐MET IHC protein expression . Sensitivity analysis confirmed that the relative risk of mortality was not influenced by cancer‐specific deaths of 6 months or less in the study cohort .Multivariable analysis found nonconcordant py Table . Patient4MET pathway is frequently dysregulated in cancer and acquired genomic aberrations can lead to treatment refractory disease with EGFR tyrosine kinase inhibitors [MET expression in CC through use of orthogonal technologies for MET quantification in a population‐representative cohort. This study demonstrates that c‐MET overexpression arises most often due to a relative increase in RNA expression in stage II/III CC. Importantly, this study identifies subgroups of patients with discordant MET RNA‐ISH/c‐MET IHC protein expression who may benefit from dual testing with RNA‐ISH and IHC and that should be considered in future clinical trials using MET inhibitors.The hibitors , 7. MET hibitors . Therapehibitors . Both gehibitors , 13. It hibitors . We therMET genomic aberration affected less than 2% of stage II and stage III CCs diagnosed in Northern Ireland, with MET amplification and mutation independently occurring in 0.42% and 1.25% of patients. MET RNA‐ISH and c‐MET IHC protein expression was found to demonstrate a moderate‐positive relationship in the absence of genetic aberration influencing relative c‐MET expressed from the tumour. In patients with genetic aberrations present, MET amplification was found to be associated with increased MET RNA‐ISH and c‐MET IHC protein expression, while MET mutations appear to randomly influence overall c‐MET expressed. However, the number of patients with MET aberrations present in this cohort are too small to draw firm conclusions from the data. In a third of patients assessed for MET expression analysis, there was a lack of agreement in dichotomised low and high groups representing MET RNA‐ISH and c‐MET IHC protein expression, with nonconcordance demonstrated in 27.91% of tumours assessed. Of these, only patients with high MET RNA‐ISH/low c‐MET IHC protein expressing tumours were found to be twice as likely to die of a colorectal‐specific death in 5 years. These patients represented 14.58% of the overall study population. No significant difference in survival was observed in patients with low MET RNA‐ISH/high c‐MET IHC protein expressing tumours compared to tumours with concordant MET RNA‐ISH/c‐MET IHC protein expression. However, these patients represent 13.33% of the study population who exhibit evidence of enhanced dimerization with tyrosine kinase receptors and upregulated production of the c‐MET IHC protein. Importantly, concordant high MET RNA‐ISH/c‐MET IHC protein expressing tumours demonstrate no significant survival compared to the low MET RNA‐ISH/c‐MET IHC protein expressing patients. This demonstrates that while MET is dysregulated in these patients, it is unlikely to be the oncogenic pathway driving tumorigenesis in those patients. Rather, it is the patient subgroup with high MET RNA‐ISH/low c‐MET IHC protein expressing tumours that would most likely benefit from MET inhibition.This study found that the incidence of MET genomic aberrations. MET amplification was only found to have an incidence of 0.4% when assessed in stage II/III CC, which was significantly lower than the 1.9–2.2% reported elsewhere [MET amplification in large studies were reported on metastatic CRC only [et al. [MET amplification was more likely to occur in patients with metastatic disease and may contribute to the lower than expected incidence of MET amplification present in the current study. Further, incidence of MET mutation reported in the current study was also significantly below the expected 2–5% demonstrated in the literature [MET mutation instead of whole genome sequencing restricted the number of MET mutations that could be called to two single point mutations. Our findings of 1% incidence in MET R970C and T992I point mutations are in line with Tyner et al. [MET exon 14 skipping in the current study, and however, this has been previously shown to not occur in CRC [This study found lower than expected incidence of lsewhere , 24, 25.CRC only . Supportterature . This war et al.  who lookr in CRC .5MET gene amplification or MET R970C and T992I point mutations, nonconcordant patterns of MET expression are associated with 5‐year CSS in CC. The impact of MET expression subgroups on efficacy of MET inhibition was not considered in the current study design and warrants investigation in future studies.In conclusion, MET inhibitors are used to target c‐MET expressing tumours. Through use of population research, this study demonstrates that in the absence of genomic aberration via either Dr MS‐T has recently received honoraria for advisory work in relation to the following companies: Incyte, MindPeak, QuanPathDerivatives and MSD. He is part of academia‐industry consortia supported by the UK government (Innovate UK). Dr JAJ is also part of the academia‐industry consortia supported by the UK government (Innovate UK). These declarations of interest have no relationship with the submitted publication. All other authors declare no competing interests.All authors contributed to interpretation of data and writing of the manuscript. RHW, SVS, JAJ and MS‐T were involved in study conception and design. SGC, SM, MPH, VB, AVP and SMcQ contributed to data acquisition. SGC, SM and VB were involved in data analysis.https://publons.com/publon/10.1002/1878‐0261.13089.The peer review history for this article is available at


Aconiti Lateralis Radix Praeparata (Fuzi) as an example, we adopted a systems pharmacology approach to investigate the mechanism of Fuzi in treating rheumatoid arthritis and in inducing cardiac toxicity and neurotoxicity. The results showed that Fuzi has 25 bioactive compounds that act holistically on 61 targets and 27 pathways to treat rheumatoid arthritis, and modulation of inflammation state is one of the main mechanisms of Fuzi. In addition, the toxicity of Fuzi is linked to 32 compounds that act on 187 targets and 4 pathways, and the targets and pathways can directly modulate the flow of Na+, Ca2+, and K+. We also found out that non-toxic compounds such as myristic acid can act on targets of toxic compounds and therefore may influence the toxicity. The results not only reveal the efficacy and toxicity mechanism of Fuzi, but also add new concept for understanding the toxicity of herbal medicines, i.e., the compounds that are not directly toxic may influence the toxicity as well. In recent decades, herbal medicines have played more and more important roles in the healthcare system in the world because of the good efficacy. However, with the increasing use of herbal medicines, the toxicity induced by herbal medicines has become a global issue. Therefore, it is needed to investigate the mechanism behind the efficacy and toxicity of herbal medicines. In this study, using  In addition, compounds isolated from herbal medicines are important resources of lead compounds in drug discovery area. For example, it is reported that 63% of anticancer small molecular drugs approved by the US Food and Drug Administration (FDA) are directly or indirectly come from herbal medicines2. Recently, herbal medicines have played significant roles in fighting against COVID-19 before the wide application of vaccines4. Predictably, herbal medicines will play more and more important roles in fighting against diseases.With the usage history over hundreds of or even thousands of years, many complementary and alternative medicines harbor rich clinical experiences. Because of the high safety and rich clinical experiences, the use of complementary and alternative medicines such as traditional Chinese medicines has increased globally over the past decades. Herbal medicines are an important resource of complementary and alternative medicines, and they play important roles in the current medical system. Reports published by the World Health Organization (WHO) showed that about 75–80% of the world population, especially those in developing countries, rely primarily on herbal medicines for healthcareAristolochia and Asarum. Since the 1990s, aristolochic acids-induced nephropathy and upper tract urothelial carcinoma have been reported in countries such as Belgium, UK, France, Japan, and China5. In 2012, the WHO cancer agency the International Agency for Research on Cancer (IARC) classified aristolochic acids as group 1 human carcinogens6. Gradually, the use of aristolochic acids-containing herbal medicines is forbidden in many countries. Except for the herbal medicines that contain aristolochic acids, there are many herbal medicines with strong toxicity that are still used in the clinic. This is because the toxicity of these herbal medicines can be eliminated with careful use. However, the reason an herbal medicine can lead to both efficacy and toxicity is not fully known.Even though herbal medicines have been demonstrated to be effective on many diseases, the toxicity caused by herbal medicines has become a global issue. For example, aristolochic acids are a group of herbal compounds that widely exist in the plant genus Aconiti Lateralis Radix Praeparata (Fuzi) is the typical herbal medicine with conspicuous efficacy and strong toxicity. Fuzi, also known as Chinese wolfsbane, Chinese aconite, monkshood, Kyeong-Po Buja, and Bushi, is the processed daughter root of Aconitum carmichaeli Debx. As one of the most well-known herbal medicines, Fuzi has been extensively used for over 2 thousand years in clinics to treat diseases7. In the clinic, Fuzi is commonly used to treat a wide variety of diseases, such as rheumatoid arthritis, acute myocardial infarction, low blood pressure, coronary heart disease, chronic heart failure, tumors, skin wounds, depression, diarrhea, gastroenteritis, and edema8. Although Fuzi has shown wide and promising therapeutic effects, its toxicity has been recognized by ancient people and has attracted widespread attention all over the world in the past decades. From 2001 to 2010, 5000 cases of acute toxicity of Fuzi were recorded in countries such as China, Japan, and Germany9. From 2004 to 2015 in mainland China, at least 40 cases of fatal Fuzi poisoning with 53 victims were recorded10. The main toxicity of Fuzi includes cardiotoxicity and neurotoxicity, and the typical symptoms of Fuzi poisoning include arrhythmia, palpitation, hypotension, shock, dizziness, coma, vomiting, and nausea11. Although the potent therapeutic effects and strong toxicity of Fuzi are well recognized, it is now still not fully understood that which compounds are responsible for the efficacy and toxicity, and the mechanism differences of Fuzi in inducing efficacy and toxicity.Among all herbal medicines, 12. The traditional wet experiment-based approach is rather difficult to identify all the bioactive compounds and their mechanisms. Fortunately, with the development of systems biology, systems pharmacology has emerged as an efficient tool to study the mechanism of herbal medicines based on the complex status of the biological system of the human body13. For this reason, it has been extensively and successfully used to study the mechanisms of herbal medicine such as Morinda officinalis14. In this work, we adopted a standard systems pharmacology approach to screen the compounds that are responsible for the efficacy and toxicity of Fuzi in treating arthritis and inducing toxicity, respectively , a systems pharmacology platform that is specialized for traditional Chinese medicines15. Considering that gut microbiota can convert glycosides in the intestinal tract by gut microbiota16, the corresponding aglycones were also included in the compound library of Fuzi. To screen the potential bioactive compounds from Fuzi, two important parameters including oral bioavailability (OB) and drug-likeness (DL) that are associated with drug absorption, distribution, metabolism, and excretion were used. The threshold for screening bioactive compounds was set as OB ≥ 30 and DL ≥ 0.1817. Because some compounds that did not pass the threshold might show significant pharmacological effects as well, compounds that were reported to exhibit strong pharmacological effects yet not meet the threshold of OB or DL were also used.All the compounds and molecular structures of Fuzi were retrieved and downloaded from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform 18. Similarity Ensemble Approach 19, TCMSP (http://ibts.hkbu.edu.hk/LSP/tcmsp.php) 15, Therapeutic Targets Database  20, PhID (http://phid.ditad.org/MetaNet/)21, and Swiss Target Prediction  22. It is noteworthy that only the targets related to Homo sapiens were reserved for further analysis. P-value less than 0.05 were used for screening of targets in on-line serves and database where applicable. The other parameters for target screening were defaultly settled, for example, Score cutoff with 20 was used for BATMAN-TCM.To screen the whole targets of bioactive compounds in Fuzi, a chemometric method and information integration approach were used. First, the screened bioactive compounds were submitted to various on-line servers and databases, including Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine . Compound structures of coryneine, denudatine, norcoclaurine, songorine were downloaded from TCMSP. Compound structures were performed energy minimization calculation by Chem 3D software and then imported into AutoDockTools for adding hydrogen and computing gasteiger. Crystal structures were separated from original ligands and were prepared by AutoDockTools through removing water, adding hydrogen and computing gasteiger. The virtual docking was implemented in the AutoDock Vina. The best docking pose were predicated based on the docked free energy and inhibition constant. The 3D binding model was shown by Pymol, 2D shown by LigPlus.https://www.omicshare.com/tools/). The protein–protein interaction (PPI) network of targets acquired from database STRING . For visual of the PPI network, the line color indicated the type of interaction evidence, the active interaction sources were based on experiments, and the disconnected nodes were excluded in the final network.To gain the biological, molecular, and cellular function of target genes, gene ontology (GO) analysis including biological process (BP), cellular component (CC), and molecular function (MF) was performed. An on-line web tool OmicShare was used to carry GO analysis (http://ctdbase.org/ )23 and DrugBank (https://go.drugbank.com/) to obtain the efficacy-related targets24, and only those genes with direct evidence to rheumatoid arthritis were reserved. The efficacy-related targets of Fuzi are those genes that belong to the targets of Fuzi and are directly linked to rheumatoid arthritis. To obtain the mechanism of Fuzi in treating rheumatoid arthritis, the efficacy-related targets of Fuzi were first subjected to Webserver the Database for Annotation, Visualization and Integrated Discovery , and only the no disease pathways with P < 0.05 were reserved25. To obtain the targets and mechanisms of the toxicity of Fuzi, all the target genes were subjected to DAVID. Because the main toxicity of Fuzi is cardiac toxicity and neurotoxicity, and ion channels are involved in the onset of toxicity26, the enriched pathways including calcium signaling, adrenergic signaling in cardiomyocytes, neuroactive ligand-receptor interaction, dopaminergic synapse were considered as the toxic pathways of Fuzi, and all the target genes on those pathways were reserved.The genes and proteins related to rheumatoid arthritis were retrieved from Comparative Toxicogenomics Database  was used for the visualization of networks.After screening of bioactive compounds by OB and DL, a total of 22 compounds were screened. In addition, the compounds that did not meet the criteria of OB and DL but were reported to show pharmacological effects were also preserved. These compounds include myristic acid, mesaconitine, etc. As a result, a total of 32 bioactive compounds were screened Table . These cTo verify the reliability of targets screened, molecular docking was utilized to estimate the binding ability between compounds of Fuzi and targets, and to view the compound-target binding interactions. The bioactive ingredients in Fuzi exhibited a strong binding ability towards the predicted genes Fig. . The resTo visualize the properties of targets, first, a PPI network was constructed with the help of String database. Results showed that a total of 902 targets were integrated into the whole PPI network. The number of edges was 2095, and the average node degree was 4.64 Fig. . Notewor28. To help visualize the mechanism of Fuzi, a compound-target-pathway network was constructed , benzoylnapelline (M18), denudatine (M20), songorine (M30) can act on AKT1, and AKT1, ATP1A1, ATP2B4, BCL2, and other targets in adrenergic signaling in cardiomyocytes pathway to induce toxicity. In addition, the compounds that are usually believed to be non-toxic also involved in the toxicity of Fuzi. For example, myristic acid, a compound widely used in the food industry as a flavor ingredient, does not pose a health risk to humans34. In our study, myristic acid can act on hypaconitine targets TRPV1, THRA, MAPK1, etc. However, whether this action can lead to increase of toxicity, or decrease of toxicity, or no change of toxicity is known. Further animal studies are needed to confirm the three possible results. Although the effects of non-toxic compounds on final toxicity of Fuzi is known, the results can add new knowledge for our understanding of the toxicity of herbal medicines, i.e., non-toxic compounds can act on targets of toxic compounds, and therefore may influence the toxicity of Fuzi.The toxicity of Fuzi poses a great threat to the patients and can even lead to the death of patients. Studies have demonstrated that the toxicity of Fuzi mainly derives from diester diterpene alkaloids including aconitine, mesaconitine, and hypaconitinePaozhi). The main processing method includes boiling Fuzi in water for a long period of time. In this process, the main toxic diester-diterpenoid alkaloids including aconitine, mesaconitine, and hypaconitine can be first transformed into monoester-diterpenoid alkaloids and finally into unesterified compounds35. The unesterified diterpenoid alkaloids showed no toxicity in the clinic, but at the same time, their pharmacological activities are not influenced30. Although Fuzi must be processed and boiled for a long time before orally taken, there are many cases of using unprocessed Fuzi and Fuzi that are not boiled for enough time36. In addition, many Fuzi victims used Fuzi in medicinal liquors. As a result, there are many cases that toxic alkaloids lead to the Fuzi poisoning in clinic36. Therefore, in our study, we collected the compounds of processed and raw Fuzi, including diester-diterpenoid alkaloids and unesterified compounds in our study to investigate the efficacy-toxicity relationship of Fuzi. We found out that non-toxic compounds can act on targets of toxic compounds and therefore may influence the toxicity. Clinically, the processed Fuzi showed almost no toxicity. Therefore, our study also indicates that boiling Fuzi in water for a long period of time is a necessary step to avoid the toxicity of Fuzi, and the non-toxic compounds alone cannot induce toxicity.In the clinic, the major way to avoid the toxicity of Fuzi is processing (In our study, some drawbacks should be noted. Although many targets of Fuzi have been validated by literatures, many targets identified in this study are needed to be further validated by in vivo studies. In addition, system pharmacology is intrinsically flawed in several aspects. First, the efficacy and toxicity of a drug are directly related to the dosage, and systems pharmacology currently cannot give the direct dosage- phenotype outcome. Secondly, an herbal medicine can contain hundreds or even thousands of different compounds, and it is now not possible to identify all the compounds that exist in Fuzi. Therefore, not all bioactive compounds and targets in our study can be screened currently. Thirdly, herbal compounds can be metabolized by gut microbiota and liver, and the metabolite can act different targets compared with their parent compounds. Even though we have predicted the aglycones of glycosides in our study, not all the metabolites can be predicted based on the structure of compounds. Therefore, not all the bioactive metabolites in the body can be predicted as well. Nevertheless, as an efficient and new tool to study the mechanisms of herbal medicines systemically, systems pharmacology enables us to understand the relationships between efficacy and toxicity.Prevotella histicola is involved in the pathogenesis of rheumatoid arthritis40. Gut microbiota can synthesis and release a large number of metabolites with anti-inflammatory effects such as short-chain fatty acids to ameliorate diseases43. In our study, some compounds in Fuzi are not classified as bioactive compounds according to our screen standard, such as arachic acid. However, arachic acid is reported to be able to modulate the composition of gut microbiota45. Therefore, other compounds that are omitted in our study may act on gut microbiota to ameliorate rheumatoid arthritis. In addition to small molecular compounds, big molecular compounds that mainly include polysaccharides are also the bioactive compounds of herbal medicine. Polysaccharides can modulate the composition of gut microbiota and can be metabolized into short-chain fatty acids to modulate the immune system of hosts48. Currently, the in silicon approach can only screen small molecules, and polysaccharides are usually omitted. Therefore, like other compounds such as arachic acid, polysaccharides of Fuzi may also be effective on rheumatoid arthritis, and further studies are needed to confirm this hypothesis.In recent years, gut microbiota has emerged as a new frontier to understand the development and progress of diseases. Dysbiosis of gut microbiota such as Many herbal medicines with toxicity are extensively used in clinic to treat diseases, however, the mechanism relationships between the toxicity and efficacy of herbal medicines remain unknown. Fuzi is the typical toxic herbal medicine with remarkable clinical efficacy. Using Fuzi in treating rheumatoid arthritis as an example, we demonstrated that the efficacy of Fuzi can be attributed to 25 bioactive compounds that act holistically on 61 targets and 27 pathways, and the toxicity of Fuzi can be attributed to 32 compounds that act holistically on 187 targets and 4 pathways. In addition, non-toxic compound such as myristic acid can act on targets of toxic compounds and therefore may influence the toxicity. However, the effects of non-toxic compounds on the final toxicity of Fuzi remain to be studied further. The results suggested that removing of toxic compounds by processing and boiling is a necessary procedure to avoid the toxicity of Fuzi.Supplementary Information.


The fabrication of bricks commonly consumes relatively high natural resources. To reduce the carbon footprint in the brick production industry, repurposing industrial wastes in the making of sustainable bricks is a recent trend in research and application. Local wastes, such as oil palm shell (OPS), palm oil fuel ash (POFA), and quarry dust (QD), are massively produced annually in the palm oil-exporting countries. Moreover, QD from mining industries is hazardous to both water and air quality. For better waste management in marching towards sustainability, these wastes should be given their second life as construction materials. Therefore, this paper investigates the possibility of incorporating agro-industrial wastes into the brick mixture by examining their properties by means of several standardized tests. For the mix design, a 100% replacement of coarse aggregate with OPS, 20% replacement of cement with POFA, 20% cement weight of limestone as admixture, and 0 to 50% replacements of fine aggregate with QD are experimentally considered. The optimum mix of these wastes is preliminarily determined by focusing on high compressive strength as an indicator. Other examinations include splitting tensile, flexural strength, water absorption, and efflorescence tests. Although the agro-industrial waste cement brick is 18% lower in the strength to weight ratio compared to that of conventional, it is observed that it has better late strength development due to its POFA pozzolanic properties. Moreover, the proposed green cement brick is further checked for compliance with several standards for feasible use in the construction industry. Financially, the cost for the brick with the new mix design is almost equivalent to that of conventional. Hence, this green cement brick is reasonable to be employed in the construction industry to promote material sustainability for better waste management. Bricks are common building materials actively applied in the vast majority of construction works. Because of their remarkable features, such as high strength, low production costs, and durability, bricks have played an important role in construction for thousands of years ,2,3. BriThe building and demolition industry accounts for 40% of global energy consumption and one-third of greenhouse gas emissions ,16. AccoBecause of its mechanical and physical characteristics, as well as the creativity of combining diverse wastes in its manufacturing, the masonry block is one of the most comprehensive building material components . DespiteNatural resource shortages and the formation of solid waste without effective disposal are global concerns ,32,33,34There is a high demand for cement bricks in the current construction industry, and Malaysia produces tons of waste from industries. In order to incorporate all these local wastes into the concrete matrix and better waste management, this paper investigates, therefore, the design mix feasible to be applied in this proposed green cement bricks towards achieving the aim of manufacturing sustainable construction materials. The main focus is to utilize local all wastes to fully replace coarse aggregate in cement brick which is rarely found in current research trends. To obtain the mechanical properties of the proposed mixes, the experimental program for this study includes compressive, splitting tensile, flexural strengths, water absorption, and efflorescence tests. Additionally, their feasibility as sustainable construction materials is identified through appraisal of compliance with the current local codes. A cost analysis has also been considered comparable to that of conventional to seek its production financial impact. The outcomes should benefit and promote sustainability in the construction industry.The current experimental program for studying the feasibility of local wastes for brick production was carried out in three phases. The mixture was designed and identified in Phase 1, properties characterization was performed in Phase 2, while Phase 3 was devoted to the code compliance and cost analysis.The identified wastes were POFA, OPS, and QD, where they were, respectively, functioned as the cement, coarse aggregate, and fine aggregate replacements to produce a lightweight concrete mix for brick production.3 and sieved to maintain its fineness modulus range of 2.3 to 3.1  and calcium carbonate powder  as the admixture to promote early strength to the overall concrete matrix. Additional details for the investigated materials are: Cement—Ordinary Portland cement (OPC), CMC Engineering Sdn Bhd, Selangor, Malaysia; a local product that complied with MS EN 197-1 and ASTM C150; POFA  was considered as the supplementary cementitious material with sizes passing through 45 μm sieve and heat-treated at 500 °C; OPS  complied with ASTM C33 for the sizing; QD  with the density of 1350 kg/mThe main purpose of the sieve analysis was to determine the particle size distribution for each material used to cast the concrete in complying with the ASTM C330M  and C33MThe mix design was fixed at 1:1:1 for cement, fine, and coarse aggregates according to . In PhasIn the following, the conducted tests for the characterization of the concrete specimens are described. All specimens were tested according to test procedures in codes of practice. Only one specimen was tested for slump, density, efflorescence, and water absorption tests. However, average values of three specimens were obtained from the strength tests.Slump tests were carried out in accordance with ASTM C143 . Three lCompressive strength was determined according to ASTM C140  at 7, 14At the concrete curing age of 28 days, efflorescence and water absorption tests were carried out according to the ASTM specifications ,69. OnlyFrom Phase 1, the optimum superplasticizer was determined at 1% of the cement weight with a water-cement ratio of 0.45. This was then applied in Phase 2 investigation for various fine aggregate replacements with different proportions of QD. The properties of the design mixes in Phase 2 are discussed in detail in the following sections.From From From the test observation, due to the low specific gravity of OPS, the design mixes showed tendency to segregate thereby potentially affecting their compressive strength . Compare2 in POFA can react with the Ca(OH)2 from cement hydration to produce extra C-S-H bonding [It can be seen in  bonding .3 [tf is the splitting tensile strength, cuf is the characteristic compressive strength, cyf is the compressive strength (concrete age is not specified).3 , and Equ3 . Making As summarized in rf is the flexural strength, cuf is the characteristic compressive strength.Several equations are readily available to estimate the flexural strength, including Equation (5) for OPS concretes and Equations (6)–(8) for lightweight concretes:From 2 from the hydration. Ca(OH)2 was transported out to the 25 mm distilled water layer as the salt content was lower in the distilled water. After the immersion process, the samples were taken out and dried, during which the precipitation happened as Ca(OH)2 was in contact with carbon dioxide to form CaCO3.All specimens were effloresced where a layer of mineral salt precipitation was noticed on their immersed surfaces, as shown in 2 in the concrete matrix with the pozzolans (POFA) to form C-S-H gels and thereby increase the density of the concrete while pushing out the excess insoluble CaCO3 through the capillaries of the concrete. As the specimens with limestone powder experienced two phases of precipitation, it is justified that the efflorescence visibility was more obvious as compared to the control specimen.Specimens with limestone powder may cause extra precipitation due to the reaction of Ca(OH)Drying shrinkage is one of the most common issues in the concrete industry attributed to its deteriorating effects on concrete strength. Drying shrinkage can be caused by the type of aggregate used, concrete rigidity, water-cement ratio, curing method, environment, etc. According to , the norA higher replacement of quarry dust requires a larger amount of water for the concrete mix to be workable as quarry dust has a high affinity for water, creating a greater moisture suction scenario within the concrete matrix and hence the occurrence of drying shrinkage. This can be solved by prewetting the quarry dust before using it for casting purposes . FurtherPrewetted aggregates or saturated surface dried aggregates are the methods used to saturate the inner pores of the aggregates before casting. The process is executed by soaking the aggregates for 24 h before drying them under the sun or any way appropriate to achieve a dry surface, as suggested by . This prAccording to Malaysian standards, all mixes considered in the current study do not meet the specifications of engineering blocks. However, they are within the limit of load-bearing brick Classes 1 to 4. For Singapore standards, these mixes comply with the common bricks of Grade 2 and 3 specifications. Generally, the proposed design mixes are only recommended for moderate weather conditions but not severe ones. This is due to the water absorption issue. Water absorption from the surrounding environment is another important performance indicator as this property may also reflect the absorbance of chemicals from the building outdoor to indoor, thus endangering its occupants. In order to address this concern, it is suggested here to apply one impermeable layer of coating to prevent the concrete from absorbing water from the surrounding environment. For a general comparison, The preliminary costing of a brick fabrication has been calculated and compared to ordinary OPC brick. Depending on the code specifications, these investigated design mixes serve a wide range of applications for cement brick, as shown in −All design mixes in the second phase met the minimal structural compressive strength of 17 MPa, whereas only 30% QD + 70% RS achieved the minimal tensile splitting strength of 2 MPa for a structural requirement. The compressive strength has been improved as compared to those in .−3. All design mixes were effloresced where a layer of mineral salt precipitation was found within the specimens.The water absorptions of all specimens were between 14.9 to 18.3%, with densities of lesser than 1700 kg/m−It is found that the grounded POFA is able to slip in the gaps between coarse aggregates and undergo pozzolanic reactions. Moreover, these pozzolanic reactions are able to reduce the water pockets in the concrete matrices and hence reduce the drying shrinkage, resulting in a more dense and solid concrete product.−All design mixes complied with Malaysian (load-bearing brick of Classes 1 to 4), Singapore (common brick of Grades 2 and 3), and ASTM (building and facing bricks at moderate weather conditions) specifications.−It is revealed that the specimens with limestone powder have experienced two phases of precipitation. This justified that the efflorescence visibility was more obvious as compared to the control specimen.−The cost to fabricate a single brick was found almost equivalent to a conventional cement brick.This study was devoted to the mechanical and physical properties examination of concrete mix with the full replacement of coarse aggregate with OPS, 20% of cement replacement with POFA, and different replacements of fine aggregate with QD as design mixes. Several conclusions can be drawn from the study:Hence, the proposed green cement brick shows good feasibility to be applied in the construction industry in promoting materials sustainability and better waste management. It is also recommended to conduct a more comprehensive testing program to assess the potential use of the new bricks, such as length change, sound, and thermal insulation properties.