Current research into alcohol consumption focuses predominantly on problematic drinkers and populations considered likely to engage in risky behaviours. Middle-aged drinkers are an under-researched group, despite emerging evidence that their regular drinking patterns may carry some risk.We searched Scopus, Ovid Medline, and Ovid PsycInfo for peer-reviewed, English-language publications appearing prior to 31 December 2015 and relating to the construction of alcohol consumption by middle-aged non-problematised drinkers. Thirteen papers were included in our thematic analysis.Middle-aged non-problematised drinkers constructed their drinking practices by creating a narrative of normative drinking via discourses of gender, identity, play, and learning to drink. They also used drinking norms to construct their gender and identity. Health was not identified as a significant consideration for the population of interest when constructing alcohol consumption, except where drinking behaviours were likely to harm another.These results suggest that public health campaigns aimed at reducing alcohol consumption may be more effective if they focus on unacceptable drinking behaviours instead of personal health outcomes.The online version of this article (10.1186/s12889-018-5948-x) contains supplementary material, which is available to authorized users. Alcohol drinking and non-drinking is a complex social process influenced by a variety of factors and deeply embedded in the social environment. The current body of research into alcohol consumption focuses overwhelmingly on problematised drinkers such as young drinkers and binge drinkers. Low-level drinking is considered unproblematic in many alcohol studies  and in society more broadly . In thisOne such group is middle-aged drinkers, whom we have defined as 30–65 year olds. While drinking in this age group has sometimes been problematised, such as in the case of alcoholism or binge drinking , 5, thisDespite the increasing evidence that alcohol consumption among older drinkers is increasing over time, and that older drinkers are consuming more overall than younger drinkers, we know very little about the motivations and decision-making processes of non-problematised middle-aged drinkers when it comes to their alcohol consumption. We sought to fill this gap by systematically reviewing and synthesising qualitative literature that describes the ways in which non-problematised middle-aged drinkers construct their consumption of alcohol. Understanding alcohol consumption in this group will support more effective health interventions by, for instance, enabling health promotion campaigns aimed at reducing alcohol consumption among this group to speak to their greatest concerns and priorities.significantly harmful consumption as:consumption that significantly increases the risk of ill health or injury to oneself or others, such as binge drinking, drink driving, drinking while pregnant, drinking after being diagnosed with a medical condition adversely affected by alcohol consumption , and drinking that constitutes a substance use disorder .We further defined socio-legally proscribed consumption as:consumption that occurs against the constraints of prohibitive cultures (e.g. where religious conviction requires or strongly commends abstinence) or illegal consumption (e.g. where consumption is prohibited in specific locations).We included studies that included alcohol consumption beyond recommended guidelines unless the drinkers either self-identified their drinking as problematic or it was presented by the authors as such.The purpose of this study was to describe the constructions of the consumption of alcohol evident in academic analysis of accounts of non-problematised middle-aged drinking. We defined non-problematised alcohol consumption as alcohol consumption that is neither significantly harmful, nor socio-legally proscribed. We defined We searched three databases: Scopus, Ovid Medline, and Ovid PsycInfo, on the advice of the university’s discipline search specialist  analysis?Did this study include the population of non-problematised, middle-aged (30–65 year-olds) consumers of alcohol?We assessed studies for inclusion against the following questions:Papers that did not meet these criteria were excluded see Fig. . Where pAn initial pool of 9813 search results was assessed by EM and NM against title, keyword and abstract. NM’s initial assessments  were also reviewed by EM who identified no inappropriate exclusions, and so subsequent papers were checked independently by either EM or NM with a combined total of 245 papers for further analysis .During the data extraction and synthesis phases, some papers were identified as inappropriate for the aims of the study . ReasonsThese papers were rejected as they did not meet inclusion criteria regarding participants’ participants own voices discussing their experience of alcohol consumption.EM read and re-read each paper, extracting key findings using a data extraction form , with Scotland (4) and England (4) being heavily represented. The remaining four papers are from Norway (2), Australia (1) and Japan (1). The Scottish papers had a specific focus on mid-life drinking. More information about the study aims, lenses of inquiry, populations, methods and key findings is appended in an attachment . One study participant consumed masculinised drinks such as beer and whisky to redefine her identity: by transgressing gendered drinking norms, she adopted masculinised traits that enhanced her identity as a  manager in charge of a male-dominated sales team [Just as gendered norms of drinking affected the mode of play, so gendered drinking norms could construct an identity that moved beyond simplistic binaries of gender-appropriate behaviour. A study of playgroup mothers in Australia showed how they consumed and discussed alcohol in ways that skirted the edges of gender expectations without actually transgressing them: the mothers drank, but not too much, or they talked about drinking, rather than actually drinking . In thisles team . Similarles team  describeles team  when drirejection of connoisseurship. By declaring a preference for mid-range wines and simultaneously repudiating the label of aficionado, respondents successfully navigated around the possibility of being seen as pretentious while still accessing the cultural capital associated with wine consumption [Participants in various studies used the learned aspects of normative drinking to construct their identity. In Emslie et al. , connoissumption .Group identities were also constructed through how individuals learnt to drink. In Nesvåg and Duckert , the “knMost papers we reviewed reported studies carried out in the Anglosphere (predominantly the United Kingdom), possibly because of our inclusion requirement that publications be in English. Thus, our findings may not have captured all research around middle-aged drinking and may be limited in cross-cultural applicability.Drinking Attitudes in Midlife (DrAM) study [Several reviewed papers shared authorship or a data source. We regarded papers from the M) study , 17, 21 M) study , 22 wereM) study , 18 we rAlcohol drinking and non-drinking is a complex social process that is influenced by a variety of factors and deeply embedded in the social environment. Based on the literature, we have articulated five themes that explain how non-problematised middle-aged drinkers construct their drinking. Participants in reviewed studies distinguished between acceptable and unacceptable drinking practices in nuanced ways to produce their version of normative drinking. Normative drinking was influenced by play, gender, identity, and learning to drink, as well as by interactions between these concepts.Our results offer insights into how public health messages about the health effects of alcohol consumption may be received by middle-aged non-problematised drinkers, and the barriers that may prevent this group from receiving and acting on these messages. In Ling et al. , public We have shown how participants maintain their status as non-problematised drinkers partly by setting boundaries around drinking behaviours and adhering to certain norms. There are some parallels between these practices and the normalisation of substance abuse by drug users. For example, “non-problem” drug use is normalised –28 and “Several themes in this paper may be helpful when formulating interventions to limit or moderate alcohol consumption. Narratives of unacceptable drinking practices may be useful in framing public health messaging of relevance to this demographic. For example, public health strategies can focus on meeting responsibilities to others, the possibility of causing harm to others, the requirement for respectability in drinking, the physical limits of ageing bodies and subsequent physical consequences, and gendered expectations of behaviour. One example of a campaign using notions of respectability and behaviour appropriate to one’s age and stage of life is the Motor Accident Commission of South Australia’s “Drink Driving—Grow Up” campaign, which relies on notions of moderate drinking being respectable, and excessive drinking being inappropriate for mature adults, when it suggests that drink-driving is ‘childish’ behaviour by using child actors in adult roles .How people define themselves as drinkers—their identities—can also be used in public health campaigns. Some identities, such as the connoisseur and the “real ale drinker,” are closely tied to consumption—without consumption, the identity does not exist. However, these specific identities are concerned with a specific type of drinking, rather than with consuming a lot of alcohol. These identities could be framed in ways that reject higher levels of consumption and emphasise quality over quantity in consumption. There is a danger, however, that this message of gaining social capital through moderate drinking could be exploited. For example, the DrinkWise “Drinking: Do It Properly” campaign was promoted as influencing “young adults (18-24) to drink responsibly – by moderating the intensity and frequency of binge drinking occasions” . HoweverIt is also possible for the themes we have identified in this paper to be used in public health campaigns in ethically problematic ways. For example, the use of gendered public health messaging to encourage or discourage particular modes of consumption is problematic, since many of the gendered drinking norms are closely tied to traditional and potentially oppressive notions of masculinity and femininity. For example, the “Think Twice” campaign of Balance (the North East UK Alcohol Office) and Breakthrough Breast Cancer included an image of two glasses of rosé swirled to resemble a woman’s cleavage.  This imaIn contrast to this, the DrinkWise “Kids Absorb your Drinking” campaign  used parFor middle-aged non-problematised drinkers, alcohol drinking and non-drinking is a complex social process that is influenced by a variety of factors and deeply embedded in the social environment. We found that middle-aged drinkers constructed their alcohol consumption within a framework of Normative Drinking. This key concept was expressed through the four interrelated themes of Gender, Play, Identity, and Learning to Drink. Normative drinking was also used to offer alternative interpretations of gender and identity.Concerns about health and healthy behaviour, however, were either minor or non-existent. For these drinkers, alcohol was both a tool for relaxation described by learned norms of behaviour around gender and identity, and a means by which the self could be expressed through deliberate adherence to and rejection of those norms. This offers possible narrative frameworks for public health interventions around alcohol consumption, although care must be taken to ensure that ethically problematic issues around gender and identity are considered.Additional file 1:Search matrix exemplar. (DOCX 16 kb)Additional file 2:Evaluation & extraction tool. (DOCX 15 kb)Additional file 3:Metadata and paper summaries. (XLSX 16 kb)


As international travel, migration, and mass media increase cross-national influences on alcohol use and alcohol-related problems, better understanding of the sociocultural influences on women’s and men’s drinking will become increasingly valuable for improving national and international efforts to reduce problems caused by alcohol consumption. This article summarizes international collaborative research conducted during the past decade on gender-related influences on alcohol use, identifies future directions for research in this area, and suggests possible implications of this research for the development of more gender-sensitive national and international alcohol policy.International comparative research on alcohol use (comparing two or more countries or cultures) developed in the 1970s, initially to examine alcohol control policies  and commParalleling the development of international research on drinking behavior, gender’s influence on drinking patterns and problems has also been the subject of increased attention as a result of research in the United States and Canada  . In all One topic of discussion throughout IRGGA’s history has been how quantitative and qualitative methods can be used together to improve the design of survey instruments and to better understand the psychological and social processes that underlie quantitative research findings. Qualitative research in IRGGA has included a study in which members conducted in-depth interviews with women and men in six countries about their reactions to gender-related images in alcohol beverage advertisements, and a workshop on the use of qualitative methods in international gender and alcohol research. Directors of country surveys for the group’s new GENACIS project (described below) have been encouraged to incorporate qualitative substudies to aid in interpreting their quantitative survey findings.In 1996 a subset of IRGGA members from European Union (EU) countries conducted a comparative study of women’s and men’s drinking patterns, and their acute and chronic consequences, in nine European countries: Finland, France, Germany, Great Britain (Scotland), Italy, the Netherlands, Sweden, Switzerland, and the Czech Republic. The EU group investigated whether European women’s and men’s drinking patterns were becoming more similar (the “convergence” hypothesis). The researchers concluded that any such convergence has been small, inconsistent, and statistically uncertain in most European countries, with the exception of Finland . In none of the European countries studied did women’s drinking frequencies or quantities exceed those of men .heavily (12 or more drinks per week) . The data showed that women with higher levels of education were more likely to drink at least occasionally  . Howeverer week) .The EU study also addressed the question of how survey measures of drinking behavior could be made more gender-sensitive. The investigators concluded that oversimplified questions  should be avoided because they underestimate women’s drinking more than men’s, leading to overestimation of gender differences. They also recommended the evaluation of cultural effects on the reliability and validity of drinking measures .International research using secondary analyses of existing data sets has been hampered by differences in how drinking is measured  and by differences in how drinking consequences are measured (with different countries emphasizing different types of consequences). These differences make it difficult to compare drinking behavior and its consequences across countries.Drawing on their experience in collaborative research, IRGGA members have designed a new international study, known as GENACIS . GENACIS is obtaining new survey data using a standardized set of questions. At the time of this writing, 30 countries have completed, are conducting, or plan to conduct surveys as part of GENACIS (see table). Some surveys are being conducted specifically for GENACIS, and some surveys designed for other purposes  will include sets of GENACIS questions. Funding from the European Union is supporting coordination and centralized analysis of data from GENACIS surveys in 13 EU member states and associated states. The WHO has provided funding for GENACIS surveys in several countries, presently including Argentina, Costa Rica, India, Kazakhstan, Nigeria, Sri Lanka, and Uganda. Additional funding is provided by the National Institute on Alcohol Abuse and Alcoholism to support worldwide coordination of GENACIS and to assist countries not eligible for funding from the EU or WHO.societal-level characteristics such as (1) gender inequality , and (2) differences between “wet” drinking cultures  and “dry” drinking cultures  , and drinking consequences (as perceived both by the respondent and by other people), and includes alcohol dependence questions from the Alcohol Use Disorders Identification Test (AUDIT) . The queg norms) .Centralized analyses of GENACIS data are designed through collaboration of survey directors and other project partners interested in specific research questions. Data analysis is expected to continue for several years, examining cross-national similarities and differences in women’s and men’s drinking patterns and drinking-related problems, and in the individual- and societal-level predictors of gender-specific drinking patterns and drinking problems. The unique GENACIS data will be archived for future use to study such things as global time trends in women’s and men’s alcohol use, its predictors, and its consequences.Research examining alcohol and gender issues worldwide, as described above, reveals several issues that have important implications for international alcohol policy.and culturally defined gender roles that specify expected and tolerated drinking behavior for women and men. For example, prevention strategies targeting social norms that promote high-risk drinking behaviors among males may be particularly appropriate and needed in countries with highly gender-differentiated drinking behavior  might be much too high for chronic daily alcohol consumption. Demographic and cultural characteristics may also affect “safe” levels of drinking: consumption levels that are risky for elderly retirees in Florida may not be as risky for young businesswomen in Oslo or Prague.Publicized guidelines for high- and low-risk drinking should be better informed by research on gender and alcohol. Most countries with such standards recommend lower limits of alcohol consumption for women than for men, but the levels of consumption defined as “safe” vary considerably across countries. For example, Australia defines low-risk consumption as no more than 28 drinks per week for men or 14 drinks per week for women , whereasDouble standards of drinking that judge excessive (or any) alcohol use more harshly for women than for men have been reported in many cultures throughout history e.g., . This stnot imply that women with higher levels of education and higher status jobs are in greater danger of becoming problem drinkers. It does imply that where women gain increasing education, income, and status, prevention of problem drinking will have to be achieved primarily at the individual level, because social environments will no longer inhibit all drinking by all women.A final implication of international gender and alcohol research for alcohol policy is that changes in women’s and men’s roles are likely to be accompanied by changes in women’s and men’s drinking behavior. When women improve their education, employment, and status, they are likely also to have more opportunities to drink. However, this does men’s drinking? If improvements in women’s roles threaten the self-worth of some men, or if women have better employment opportunities than men do, will changes in women’s roles increase risks of problem drinking among men? This question may deserve greater attention from alcohol problem prevention specialists as gender roles become defined more by global standards and less by local traditions.Finally, if women’s education and employment improve, what will happen to As the world becomes “smaller,” it will become increasingly important and valuable to compare the patterns, predictors, and consequences of women’s and men’s alcohol use across varying cultural contexts. In addition to improving the culture- and gender-sensitivity of measures of alcohol use and alcohol problems, cross-national research can help to explicate the complex interactions of biology with other individual-level and societal-level variables that influence the drinking behavior of women and men. Such research can ultimately inform the design of better-targeted and more effective prevention and intervention efforts for women and men worldwide.


This study aimed at assessing the nutritional status among the elderly population and factors associated with malnutrition in the community setting in rural Nepal.Dalit community, being unemployed, having experience of any form of mistreatment, lack of physical exercise, experiencing problems with concentration in past 30 days and taking medication for more than one co-morbidity was significantly associated with the malnutrition status of the elderly.Out of 339 participants, 24.8% (95% CI 20.21–29.30) fell into the normal nutritional status range; 49.6% (95% CI 44.29–54.91) were at risk for malnutrition while 24.8% (95% CI 20.21–29.30) were in the malnourished range, based on Mini Nutritional Assessment scores. Our findings revealed that belonging to a  Every country in the world is facing a demographic challenge due to the drastic growth of population over 60 years. The proportion of this population is projected to grow by 56% between 2015 and 2030, and by 2050 the global proportion of elder populations is projected to be double than its size in 2015. The pace of population aging in many developing countries today is noticeably faster than occurred in developed countries in the past , having concentration problems in the past 30 days  and those taking medication for more than one co-morbidity, i.e., polypharmacy  had higher risk of falling into the malnutrition range.Nutritional status of older people results from a complex interplay between dietary, socio-economic, physical and psychological factors . Long reOur study has revealed that 24.8% of rural elderly people are malnourished and 49.6% of elderly are at risk of malnutrition. Thus, it appears that malnutrition is much higher among the Nepalese elderly residing in the rural community. This finding is consistent with the results of earlier studies conducted in Nepal, India and Bangladesh –22. ConsOur findings showed that elderly from the Dalit community were at more than two times at higher risk of malnutrition compared to elders of a higher caste. Our study aligns with findings from study conducted in Nepal  where etIn the current study, unemployed elderly population were at 3.23 times more risk of malnutrition compared to those who were employed. This can be explained by the fact that the employed elderly possibly could have good financial status, resulting in good access to nutritional foods. In addition, they have to depend economically less on the caregivers for the fulfillment of dietary and basic needs needed for good health. Consistent with our findings, study from Egypt  have ideIn this study, the elderly who reported a lack of physical activity were at 4.67 times likelihood of being malnourished, as has been reported in other studies , 31. ThiThe elderly who had history of polypharmacy were at 3.01 times more risk of malnutrition compared to their counterparts who did not had history of polypharmacy. The could be explained by the fact that, polypharmacy is chosen by the practitioners for the treatment of such multiple co-morbidities, can indirectly result in malnutrition because of side effects like poor appetite, nausea, dry mouth and constipation. Evidence shows that moderate intakes have a protective effect on malnutrition while an excessive intake may have a negative effect on the health of elders . This fiA limitation of this study is the cross-sectional design, no conclusions can be drawn in line with the cause-effect model. In addition, the study was conducted in one district, so its findings could not be generalized to other setting of Nepal.


The rubber hand illusion (RHI) is a perceptual phenomenon in which participants experience ownership over a fake model hand through synchronous visuotactile stimulation. Several studies have shown that the illusion occurs only when both hands are in close proximity to each other. In the present study, we systematically examined the role of relative position  and distance (13–75 cm) of the model hand  on illusion experience across both lateral and distal positions. Furthermore, we also compared different facets of the subjective illusion experience; the experience of the model hand being part of one’s body  and the perceptual fusion of vision and touch . In two experiments we observed indications for a stronger illusion experiences in distal compared to lateral positions of identical distances, indicating that the illusory effects may vary as a function of the relative position of the hand. Our results also showed that manipulations of distance differently modulated both facets of the illusion. While ownership was restricted to near distances, referral of touch sensations remained stable at farther distances. These results are interpreted in relation to variations in sensory weighting across different planes.The online version of this article (10.1007/s00221-019-05539-6) contains supplementary material, which is available to authorized users. The rubber hand illusion  might therefore depend upon the effective merging of sensory information across different modalities, leading to higher order experiences of ownership. Distance effects have provided evidence for a spatial boundary in the experience of the RHI and supports the idea that the illusion might be based on processes akin to multisensory integration that follows basic temporal and spatial rules  participated in the study. Written informed consent was obtained from all participants prior to participation. All participants had normal or corrected vision, and none reported any sensory deficits. The study was approved by the University of Reading Research Ethics Committee and has been conducted in accordance to the declaration of Helsinki. Participants received a monetary compensation for their participation.Participants were comfortably seated in front of a table on a height-adjustable chair with their right arm maximally stretched out and placed on the table. The right arm was positioned such that it was occluded from view by a wooden board and the right shoulder was covered with a black cloth. The fingers were placed comfortably on a wooden cube to elevate them and make the fingers more visible at the distal position. Participants sat close to the table ensuring that their upper body leaned close against the table. This prevented participants from moving and ensured no arm movement. A life-size model hand was then placed in one of three positions: distal-near, lateral-near both 13 cm), and lateral-far (38 cm) . All reported results are two-tailed, and all tests are based on a priori hypotheses unless otherwise stated.Overall illusion scores and control scores were calculated by averaging the four illusion-related statements  and control statements for each condition separately. Additionally, sub-scores for ownership and referral of touch were computed by separately averaging statements 1 and 2 (ownership) and 3 and 4 . In line with previous studies, we considered a participant with an average illusion score of ≥ 1 as an illusion responder (Petkova and Ehrsson M: 1.5), compared to the asynchronous condition . A significant difference was also observed between illusion and control statement scores (M: 0) in the synchronous condition with illusion statements being higher , and negative ratings in the asynchronous condition (M: − 0.75). This difference was significant . Ratings to illusion statements were significantly higher than the control statements in the synchronous condition , but not in the asynchronous condition (M: − 0.75). This difference was significant . Participants also gave significantly higher ratings to illusion statements compared to the control statements in the synchronous condition . The difference between the lateral-near and the distal-near positions was not significant .We compared illusion scores of the three synchronous conditions and found significant differences in illusion ratings , lateral-far  and distal-near . In all synchronous conditions, the referral of touch scores were higher than ownership scores  and distal-near  conditions. The difference between the lateral-near and distal-near positions was not found to be significant . No significant differences for referral of touch scores were found across the conditions , and no subsequent pairwise comparisons were performed.Next, two Friedman tests on ownership and referral of touch scores were independently conducted. A significant main effect for ownership scores was observed: In line with previous studies . All participants provided written informed consent, had normal or fully corrected vision and reported no sensory deficits.The materials used, and experimental procedures were identical to Experiment 1. Both synchronous and asynchronous stimulation conditions were tested across all three distal positions see Fig. . The synData collation and analyses were identical to Experiment 1.M: 1.5) were higher than the asynchronous condition . Overall illusion ratings were also significantly higher compared to control statement scores (M: − 0.25) in the synchronous condition  and negative (M: − 1) in the asynchronous condition. This difference was significant . Here again, significantly higher ratings to the illusion statements compared to the control statements  for illusion scores in the synchronous condition compared to the asynchronous condition, in which overall illusion scores were negatively rated (M: − 1). This difference was significant . Ratings to illusion statements were significantly higher than control statements  compared the control statements (M: − 0.63) in the synchronous condition .Participants gave significantly higher ratings for illusion . Post hoc pairwise comparisons demonstrated significant differences in illusion scores between the distal-near and distal-far position , the distal-near and distal-very far position  as well as the distal-near and lateral-near position . No other comparisons were significant at a Bonferroni corrected significance level of p = 0.008.A significant main effect for overall illusion ratings were observed across the three distal conditions and lateral-synchronous condition , distal-far , distal-very far , and lateral-near  conditions , distal-far , distal-very far  and lateral-near . In line with Experiment 1, we compared ownership and referral touch scores and observed significantly higher referral of touch scores in the distal-near . Follow-up, pairwise comparisons demonstrated significant differences between the distal-near and the two farther distal positions; . No significant differences between the distal-far and distal-very far positions  as well as between the distal-near and lateral-near positions  were observed (Bonferroni corrected p = 0.008). A second Friedman test on referral of touch scores also revealed a significant difference across the conditions . Follow-up pairwise comparisons indicated significant differences between distal-near position and distal-far  as well as the distal-near and lateral-near positions  positions. Significant differences were also not observed between the distal-near vs. distal-very far  and distal-far vs. distal-very far conditions .A Friedman test conducted across the distal positions and lateral-near position showed a main effect for ownership scores  even at the farther distal distances should not be ignored. Indeed, as with Experiment 1, referral of touch scores remained significantly higher than ownership scores across at all distances. The overall response patterns may therefore, suggest that although stronger integrative processes may occur at near-spaces, visuotactile process might still be at operation even at farther spaces from a body.The present study measured the effect of distance and position on the rubber hand illusion. We observed higher illusion scores in distal positions compared to lateral positions when distance was kept the same. Our results also showed that distance generally affects the illusion, and provides a detailed account of the role of distance on RHI experiences. While increasing distances led to weaker subjective illusory reports, this effect was specific to the subjective component of the illusion assessed, i.e., ownership experiences decreased when the model hand was placed more than 27 cm away while referral of touch sensations were rated high  at all distances.Our observation of a general distance effect is in agreement with previous studies showing that as distance increases, the illusion significantly decreases Lloyd . Hence, We observed a stronger illusion at the distal-near position compared to the lateral-near position of 13 cm see “”. Such aAt a mechanistic level, differences between the distal and lateral positions can be related to alterations in the perceptual processes underlying the illusion. The multisensory representation of the space surrounding the hand is coded for by visuotactile bimodal neurons found in the parietal and premotor regions as well as the putamen  to be differently influenced by manipulations of spatial disparity, and thus raises theoretical and methodological concerns regarding what constitutes the experience of ownership and the establishment of the rubber hand illusion.Supplementary material 1 (DOCX 4867 kb)Below is the link to the electronic supplementary material.


Most microbes that are capable of mediating Mg-rich carbonates have been isolated from evaporitic environments in which temperature and salinity are higher than those of average marine environments. However, how such physicochemical factors affect and concur with microbial activity influencing mineral precipitation remains poorly constrained. Here, we report the results of laboratory precipitation experiments using two mineral-forming Virgibacillus strains and one non-mineral-forming strain of Bacillus licheniformis, all isolated from the Dohat Faishakh sabkha in Qatar. They were grown under different combinations of temperature , salinity , and Mg2+:Ca2+ ratios . Our results show that the incorporation of Mg2+ into the carbonate minerals is significantly affected by all of the three tested factors. With a Mg2+:Ca2+ ratio of 1, no Mg-rich carbonates formed during the experiments. With a Mg2+:Ca2+ ratios of 6 and 12, multivariate analysis indicates that temperature has the highest impact followed by salinity and Mg2+:Ca2+ ratio. The outcome of this study suggests that warm and saline environments are particularly favourable for microbially mediated formation of Mg-rich carbonates and provides new insight for interpreting ancient dolomite formations.Studies have demonstrated that microbes facilitate the incorporation of Mg These studies are relevant to understand the origin of sedimentary dolomite11, a mineral that is abundant in ancient sedimentary rocks, but whose formation may result from substantially different and not fully understood processes13. Because sedimentary sequences rich in dolomite are often studied for paleoclimatic and paleoenvironmental reconstructions and because dolomite comprises many economically important gas and oil reservoirs14 much effort has been, and is still being invested in understanding the processes that leads to its formation.Studies conducted during the last 20 years in modern natural environments and in the laboratory have shown that some microorganisms promote the formation of Mg-rich carbonate minerals, including phases that may be precursor of ordered dolomite  present in extracellular polymeric substances (EPS) or in the cell wall adsorb ions , creating a supersaturated local environment27 and also reducing the activation energy necessary for the initiation of crystal growth 28.However, conditions of supersaturation with respect to dolomite are insufficent to explain its precipitation at low temeperature, which is likely inhibited by kinetic factors. An increasing number of studies suggest that microbially-produced organic molecules may play a key role in overcoming such kinetic barriers2+: Ca2+ ratio (above 8)29. From a purely physicochemical rather than geobiological perspective, all these factors have been proposed as important –although not necessarily essential30 – for the formation of carbonate minerals33. Temperature is known to promote the incorporation of Mg into carbonates35. Salinity and the increased concentration of Mg2+ (and the Mg2+:Ca2+ ratio) can also cause supersaturation with respect to dolomite36. It is therefore likely that a combination of abiotic parameters determines the optimum conditions for the formation of microbially mediated Mg-rich carbonates37. For instance, sulfate reducing bacteria are virtually ubiquitous in the marine realm38, but their ability to mediate the formation of dolomite may be expressed only in a hypersaline environment in waters that have a high Mg2+:Ca2+ ratio 39. Indeed, extensive evaporation leads to the precipitation of aragonite and subsequently of gypsum, which results in Ca2+ removal from solution and an increased Mg2+: Ca2+ ratio29. On the other hand, in evaporitic environments the positive effect of a high Mg2+:Ca2+ ratio might be thwarted by a salinity that is so high to inhibit microbial growth40. Knowing the optimal conditions for microbially mediated formation of very high Mg calcite —often considered as a potential dolomite precursor41— would be very helpful for interpreting the paleoenvironment associated with this type of dolomite in ancient sedimentary sequences. Because microbial species react differently to changes in physiochemical parameters42, the identification of optimal conditions for the microbial formation of Mg-rich carbonates is very challenging and caution is required before generalized conclusions can be made. There is as yet, a very limited number of studies evaluating the combined effect of several environmental factors on microbial (eco)physiology44.Most of the microbial “protagonists” in the studies mentioned above were isolated from environments characterized by high temperature , salinity (twice seawater or more) and a high Mg2+:Ca2+ ratio on microbially mediated formation of Mg-rich carbonates at aerobic conditions by the bacterium Virgibacillus were investigated. Two strains of this bacterium, known to mediate the formation of Mg-rich carbonates5 and one non-mineral forming bacterial Bacillus, all previously isolated from the Dohat Faishakh sabkha (a modern dolomite-forming environment located in Qatar) were used for the experiments.To contribute in understanding such a complex process, here, the interactive effects of temperature, salinity and MgVirgibacillus and crystal formation, two strains previously isolated, identified, and characterized as high magnesium calcite (HMC) forming strains5, were compared to a non-crystal producing bacterium Bacillus lechinforms , regardless of their capability to mediate mineral formation. No indications about the mineral composition could be drawn at this stage. No mineral formation was observed in any negative control cultures .In order to investigate the impact of the three abiotic factors on the growth of ms Table . The ear2+:Ca2+ ratios showed the presence of peloids composed of carbonate minerals  investigations of the minerals formed in the DF112 and DF2141 cultures incubated at various NaCl (%w/v), temperatures, and Mgals Fig. . The dom2+, and Mg2+ but at various ratios  and data reported in the literature, the ν1, symmetric stretching mode of the carbonate observed at 1086 corresponds to calcite45, the increase in the carbonate v1 peak position at 1093 may be attributed to an increase in magnesium content46. The 1121 cm−1 Raman band is consistent with the v1 band of hydromagnesite, which is characterized by frequencies ranging from 1117 to 1120 cm−1 47.The observed bands in Raman shift spectroscopy confirm the occurrence of calcite, high magnesium calcite and hydromagnesite Fig. . Based o2+:Ca2+ ratios of 6 and 12  with a variable mol% of Mg  in cultures that had initial Mg2+: Ca2+ ratio of 1; these cultures only produced calcite and halite peaks  and/or higher temperature . However, the differences between the two Virgibacillus strains were not significant . The multilinear regression revealed that the effects of the combined three studied parameters were statistically significant with respect to the incorporation of Mg into carbonate minerals  revealed significant differences in the incorporation of Mgls Table .Table 3R2+: Ca2+ ratios respectively, suggesting that temperature had the highest impact on Mg incorporation, while the Mg2+: Ca2+ ratio had the lowest impact. Among the three parameters, the interaction between temperature and Mg2+: Ca2+ ratio was significant.Indeed, the adjusted R-squared value of 0.584 indicates that 58% of variation in the data is explained by the model, with temperature producing the largest R-squared increase. The standardized coefficients (Beta) values were  for temperature, NaCl (%w/v) and MgBased on microscopic and SEM observations, Raman spectroscopy and confirmation by XRD analysis, no mineral formation was detected in the negative control cultures .48 and ammonification . Previous studies using microbial culture experiments showed the existence of a positive correlation between temperature and the amount of Mg incorporated into carbonate minerals3. High and low temperatures - beyond optimum range of growth - are a source of stress for the microorganisms54. In response to such environmental stress, it is known that microbes may react by secreting specific organic molecules –  that, in turn, might favour mineral nucleation (i.e.55). For instance, the optimal growth temperature for Virgibacillus bacteria genetically close to that used in the present study was reported to be approximately 37 °C, though they can tolerate temperatures between 15–50 °C56. With respect to microbially mediated formation of Mg-rich carbonates, the role of temperature might therefore be double: one relates to the thermodynamic and kinetic of the reaction, the other to an ecological stress that causes microbes to increase the production of EPS, which in turn promotes mineral formation. All these considerations are consistent with the results of our experiments carried out at 20, 30, and 40 °C, which showed a positive correlation between temperature and the Mg mol% content of the carbonate mineral  with the saturation index of the carbonate minerals. However, in our experiments, the Na+ and Cl− concentrations increased independently of the concentrations of Ca2+ and Mg2+, allowing the separate evaluation of the effect of salinity and the Mg2+:Ca2+ ratio. The effect of salinity and ionic strength on the incorporation of Mg into the carbonate minerals are not fully resolved, and existing studies show contrasting results. Stephenson et al.58 reported an inverse relationship between the ionic strength and the Mg content in Mg-rich calcite obtained abiotically. In addition, a theoretical geochemical model for dolomite formation predicted a higher saturation index (SI) at higher salinity59. However, various microbial mediation experiments conducted at surface temperatures suggested that high salinity  generally promotes the formation of Mg-rich carbonates50. This correlation was also clearly observed in our study. For example, the Mg mol% content increased from 27.70 ± 2.00 to 31.36 ± 0.62 to 41.52 ± 1.71 when the NaCl (%w/v) increased from 3.5% to 7.5% to 10% respectively, while the temperature was maintained at 30 °C and the Mg2+:Ca2+ ratio at 6 60. Our findings using Virgibcillus support this hypothesis: a Mg2+:Ca2+ ratio higher than 1 is vital for the incorporation of Mg2+. However, above the tested values of 6 (that corresponds to a free Mg2+:Ca2+ ratio at the beginning of the experiment of about 5 (see method section)), increasing the temperature resulted to be more important than further increasing the Mg2+:Ca2+ ratio from 6 to 12.A similar positive correlation as observed between the mol% of Mg and temperature and NaCl (%w/v) was observed for the Mg+ and Cl− is intuitively not so direct and requires some discussion. An increase in ionic strength does not enhance incorporation of Mg into the carbonate under abiotic conditions but has rather the opposite effect58. We therefore propose that to understand this correlation also a biological factor needs to be considered. Several of the most recent studies on microbially mediated formation of dolomite suggest that EPS have key importance in the mineralization process25. EPS include functional groups that, by affecting the kinetics of the nucleation process, were shown to promote incorporation of Mg into carbonate minerals at low temperature. Several studies, indeed, suggest that the chemistry of the cell walls and the associated EPS play a crucial role61. Because EPS is abundantly produced when microbial communities are under ecological stress62, we hypothesize that more EPS were formed in the experiments carried out with high concentrations of dissolved Na+ and Cl−, which in turn resulted in the mediation of carbonate minerals with a higher mol% Mg. EPS compounds bind a variety of divalent cations. Ca2+ ions are preferentially absorbed on EPS compared to Mg2+ ions due to the fact that the energy used to remove the hydration membrane of Ca2+ ions is smaller than that of Mg2+ ions. Consequently, this differential binding of Mg2+ and Ca2+ may result in a Mg2+:Ca2+ ratio that differs from that of the surrounding environment, influencing the degree of Mg incorporation into the mineral63.The observed link between the mol% of Mg into the carbonate mineral and high concentrations of dissolved Na64. It has been proposed that such peculiar rounded shape is mostly controlled by mineral nucleation and growth within a high viscosity solution, which is in turn due to the presence of microbially produced EPS and amino acids65. The morphology of the minerals produced in the experiments is therefore not caused by a process whereby a pre-existing grain is progressively coated by concentric layers of authigenic carbonates, as it is the case with ooids – another common facies of spherical carbonates that occur in evaporitic environments69.The hypothesis that EPS played an important role in our experiments is also consistent with the morphology of the precipitates. Similar morphologies have been described in previous studies on microbial mediation of carbonate mineralsFinally, the observation that different minerals  were formed simultaneously in the same pure culture is consistent with mineralization under the influence of heterogenic functional groups present in EPS and/or bacterial cell walls. Organic molecules with different cation-adsorption properties would indeed explain the co/occurrence of minerals with different morphologies and chemical compositions.2+:Ca2+ ratio—had an impact on microbially mediated formation of high-Mg calcite. Bacterial growth and EPS production, which in turn affected by temperature and NaCl (%w/v), caused an increase in magnesium incorporation into the precipitated minerals. The highest incorporation of Mg2+in the carbonate crystals was obtained with a salinity of 10% rather than 3.5% or 7.5% NaCl (%w/v), while calcium carbonates were mostly detected in experiments with 3.5% NaCl (%w/v). With a Mg2+:Ca2+ ratio of 1, the dominant mineral phase was calcium carbonate; no or rare magnesium calcium carbonates were observed. Increasing the Mg2+:Ca2+ ratio from 6 to 12 resulted in an increase in Mg2+ incorporation into the carbonate crystals. Thus, a high Mg2+:Ca2+ ratio seems to be important as proposed in most previous models explaining dolomite formation in sabkha environments29. In summary, at the tested conditions, temperature had the highest impact for the incorporation of Mg2+ during the microbially mediated mineralization process, followed by NaCl (%w/v) and Mg2+:Ca2+ ratio.In our experiments that simulate natural sabkhas environment, all the three tested parameters—temperature, NaCl (%w/v), and MgVirgibacillus strains suggest that temperatures, NaCl concentrations and Mg2+:Ca2+ ratios higher than those of average seawater favour microbially mediated formation of Mg-rich carbonates, which are often considered as potential dolomite precursors.Although our experimental approach is far too simple to simulate the complexity of natural environments, the results obtained with two different Virgibacillus strains (Virgibacillus martsimiure DF112 and Virgibacillus sp. DF2141) and one Bacillus strain (Bacillus licheniformis DF141) were used in this study. Bacillus and Virgibacillus are aerobic or facultatively anaerobic and spore forming bacteria. They are Gram-staining-positive motile rods, that may occur in single, pairs or chains. The studied strains were previously isolated in our laboratory from core samples collected from the Dohat Faishakh sabkha5. Bacterial identification was performed by sequencing the 16 S rRNA gene. The strains used were initially recovered from the established Qatari Mineral Precipitating Strains Bank, where they had been preserved at −80 °C in 30% glycerol. The strains were streaked on solid Luria-Bertani (LB) medium prior to each experiment to obtain viable, fresh, and pure cells.Two 2H3O2)2, and (CH3COO) 2Mg 4H2O, . The NaCl (%w/v) was adjusted with different concentrations of NaCl (Table The nine types of culture media used in this study were labelled MD1–9. The media were composed of 1% (w/v) yeast extract, 0.5% (w/v) peptone, and 0.1% (w/v) glucose. They were supplemented with different concentrations of acetate salts, Ca  with a resolution of 5 nm and a magnification of 200,000× equipped with an energy-dispersive X-ray microanalysis system (model 2011-Netherlands). The EDS was carried out following “ASTM standard method E1508–12a”, with spot size 5 at an accelerating voltage of 20 kV and a 4% error.−1 in the range between 50 and 3500 cm−1. The Omnic D/S software was used to perform spectral baseline correction/adjustment and smoothing.The recovered crystals were placed and oriented on the stage of an Olympus BHSM microscope, equipped with 10×, 20× and 50× objectives that was part of a DXR Thermo-Scientific DXR Raman imaging microscope, which includes a monochromator, a filter system and a charge coupled device (CCD). Raman spectra were excited by a 532 nm laser at a nominal resolution of 2 cmTo determine their mineralogical composition, particles were selected and targeted for discrete X-ray analysis. The bulk mineralogical composition was performed using a PANalytical- multipurpose Empyrean X-ray diffractometer. The analysis of XRD spectra was carried out using MATCH! Version 3.5.2.104, CRYSTAL IMPACT, Kreuzherrenstr. 102, 53227 Bonn, Germany.et al.70, which is based on the position of the (d104) peak in the XRD pattern.The Mg mol% of the carbonate minerals formed in the experiments was calculated using the formula of Goldsmith 2+:Ca2+ ratios) and the mol% Mg and to determine which predictor variable(s) are the most important. Statistical analyses were carried out at the 95% confidence level using the software IBM SPSS Statistics Version 24.All the experiments were carried out in triplicates. The significance of differences between conditions was analysed using a one-way analysis of variance (ANOVA). Multilinear regression analysis was performed to estimate the association between the three independent variables  and Mg


SPC@AVM also prevented UV photolysis, wherein the half-life was extended from 18 to 68 min when exposed to UV light. Moreover, toxicity tests showed that the encapsulation of AVM was beneficial to retain the insecticidal effect of AVM in the presence of ultraviolet light. The release rate of AVM showed pH responsiveness and the release rate under neutral conditions was faster than acidic and alkaline conditions. Moreover, the process conformed to the Weibull model.Carboxymethyl cellulose (CMC) was grafted onto the surface of soy protein isolate (SPI) to obtain soy protein isolate-carboxymethyl cellulose conjugate (SPC). Avermectin (AVM) was hydrophobically encapsulated as a model drug to obtain SPC@AVM. The reaction between SPI and CMC was confirmed by infrared spectroscopy, thermal analysis and SDS-PAGE electrophoresis. The results of scanning electron microscopy showed that the average particle size of the drug-loaded microspheres was 129 nm and the shape of microspheres changed from block to spherical after the addition of AVM. After encapsulation of AVM, the absolute value of zeta potential was greater than 15 mV, which indicated better stability. Compared to AVM solution, SPC@AVM showed more wettability on the leaf surface and the contact angle on the leaves decreased from 71.64° to 57.33°. The maximum liquid holding capacity increased by 41.41%, from 8.85 to 12.52 mg cm Annual et al. [Scientists have proposed two different approaches to solve this problem. The first one is to replace highly toxic organochlorine pesticides with bio-pesticides, which are biodegradable . Some stet al.  grafted et al. [The nano-material as a carrier of active ingredient possesses better permeability, thermal stability and biodegradability compared to conventional pesticide formulation. It improves the solubility of the active ingredient and facilitates its intelligent release ,16. Compet al.  synthesiPolysaccharide–protein complexes and coacervates are biopolymeric systems that have been used over the past decades for encapsulation of numerous active ingredients . In our N'-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) was used as the catalyst to form a stable amide bond between them, after which AVM was encapsulated as a model drug. CMC was obtained by modification of cellulose, which is the most abundant resource in nature. The molecular chain contained a large number of hydroxyl groups and so it was highly viscous in aqueous solution [In this work, soy protein isolate (SPI) and CMC were used as raw materials and solution . Using Csolution . The CMC2.2.1.N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and the N-hydroxysuccinimide (NHS) were purchased from Aladdin Reagent Co. Ltd . The molecular weight of CMC, measured by gel permeation chromatography, was 216 kDa. AVM (96%) was provided by Hebei Weiyuan Biochemical Co. Ltd . Hydrochloric acid, sodium hydroxide, absolute ethanol, methanol, potassium bromide and other chemicals were purchased from Tianjin Damao Chemical Reagent Co. Ltd , all of which were of analytical grade. Acrylamide (30%), Coomassie Brilliant Blue fast staining solution, Rainbow 245 plus Spectral standard protein (Maker) were purchased from Solarbio Technology Co. Ltd  Tris–HCl buffer , Tris–HCl buffer  and sample buffer were purchased from Biosharp Biotechnology Co. Ltd .SPI was obtained by extraction of de-fatted soy protein powder, provided by Tianli Food Co. Ltd , according to previously reported methods . CMC (US2.2.EDC (0.25 g) and NHS (0.125 g) were weighed accurately and dissolved in deionized water to obtain 25 ml EDC/NHS solution. SPI (0.5 g) was dissolved in 50 ml deionized water and filtered to remove insoluble particles. Then CMC, with a certain mass ratio with respect to SPI, was added and dissolved by stirring. The pH of the above mixture was adjusted to 5.5 using dilute hydrochloric acid, followed by addition of 5 ml of EDC/NHS solution. The reaction was stirred for 24 h. The reaction solution was then transferred to a dialysis bag with a molecular weight cut-off of 5000 Da for 24 h to remove EDC and NHS. To obtain a pure SPC, the dialysed solution was placed in a refrigerator at −40°C for 24 h and then freeze-dried. The mechanism of SPC synthesis is shown in −1, the pH was adjusted to 7 and it was heated to 80°C for 1 h. When the temperature of the solution reached room temperature, 2 ml of AVM ethanol solution was added to it and stirred for 0.5 h to obtain SPC@AVM solution. The route for the synthesis of SPC@AVM was shown in AVM (0.25 g) was dissolved in 25 ml of absolute ethanol. SPC was dissolved in 50 ml of deionized water to obtain a solution with SPC concentration of 1 mg ml2.3.m2 is the feeding amount of CMC, m1 is the feeding amount of SPI and m3 is the mass of SPI not reacting with CMC.Graft ratio (GR): after completion of the reaction between SPI and CMC, the pH of the solution was adjusted to 4.5 (isoelectric point of SPI) by hydrochloric acid, and the suspension was centrifuged at 12 000 r.p.m. for 10 min, the supernatant was removed, freeze-dried for 24 h. The GR was calculated according to the formula (2.1). As shown in Mw) of a protein can be determined based on the extent of migration of a protein, after the complexation with a strong cationic detergent SDS separated on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) [Molecular weight (DS-PAGE) . Sample −1. The samples for scanning electron microscopy (SEM) were prepared in the following manner: the sample solution was diluted 10 times, dropped on a substrate and then sprayed with gold, and observed under a scanning electron microscope . Thermal stabilities of the materials were determined by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC), using TGA2  from 40°C to 600°C at a heating rate of 10°C min−1 under a nitrogen atmosphere (20 ml min−1), using Q200 differential scanning calorimeter  from 40°C to 180°C at a heating rate of 10°C min−1 under a nitrogen atmosphere (20 ml min−1). Zeta potentials and particle sizes of SPC@AVM were measured using laser particle size analyser  after 10-fold dilution of the samples. To determine the contact angles of the samples, SPC@AVM solution was diluted to an AVM concentration of 100 mg l−1, and the same concentration of AVM solution was used as control. About 5–10 µl of the sample was dropped onto the leaf surface, and the contact angle was measured using a contact angle meter .Fourier-transform infrared (FTIR) spectra of the samples were obtained on a Spectrum-100 instrument , using KBr pellet method, in the spectral range of 4000–450 cm2.4.A = 0.03319C − 0.00591 (R2 = 0.999). The amount of free AVM was calculated, and the entrapment efficiency EE of AVM was calculated using the formulamtotal AVM is the total mass of AVM, and mfree AVM is the mass of AVM after centrifugation and filtration.According to a previous method , SPC@AVM2.5.M0 and M1 represent the weights of the leaves before and after soaking, respectively, and A represents the area of the leaves.Fresh leaves of vegetables were washed with deionized water and then cut into pieces of 2 × 2 cm dimensions for testing. The evenly sized leaves were soaked in the sample solution for 5 s and then lifted vertically using tweezers. They were weighed after the liquid stopped dripping. The liquid holding capacity (LHC) per unit area was calculated using the formula2.6.−1 AVM ethanol solutions were diluted to 100 mg l−1 with deionized water. The source for photolysis of AVM was a photochemical reactor . The sample solution (50 ml) was placed under a 300 W mercury lamp (Emax = 365 nm) at a distance of 15 cm from the source. At specific intervals, 1 ml of the sample was transferred to a brown volumetric flask, diluted to 25 ml with absolute ethanol and its absorbance was measured at 245 nm. The concentration of AVM in the sample at different time intervals was determined from the standard curve A = 0.03319C − 0.00591 (R2 = 0.999). The remaining ratio (RR) of AVM in the sample was calculated using the formulac0 is the initial concentration of AVM, and ic is the concentration of AVM at different time points.The SPC@AVM and 10 mg ml2.7.t), 1 ml aliquot of the solution was transferred to a 25 ml volumetric flask. An equal volume of 40% aqueous ethanol solution was then added to the conical flask. The sample aliquot taken in the volumetric flask was diluted with 40% aqueous ethanol to the mark. The absorbance of the diluted sample solution was measured by an ultraviolet spectrophotometer at 245 nm. The AVM concentration of sample solution was calculated from the standard curve A = 0.0299C + 0.00527 (R2 = 0.999), and the cumulative release rate was calculated using formula (2.5) [ic is mass concentration (mg l−1) of AVM for each sample at different time intervals, mAVM is the total mass of AVM in a conical flask.SPC@AVM solution (5 ml) was transferred into a dialysis bag and placed in a conical flask. Then, 50% ethanol–water solution (50 ml) was added at 36°C. At different intervals of time (f1) and the similarity factor (f2), were proposed to compare the differences of release curves [f1 represents the per cent difference between the two curves at every point of time. It measures the relative error between the two curves, while f2 is a logarithmic reciprocal square root transformation of the sum of squared error and is a measure of similarity in per cent dissolution between the curves. f1 and f2 were obtained from formulae (2.6) and (2.7) [n is the number of intervals in the experimentation, R0 is the release rate from the reference group at specific time intervals and iR is the release rate from the control group at specific time intervals.Two factors, difference factor (e curves . f1 reprnd (2.7) .2.6f1=102.8.−1 concentration. They were both exposed to 300 W using a mercury lamp (Emax = 365 nm) for 15 min to obtain corresponding UV/SPC@AVM and UV/AVM solutions. Using them, solutions were prepared which had AVM concentrations of 200, 100, 50, 25, 12.5 and 6.25 mg l−1 and insecticidal activities of these formulations were tested. The leaves were cut into homogeneous pieces and dipped into the sample solution for 5 min before they were removed and dried naturally. Then, the dried leaves along with 10 Plutella xylostella moths were placed in a Petri dish. These untreated leaves served as the blank experimental groups. The survival rate of plutella xylostella moths was evaluated after 48 h. Mortality and corrected mortality were calculated according to the Abbott formula, and median lethal concentration (LC50) and toxicity regression equations were calculated based on the probabilistic analysis.The solutions obtained above, SPC@AVM and AVM, were diluted with deionized water to 200 mg l2.9.p < 0.05) using the SPSS software package.All measurements were performed in triplicates. The results obtained were presented as means ± s.e. Data were analysed by analysis of variance  of SPI, CMC and SPC was studied, and the results are shown in figure 3c. The weight loss peaks at 246°C and 290°C correspond to decomposition temperatures of SPI and CMC, respectively. However, compared to SPI and CMC, the decomposition temperature of SPC was increased. During degradation, initially, non-covalent bonds, such as intermolecular and intramolecular hydrogen bonds, electrostatic bonds and bonds due to hydrophobic interactions were broken. Subsequently, covalent bonds between C–N, C(O)-NH and C(O)-NH2 were broken as temperature increased [The mass loss curve during heating of SPI, CMC and SPC is shown in figure 3ncreased ,36. SPC d shows the DSC plots of CMC, SPI and SPC. The glass transition temperatures of SPI and CMC were 112°C and 133°C, respectively. SPC showed a single glass transition temperature at 118°C, indicating that SPC was more stable than SPI. A single glass transition in the DSC heating curves indicated that SPI and CMC were highly compatible [Variation in glass transition temperature is an effective indicator of compatibility between SPI and CMC. Figure 3mpatible .3.3.W markers (line a), SPI (line b) and SPC (line c). In the case of line b, bands corresponding to molecular weights of 75, 48, 35, 18 and 10 kDa were observed, which was consistent with previous reports by Conti et al. [i et al.  However,i et al. .Figure 3.4.a–c shows the morphologies of SPC before and after heating and of synthesized SPC@AVM (mSPI : mCMC = 1 : 0.25). Figure 5d–f, respectively, represents the particle size distributions of SPC before and after heating and SPC@AVM, which were determined using Image—Pro Plus 6.0 software. The SPC particles were spherical in shape, had an average particle diameter of 136 nm and were connected to each other. This is due to the fact that SPI is a globular protein with an outer hydrophilic group and an internal hydrophobic group [Figure 5ic group . CMC is ic group . In the ic group .Figure c, the entry of AVM into SPC was followed by the transformation of the particles into spherical shape. The SPC@AVM solution showed good dispersibility, and a significant Tyndall effect under laser irradiation, indicating that SPC@AVM formed micelles in aqueous solution. In addition, comparison of figure 5e,f showed that the addition of AVM to SPC resulted in an increase in the average particle diameter, from 125 to 129 nm. This is because the hydrophobic group of SPC itself formed a cavity in the hydrophobic action. When AVM entered this hydrophobic cavity, the original hydrophobic balance was broken and a new hydrophobic cavity was formed due to enhanced hydrophobic interactions. The reformed hydrophobic core had a greater attraction for the SPC segment, which exceeded the tendency of CMC to form a film. Therefore, the SPC was re-transformed into a spherical shape from a block shape.As shown in figure 53.5.3.5.1.3.5.2.The zeta potential value of the system varies significantly with pH. The particle size of SPC-11@AVM increased with an increase in pH. At pH 5, the repulsive forces between the particles were small, and agglomeration caused an increase in particle size. With an increase in pH, the degree of protonation of the amino group decreased, whereas the degree of deprotonation of carboxyl group increased, accompanied by an increase in negative charge of the system. Therefore, the zeta potential of SPC-11@AVM was minimal at pH 9. As repulsive forces between particles increased, the dispersibility of the particles increased, and the system was more stable. Under acidic conditions, the number of positively charged amino groups was greater and there was an electrostatic force of attraction between them and the negatively charged AVM. Hence, encapsulation efficiency was significantly improved in acidic conditions as compared to the alkaline conditions. As mentioned above, the increase in repulsive forces between different SPC fragments led to larger gaps between SPC segments. This made the entry of AVM into the interiors of the microspheres easier. However, an increase in potential also caused an increase in the electrostatic repulsion between the surfaces of the microsphere and AVM. Therefore, the encapsulation efficiency between pH 5 and pH 7 did not change significantly. As the pH was increased to 9, the electrostatic repulsions between the microspheres and AVM increased again, due to which the encapsulation rate decreased.3.6.a, the contact angle made by AVM solution on the foliage was significantly reduced as compared to the deionized aqueous solution. This was because ethanol used to dissolve AVM was compatible with the hydrophobic portion of foliage. The contact angles of SPC-11@AVM, SPC-41@AVM and SPC-81@AVM solutions were 56.45°, 62.70° and 68.15° on the leaves. They were all less than that of deionized water (92.47°) and AVM solution (72.19°), which indicates the better wettability performance after encapsulation in SPC. Further, with an increase in CMC concentration, the contact angle decreased, indicating that CMC had an affinity towards leaf surface. This was because the main leaf component is a polysaccharide, containing many hydroxyl groups. Moreover, the leaf surface is covered with a waxy substance, its main components being fatty acids, fatty alcohols and fatty aldehydes [The wettability of the sample solution on the plant surface was studied by measuring the contact angle of the sample solution on the foliage. As shown in figure 6b, the maximum LHC of SPC-11@AVM was significantly more than the AVM aqueous solution. There was an increase of 41.41%, from 8.85 to 12.52 mg cm−2. This variation of LHC with CMC concentration was consistent with a change in contact angle. When the leaves were pulled out from the sample solution vertically, the solution that remained on the hydrophobic leaf surface gathered into droplets and rolled on the leaves. The increase in contact angle increased the distance of the centre of gravity of the droplet from the leaf surface and reduced the interactive forces between the leaves and droplets.As shown in figure 63.7.3.7.1.a,b shows the AVM release curves of different SPC@AVM samples and the AVM release curves of SPC@AVM samples under different pH conditions, respectively. Initially, the release of AVM is exponential, because the unencapsulated AVM in the solution passes quickly through the dialysis bag under osmotic pressure, which would be beneficial for immediate insecticidal effects. Subsequently, the rate gradually slowed down, which would be useful for prolonging the time of insecticidal action and reducing the required amount of pesticides.Figure 7f1 less than 15 and f2 greater than 50 indicate that the difference was not significant compared to that of SPC-11@AVM, and the release rate was only slightly higher than them [han them ,47.TablUnlike the encapsulation process of drugs, when AVM molecule enters the interiors of the microspheres, in addition to constraint by hydrophobic forces, the negative charge of carboxymethyl group on microsphere surface also generates electrostatic repulsion, making it more difficult to escape. As described above, SPC-11@AVM carried a greater number of negative charges and its release rate was slower. SPC-41@AVM and SPC-81@AVM had less access to carboxymethyl groups and carried a lesser number of negative charges, and so their release rates were faster. In addition, particle size was also closely related to the release rate of AVM. A large particle size indicated that the AVM needed to travel a longer distance during the release process and the release rate was slower.3.7.2.b, the release rate of SPC@AVM at pH 7 was faster than the release rates under acidic and alkaline conditions. Statistical analysis showed that under acidic conditions, f1 less than 15, f2 greater than 50, indicating that they were not significantly different from the release rates under neutral conditions. SPC@AVM had a large particle size under acidic conditions and AVM required a longer time to pass from the inside to the outside, which resulted in the slow release rate of AVM. In addition to this, the positive charge on SPC under acidic conditions increased and its mutual attraction with the negatively charged AVM increased, which was one of the main reasons for the slower escape rate of AVM. Under neutral conditions, diameters of the particles were significantly reduced relative to those under acidic conditions. The AVM could pass through the particles in a shorter period of time, and the release rate was accelerated. Under alkaline conditions, f1 greater than 15 indicated a significant change from its release under neutral conditions. SPC@AVM had a smaller particle size under alkaline conditions than under acidic conditions, and hence AVM had a shorter transit time. However, the negative charge on SPC surface increased and the negative charge on the microsphere surface increased the electrostatic repulsion of the internal AVM, making it more difficult to escape, resulting in a slower release rate.As shown in figure 73.7.3.The release mechanisms of AVM under different pH conditions was further investigated and the corresponding data parameters were fitted to zero-order and first-order equations, Higuchi kinetic model,  and Weibc–f, the AVM release curves followed the Weibull model, wherein the R2 was greater than 0.99. When 0.35 < a0 < 0.69, the diffusion in the fractal or disordered matrix was different from the permeation group. When the a0 value was greater than 0.69 and less than 0.75, the release followed the Fick diffusion model in the Euclidean or fractal space. When 0.75 < a0 < 1, along with diffusion in the normal Euclidean matrix, there is another release mechanism [a0 was more than 0.35 and less than 0.69, indicating that the release of AVM was different from that of the percolation cluster. The release of AVM was not a movement in continuous media but has a process from the inside out, which is subject to many obstacles. At pH 9, a0 was greater than 0.75, which indicated that the release of AVM was affected not only by Fick diffusion but also by another release mechanism. In addition to concentration, the other effect was electrostatic interaction, which had a capturing effect on AVM.As shown in figure 7echanism . At pH 53.8.It is well known that AVM is susceptible to decomposition particularly under ultraviolet light. Therefore, improving the stability of AVM is the key to improve the utility of pesticides. 3.9.y = 1.2722x + 3.5760 and y = 1.2289x + 3.1356, and their LC50 values were 13.16 and 32.90 mg l−1, which indicated a significant difference in toxicity. The decline in AVM toxicity was due to photodegradation of AVM, which was the main reason for the low utilization of AVM during its use.The toxicities of AVM and UV/AVM were compared. As shown in 50 value of AVM, which was processed into a slow-release formulation was usually higher than that of the original drug due to its slower release rate [50 of UV/SPC@AVM was significantly higher compared with SPC@AVM.In previous reports, the LCase rate . As show50 value of UV/SPC@AVM was higher than that of AVM and also much smaller than UV/AVM, which indicated that the toxicity of SPC@AVM after illumination was intermediate between them. From the above anti-UV experiment, it can be confirmed that the encapsulation of AVM could effectively slow down its photo-degradation rate. Therefore, after irradiation, the amount of free AVM in SPC@AVM was greater than that in the untreated AVM, and consequently the insecticidal effect was good.The LC4.In this study, CMC was grafted onto the SPI surface to obtain SPC, which was then heated to increase its hydrophobicity. The reaction between SPI and CMC was demonstrated by different characterization methods. SPC@AVM could improve the residual amount of AVM on the leaf surface, reduce leaf loss and provide AVM with excellent UV resistance, which could effectively extend the half-life of AVM. Toxicity tests confirmed that encapsulation of AVM could effectively reduce the attenuation of AVM toxicity under UV irradiation. During the release process, SPC@AVM showed pH-responsive property, and the release of AVM was consistent with the Weibull model under different pH conditions. This study aimed to construct a water-based drug-loading system for AVM, which is an environmentally unstable and insoluble pesticide. SPC@AVM could reduce the loss and degradation of pesticide and control the release of pesticides, which could effectively improve the utilization of pesticides by reducing the amount of pesticide required.


Video feedback has been shown to be an effective teaching tool that can improve student learning when having them view their own performance. However, the literature on the effect of integrating smartphones with video feedback in fundamental nursing skills teaching is sparse.This study aimed to explore the potential effects of video feedback through smartphone-based instant messaging on teaching undergraduate nursing students fundamental nursing skills.We conducted a study on teaching fundamental nursing skills to 6 classes of second-year undergraduate nursing students. In 2 classes (the intervention group), the instructor elected to use smartphone-based video feedback to facilitate teaching; instructors in the other 4 classes (the control group) elected to use routine methods of teaching without video feedback. Scores from the final examination, in-class assignments, and the General Self-Efficacy Scale questionnaire were collected and compared between the two groups. Multiple linear regression analysis was performed to estimate the independent effect of video feedback after adjusting for gender, age, and prior experience in the use of WeChat/QQ in learning applications. An ad hoc questionnaire was used for student evaluation of the novel smartphone-based video feedback teaching method.A total of 195 nursing students (65 in the video feedback group and 130 in the control group) completed the study and were included in the final analysis. Mean and standard deviation of scores on the final examination, bed making, aseptic procedure, vital signs measurement, and oxygen therapy were 91.29 (SD 2.36), 90.52 (SD 3.18), 93.23 (SD 3.16), 91.65 (SD 4.21), and 92.06 (SD 3.58), respectively, in the video feedback group and 89.99 (SD 3.12), 81.71 (SD 8.63), 87.12 (SD 5.50), 87.45 (SD 8.00), and 90.37 (SD 6.36), respectively, in the control group . The mean and standard deviation of scores for assignments in catheterization and enema and General Self-Efficacy Scale were 89.69 (SD 3.22), 91.14 (SD 3.15), and 24.52 (SD 5.35), respectively, in the video feedback group and 88.82 (SD 7.48), 90.79 (SD 6.08), and 24.50 (SD 6.16), respectively, in the control group . The majority (over 98%) of nursing students were satisfied with this smartphone-based video feedback teaching method.Video feedback through smartphone-based instant messaging may be an effective way to improve nursing students’ academic performance and professional skills. Video feedback has been demonstrated to be an effective teaching tool that can improve student skills by having them view their own performance on video . StudentSmartphones are very popular with the youth and could be exploited to improve learning as a consequence of personal behavior changes ,4. In reAttempts have been made to use smartphones to achieve positive results for a range of medical and nursing education issues, including coordination supporting among groups , theory This study was carried out in Wuxi Taihu University School of Nursing between October 1 and November 30, 2018. There were 6 classes of second-year undergraduate nursing students who were taking the fundamental nursing skills course during that semester. There were 3 instructors, each of whom taught 2 classes with no overlap/exchanges/substitutions throughout the entire semester. The instructors were qualified nursing educators with the same seniority. At the beginning of the semester, an instructor for 2 of the 6 classes elected to use smartphone-based video feedback to facilitate her teaching. One author of the research team approached the instructors with a request to undertake a study to evaluate the effect of this new method of teaching. After the instructors agreed to participate in the study, an approval from the institutional review board of Taihu University was obtained. Students from the 2 classes whose instructor planned to use the smartphone-based video feedback comprised the intervention group (video feedback group), while students from other 4 classes whose instructors planned to use routine teaching methods (no video feedback) formed the control group. All students provided written informed consent to participate in the study. Fundamental nursing skills taught in this study were performed on manikin simulators and included bed making, aseptic procedures, vital signs measurement, oxygen therapy, catheterization, and enema administration. Students from both groups were asked to practice and complete one assignment for each skill after class. In the video feedback group, the instructor first explained the basic material and demonstrated the basic procedures in the classroom. Afterward, the nursing students worked in groups of 3 or 4 to record videos of each other on their smartphones. Each video lasted 5 to 15 minutes and took more than 10 minutes to upload and download. The students then sent the videos to the instructor via instant messaging for evaluation and feedback on their nursing practices (the instructor spent 5 to 15 minutes going through each video).Scores on the final examination and on the 6 nursing skills assignments  were the main outcomes of interest. Teaching and evaluation at Wuxi Taihu University School of Nursing were performed by different faculty members .The General Self-Efficacy (GSE) Scale , which iFinally, an ad hoc questionnaire was developed to measure the students’ own evaluations of the video feedback teaching method in the video feedback group. The questionnaire has 6 items with 5 response categories expressing the degree of agreement with each item.We first compared the distribution of baseline characteristics between the intervention and control groups. We then compared the scores on the final examination, the 6 nursing skills assignments, and GSE scores between the two groups. Multiple linear regression analysis was conducted to estimate the independent effect of video feedback on student performance after adjusting for gender, age, and prior experience in the use of WeChat/QQ in learning applications. Two-sided tests were used in all comparisons between the two study groups. Finally, student evaluations of the video feedback teaching method were presented. All analyses were performed using SAS v9.4 (SAS Institute Inc).A total of 195 nursing students (65 in the video feedback group and 130 in the control group) completed the study and were included in the final analysis. Our study found that video feedback through smartphone-based instant messaging may have the potential to improve the performance of nursing students in fundamental nursing skills, especially with respect to skills related to bed making, aseptic procedure, vital signs measurement, and oxygen therapy. Overall, most nursing students were satisfied with the smartphone-based video feedback teaching method. Although the study failed to demonstrate an improvement in overall self-efficacy, nursing students perceived that their interests and autonomous learning abilities had been improved.To our knowledge, this is the first study incorporating smartphone instant messaging with video feedback in fundamental nursing skills teaching. Although the effect of video feedback in medical education has been well established, the need for standard equipment in regular video feedback makes this teaching method not convenient to some extent. Through smartphone-based video messaging, students were able to record the videos at a time and place that was convenient to them and send their videos to the instructor for timely and precise feedback. In Wuxi Taihu University School of Nursing, faculty members not involved in the teaching of the particular course acted as the evaluators of student performances, thereby avoiding bias in the assessment. In contrast to Western countries, Chinese universities admit students as high school graduates according to their performance on the National University/College Entrance Examination. Top-ranked universities have priority to admit students with higher scores on the examination and superior academic performance in high school. Leading national universities recruit top students from all provinces across the country, while local universities like Wuxi Taihu University mainly recruit students locally. Once admitted, students are assigned to different classes by the university administration in a somewhat random fashion. In our study, students from the intervention and control groups were similar in gender, age, and previous life experience, so any differences between the groups should be attributable to the intervention and not to inherent differences between the two groups. The most important previous life experience relevant to this study is the prior experience using WeChat/QQ messaging in learning, which was not different between the two groups . We usedWe acknowledge limitations of this study. First, whether to use the smartphone-based video feedback was a choice by the course instructor. Although all 3 instructors are qualified nursing educators with the same seniority, the instructor who elected to use smartphone-based video feedback to facilitate her teaching may be more motivated and this may have resulted in better quality in her teaching. Because this was not a randomized controlled trial, we cannot be sure about this source of bias. Second, the answers to some of the GSE Scale questions may be somewhat inaccurate and imprecise because some of the participants completed the questionnaire several weeks after the course was over. Thus, recall bias may exist. Third, the questionnaire for student evaluation of the smartphone-based video feedback was developed on an ad hoc basis without formal validation or reliability assessment. Fourth, although faculty members who evaluated student performance did not participate in teaching the course, they were from the same school and knew who the course instructor was. As a result, the skills evaluation could not be considered entirely blind. Fifth, there may be a chance that some students in the intervention group may not have actually done the video or the instructor may not have actually sent feedback to some students. Either way, actual effect of video feedback may have been diluted. Unfortunately, we did not collect these data and could not assess the impact of the quality of video feedback on the observed effect. Sixth, the time needed for video uploading and downloading depended on the network speed and could have been frustrating for students and instructors alike if the network was slow.This study explored a method of improving fundamental nursing skills teaching for undergraduate nursing students. It integrated smartphone technology, a mobile app, and video feedback in facilitating teaching. Smartphone-based feedback could offer a novel, flexible study method, and the feedback could allow participants to know whether they are performing well or not . StudentSelf-efficacy is related to one’s beliefs as to whether or not they are capable of completing a certain task . StudiesMost students were satisfied with the smartphone-based video feedback teaching method, a finding consistent with a previous study . A systeOur study suggests that the use of video feedback through smartphone-based instant messaging may be an effective way to improve nursing students’ overall performance and skills. This novel teaching modality makes use of relatively inexpensive smartphone technology, which is now almost universally available and familiar to the millennials who will become tomorrow’s health professionals. Extending the use of smartphone-based video feedback teaching techniques more broadly across multi-year academic curricula and other areas of health sciences could lead to even better results than those observed in this limited study, including not only increased performance but increased self-efficacy.


Bacillus thuringiensis insecticidal proteins (Bt toxins) have been widely used in crops for agricultural pest management and to reduce the use of chemical insecticides. Here, we have engineered Bt toxin Cry2Ab30 and bioconjugated it with 4”-O-succinyl avermectin (AVM) to synthesize Cry2Ab–AVM bioconjugate. It was found that Cry2Ab–AVM showed higher insecticidal activity against Plutella xylostella, up to 154.4 times compared to Cry2Ab30. The binding results showed that Cry2Ab–AVM binds to the cadherin-like binding protein fragments, the 10th and 11th cadherin repeat domains in the P. xylostella cadherin (PxCR10–11), with a much higher affinity (dissociation equilibrium constant DK = 3.44 nM) than Cry2Ab30 (DK = 28.7 nM). Molecular docking suggested that the macrolide lactone group of Cry2Ab–AVM ligand docking into the PxCR10–11 is a potential mechanism to enhance the binding affinity of Cry2Ab–AVM to PxCR10–11. These findings offer scope for the engineering of Bt toxins by bioconjugation for improved pest management. Bacillus thuringiensis insecticidal proteins (Bt toxins) have been shown to be highly effective in agricultural pest control +.4”-O-Succinyl avermectin was synthesized according to previously reported procedures ,43. Selecry2Ab30 was transformed into E. coli BL21 (DE3) cells. The bacterial stab was inoculated onto the LB medium containing kanamycin (35 μg/mL)  and incubated at 20 °C. The expression of Cry2Ab30 was induced with 0.2 mM isopropyl-B-D-thiogalactopyranoside (IPTG)  after the OD600nm reached 0.6. After that, Cry2Ab30 was purified using a Ni-IDA prepacked column .The production and purification of Cry2Ab30 was as described in our previous study . The plaP. xylostella cadherin (PxCR10–11) binding Cry2Ab30 were expressed and purified as described in our previous study [2HPO4, 1.8 mM KH2PO4, pH 7.4). PxCR10–11 was purified using a GST prepacked gravity column .The 10th and 11th CR domains (1258–1602 aa) in the us study . Cells w−6 mol) was firstly dissolved in 1 mL of DMF . Then, triethylamine (2 × 10−5 mol)  was added to provide protons, EDC (2 × 10−5 mol)  was added to form an O-acylisourea active intermediate, and NHS (5 × 10−5 mol)  was added to form an NHS ester intermediate; this intermediate was stirred for 0.5 h at 25 °C to activate 4”-O-succinyl avermectin. Cry2Ab30 (2 × 10−8 mol) was desalted into PBS  using a size-exclusion column , and activated 4”-O-succinyl avermectin was then added to Cry2Ab30 PBS buffer. The mixture was vortexed and allowed to react at room temperature for 2 h. After the reaction was completed, Cry2Ab–AVM was purified using a PD-10 desalting column  [4”-O-Succinyl avermectin  . Cry2Ab–NJ, USA) . The fluNJ, USA) . Further10–11 were detected by a ligand blot assay [10–11 protein in TBST buffer overnight followed by three washes of TBST buffer.The binding abilities of Cry2Ab30 and Cry2Ab–AVM to PxCRot assay . Cry2Ab310–11 were assayed on an Octet Red 96 biolayer interferometer  [10–11 was first loaded onto anti-GST biosensors  for 300 s. The binding of Cry2Ab30 and Cry2Ab–AVM to immobilized PxCR10–11 was measured with a 400 s association followed by a 400 s dissociation. The buffer was 10 mM GSH, 50 mM Tris, pH 8.0. The dissociation equilibrium constant (KD) was determined by Octet System Data Analysis software  [The binding kinetics of Cry2Ab30 or Cry2Ab–AVM to PxCRCA, USA) ,49. GST-CA, USA) ,52.P. xylostella. Different doses of Cry2Ab30 and Cry2Ab–AVM solutions were spread on the surface of the diet and allowed to dry. Samples were treated with the same volume of PBS buffer as controls. Thirty individual larvae were used for each concentration. All the bioassay experiments were repeated in triplicates. Observations were recorded at 72 h, and the mortality was determined and statistically analyzed using SPSS 17.0  [The bioassays of Cry2Ab30 and Cry2Ab–AVM were performed with second-instar IL, USA) .P. xylostella were starved for 5 h and then fed an artificial diet including Cry2Ab30 or Cry2Ab–AVM for 24 h. Larvae fed with PBS buffer  were set as negative controls. The larvae were then dissected, and the midgut tissues were then isolated and immediately fixed in 2.5% glutaraldehyde  and post-fixed in 1% OsO4 . The fixed midgut tissues were then immersed into Epon for embedding. Ultrathin sections were sliced using a Leica EM UC7 ultramicrotome , and stained with uranyl acetate and then lead citrate [Third instar larvae of  citrate . The ult10–11 was built by I-TASSER servers (http://zhanglab.ccmb.med.umich.edu/I-TASSER) and the molecular structure of 4”-O-succinyl avermectin was built using ChemBioDraw Ultra 12.0 . Docking of 4”-O-succinyl avermectin to PxCR10–11 was analyzed by Molecular Operating Environment (MOE)  with default parameters; 4”-O-succinyl avermectin was selected as the ligand [The protein structure of PxCRe ligand . All stre ligand .