Despite the availability of high-quality evidence and clinical practice guidelines for the effective management of pediatric pain, this evidence is rarely used in practice for managing children’s pain from needle procedures such as vaccinations. Parents are generally unaware of pain management strategies they can use with their children.This study aimed to develop, implement, and evaluate the implementation effectiveness of a parent-directed YouTube video on evidence-based strategies to manage needle pain in children.This was a descriptive study. Analytics were extracted from YouTube to describe video reach. A Web-based survey was used to seek parent and health care professional (HCP) feedback about the video. The 2-minute 18-second video was launched on YouTube on November 4, 2013. In the video, a 4-year-old girl tells parents what they should and should not do to help needles hurt less. The key evidence-based messages shared in the video were distraction, deep breathing, and topical anesthetic creams. A group of parents (n=163) and HCPs (n=278) completed the Web-based survey. Measures of reach included number of unique views, country where the video was viewed, sex of the viewer, and length of watch time. The Web-based survey assessed implementation outcomes of the video, such as acceptability, appropriateness, penetration, and adoption.As of November 4, 2018 (5 years after launch), the video had 237,132 unique views from 182 countries, with most viewers watching an average of 55.1% (76/138 seconds) of the video. Overall, both parents and HCPs reported strong acceptance of the video  and reported significant improvements in plans to use distraction, deep breathing, and topical anesthetic creams.This parent-directed YouTube video was an acceptable and appropriate way to disseminate evidence about the procedure of pain management to a large number of parents. Despite the availability of high-quality evidence and clinical practice guidelines for effective management of pediatric pain, best available evidence is rarely used in practice for managing children’s pain ,2. More Existing studies of knowledge translation  inteSocial media brings new opportunities for KT of health information to parents and an opportunity to reach a very large group of individuals. The use of social media is growing exponentially, with recent data showing that 65% of all adults now use social media (a 10-fold increase in the last decade) -27. PareHowever, developing parent-targeted KT tools for pediatric pain management is not enough. It is important to evaluate the impact of the tool. Although traditional clinical trials focus on the efficacy of interventions in reducing pain in children, implementation studies focus on outcomes associated with the intervention that determine whether an intervention actually gets used. Implementation outcomes include acceptability , adoption , appropriateness , feasibility , fidelity , implementation cost , penetration , and sustainability  .The objective of this study was to develop, implement, and evaluate the implementation effectiveness of a parent-directed KT YouTube video to reach and inform parents on evidence-based strategies to manage needle pain in children. Video reach was measured using available Web-based analytics  captured 5 years after the public launch of the video. The effectiveness of the video was assessed via several implementation outcomes  among a sample of parents and HCPs who completed a Web-based survey.A brief (2 min 18 seconds) YouTube video for parents was developed to summarize evidence-based strategies for procedural pain management in a fun and entertaining way. The video  is hosteBefore the production of the video, a storyboard and a script were developed in collaboration with a communication company based on the synthesis of existing evidence-based information that was verified by the research team, partners, and parents. Initial drafts went through several rounds of revision and refinement before finalization. Once produced, the video was disseminated using a range of Web based and social media strategies, including emails, listservs, parenting forums, magazines, news coverage (television and newspaper), discussion groups, websites, blogs, social media, and other networking sites . To further promote the video, posters, handouts, and social media images were also created and revised, and YouTube advertising campaigns and sponsored Facebook posts were used , which Reach data were collected through social media metrics available via YouTube analytics . Although Google and its subsidiary YouTube do not publish the algorithms for generating their reports, YouTube analytics have been consistently used to report on reach statistics for videos . Data weA Web-based survey was used to collect feedback from parents and HCPs. Viewers were prompted to complete an electronic survey after watching the video available through a link in the description text on YouTube as well as through the dissemination methods mentioned above. A total of two separate surveys were created: 1 for parents and 1 for HCPs. Participants who completed the surveys were self-selected from the viewers, with no inclusion or exclusion criteria stated with survey completion implying consent. Ethics approval for the survey was obtained from the Research Ethics Board of the IWK Health Centre; the survey was left open for 2 years .not at all and 5 was very much. The survey was assessed for content validity and is available in The approximate survey completion time was less than 10 min. Questions were developed to discover acceptability, adoption, and feasibility of the evidence-based techniques shown in the video. The same survey was used to evaluate the newborn video . Aside fFive years after the launch, the video had a total of 237,132 unique views and had been viewed in 182 countries. The average view time was 1 min 16 seconds, which is 55.1% (76/138 seconds) of the video and corresponds to the end of the initial presentation of all evidence-based strategies by the little girl to her mother . The United States (42.5%) and Canada (24.8%) had the most views, followed by Australia (10.2%), New Zealand (8.5%), and the United Kingdom (5.4%). Viewers were primarily females (53.3%), aged between 25 to 34 years (36.6%) and 35 to 44 years (37.5%). Traffic to the video was through YouTube advertising , searching on YouTube , an external link , a suggested video on YouTube , or other methods . The video was primarily watched on tablets , computers , mobile devices , or other devices . The video was shared directly from YouTube a total of 801 times through various channels, including copying the link , Facebook , WhatsApp , Twitter , and other . The video was watched without subtitles 91.5%  of the time, with Arabic , French , Spanish , Russian , and Portuguese  as the top 5 most selected closed caption subtitles.HCPs also reported strong acceptance of the video  and that the information obtained would be helpful to their practice  and HCPs (n=98) were provided with the opportunity to offer an open-ended comment, seven themes arose: praise, suggestions for improvements, questions, plans to share video, identifying they already use these strategies, critiques about video or content, or other comments about thoughts and experiences. This paper summarizes an evaluation of the implementation effectiveness of a parent-directed YouTube video about strategies parents can use to minimize their children’s procedural pain. The findings of this study suggest that the video is an acceptable method for disseminating evidence-based information regarding pain management with a high likelihood of adoption.Penetration was high as the YouTube video received significant uptake during the period of analysis with 237,132 views. In similar parent-targeted pain management videos related to newborn pain, penetration rates measured at 12 months and 18 months had 65,478 views and 157,938 views, respectively, with the latter video part of this series receiving similar promotion as the current video ,40. PareMost parents and HCPs felt that the strategies were appropriate to minimize child pain during immunization. However, some concerns related to the feasibility of the strategies were noted in comments from parents and HCPs. One-third of the parents commented that the strategies provided were not practical , whereas nearly half of all HCPs identified similar barriers, such as cost and application timing for the topical anesthetic cream. Parents also commented on concerns related to their HCP’s acceptance of the strategies: “I’m not 100% sure my doctor would be ok with us blowing bubbles at this office.” This was reflected in some HCP responses as well: “may find other way to encourage deep breathing to avoid ‘bubble mess’.”In terms of the costs associated with this video, it cost Can $10,000 to professionally develop and produce this video and an additional Can $5000 to promote the video. It took considerable effort on the part of the team to promote the video, and further efforts in this area could be improved via partnering with parenting media who already have expertise in creating parenting content and an established reach to parents. Given the cost of video development and promotion in finances and time, this may be a hindrance for individual researchers or health care providers to undertake alone. However, consideration should be undertaken by public health organizations or clinical practice guideline developers that may have the financial resources and desire to create parent-targeted content. On the basis of this study and previous work, parent-targeted videos have the ability to reach a large number of people ,40 and sThis study had a number of limitations. First, those parents and HCPs who completed our Web-based survey may not be representative of all parents and HCPs; they may represent groups of individuals who had a particular interest in or willingness to adopt pain management strategies. Second, our study did not examine the actual implementation of pain management strategies by parents and HCPs; we simply assessed their report of whether they would use the strategies. Future prospective research should follow parents and HCPs over time to determine whether the strategies were effectively implemented. Finally, our reach analytics were limited in our reliance on YouTube analytic reports for viewer data .Communicating evidence-based recommendations for pain management to parents could be powerful, yet large-scale efforts to disseminate this knowledge to parents are lacking. A recent randomized trial showed that parental exposure to a 5-min educational video significantly increased parental pain management behaviors and decreased child pain . HoweverBuilding on previous work evaluating publicly available YouTube videos directed at parents for pain management ,50, thisAs identified in an earlier scoping review on publicly available videos on pediatric needle pain management , there iThis YouTube video benefited knowledge users, in this case, parents and HCPs, directly by providing them with evidence-based information about pain management for procedures in an engaging and accessible way. Owing to the dissemination method , the video reached a very wide, international audience. Making evidence-based information on pediatrics available in this way has the potential to result in improved awareness and use of evidence-based practices, including, in this case, reduced pain and distress in children undergoing painful medical procedures and their families. Given the evidence of success in parent-targeted videos to improve pain management, opportunities exist to expand this work to other areas of pediatric health. Future research should consider conducting randomized controlled trials that explore the impact of such videos on behavior change outcomes.


Malnutrition and poor physical performance are both conditions that increase in prevalence with age; however, their interrelation in a clinically relevant population has not been thoroughly studied.This study aimed to determine the strength of the association between malnutrition and measures of both static and dynamic physical performance in a cohort of geriatric outpatients.This cross-sectional study included 286 older adults  who were referred to geriatric outpatient mobility clinics. The presence of malnutrition was determined using the Short Nutritional Assessment Questionnaire . Measures of dynamic physical performance included timed up and go (TUG), 4-m walk test, and chair stand test (CST). Static performance encompassed balance tests and hand grip strength (HGS). Physical performance was standardized into sex-specific Z-scores. The association between malnutrition and each individual measure of physical performance was assessed using linear regression analysis.19.9% of the cohort was identified as malnourished. Malnutrition was most strongly associated with CST and gait speed; less strong but significant associations were found between malnutrition and TUG. There was no significant association between malnutrition and HGS or balance.Physical performance was associated with malnutrition, specifically, dynamic rather than static measures. This may reflect muscle power being more impacted by nutritional status than muscle strength; however, this needs to be further addressed.Malnutrition is associated with dynamic physical performance in geriatric outpatients, which should inform diagnosis and treatment/prevention strategies. Malnutrition and poor physical performance are two highly prevalent conditions in older adults that are associated with poor health outcomes, such as higher morbidity, mortality, and lower quality of life –4. Both Muscle is a key linking substrate between malnutrition and physical performance. Decreased muscle mass has been found to be an outcome of malnutrition  and decrMalnutrition has been defined “a state resulting from lack of uptake or intake of nutrition that leads to altered body composition (decreased fat free mass) and body cell mass leading to diminished physical and mental function and impaired clinical outcome from disease” and, in this definition, is synonymous with undernutrition . In a chThis cross-sectional inception cohort study aimed to determine the strength of association between common clinically used measures of malnutrition and measures reflecting both dynamic and static physical performance in a population of geriatric outpatients referred to a mobility clinic.Older adults from two Dutch inception cohorts were included: the Bronovo cohort and the Center of Geriatrics Amsterdam (COGA) cohort. The Bronovo cohort included community-dwelling older adults referred to the geriatric outpatient mobility clinic  from March 2011 to January 2012. The COGA cohort included patients referred to the geriatric outpatient mobility clinic  from January 2014 to December 2015. Individuals were included if they completed a nutritional assessment questionnaire  and completed at least one out of the five continuously measured physical performance tests . All patients underwent a comprehensive geriatric assessment (CGA). Patient characteristics were derived by questionnaires or obtained from medical records. Questionnaires and measurements were performed as part of CGA within routine care. The protocol and study design of the Bronovo cohort  and the Each study has been approved by the local ethical committees and has been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki. The need for individual informed consent was waived as the research was based on regular patient care.The presence of malnutrition was determined in the Bronovo cohort using the Short Nutritional Assessment Questionnaire (SNAQ) score, including questions about unintentional weight loss, decrease in appetite, and the use of supplemental drinks or tube feeding over the last month and has been further described elsewhere . SNAQ scMeasures of dynamic physical performance included 4-m walk test from which gait speed can be obtained and chair stand test (CST), as sub-tests of the Short Physical Performance Battery (SPPB)  as well Measures of static physical performance encompassed the third subtest of the SPPB, i.e., balance tests   as well n) and the percentage (%).Descriptive statistics for continuous variables were presented as mean and standard deviation (SD) when data were distributed normally or as median interquartile range [IQR] if the data had a skewed distribution. Dichotomous variables were reported by the sample size/number  and adjusted for age, sex, and the presence of multimorbidity. Interpretation of the results of this linear regression analysis with standardized outcome measures was as follows: the effect size represents the average difference in effect of the presence of malnutrition on the physical performance test compared to the absence of malnutrition in standard deviations.For each measure of physical performance, respectively, individuals were included if they were able to perform the test. Continuous physical performance measures were standardized into sex-specific Z-scores to allow for direct comparison of effect sizes of malnutrition with physical performance tests. Variables with a skewed distribution were log transformed prior to making Z-scores. Linear regression analysis was used to study the association between malnutrition with both dynamic and static measures of physical performance. Balance tests were dichotomized into two groups, unable to maintain for 10 s and able to maintain for 10 s, and were included in the analysis to determine the presence of an association using a binary logistic regression. Results for the linear regression analysis are presented as beta, or for the logistic regression as odds ratios (OR), and 95% confidence intervals with p value of less than 0.05 was considered statistically significant. Visualization of results was performed using GraphPad Prism 5.01.All statistical analyses were conducted using SPSS , version 24.0 . A n = 107) of the outpatients. Mean body mass index (BMI) was 25.4 kg/m2 (SD 4.3). The prevalence of malnutrition was 19.9%.This study included 286 geriatric outpatients, with a mean age of 81.8 years (SD 7.2). Characteristics of the outpatients are shown in Table Table In a cohort of geriatric outpatients, malnutrition as defined by SNAQ score was most strongly associated with CST and gait speed; less strong associations were found between malnutrition and TUG and the compound SPPB score. There was no significant association between HGS or balance.Previous studies have classified physical performance into two groups: dynamic physical performance  and static physical performance  . In the Our study confirms the previously reported associations between malnutrition and CST, gait speed, TUG, and SPPB Score –32, withAs hypothesized, none of three balance tests were associated with malnutrition. A previous study showed that muscle strength, measured by HGS and KES, rather than muscle mass, were positively associated with ability to maintain standing balance . This reThere is no gold standard for the diagnosis, screening, or definition of malnutrition, so the results may be highly dependent on the measure used to define malnutrition. The SNAQ, which was used in this study, adopts a broad definition of malnutrition that specifically reflects undernutrition by emphasis on unintentional weight loss and loss of appetite , 18, 41.To our knowledge, this is the first study to compare the effects of the association of malnutrition with different measures of physical performance in geriatric outpatients. With the methods used in this cross-sectional study, it is difficult to distinguish the underlying systems involved in physical performance that may have been affected by malnutrition because of mutual interaction. Future studies should aim to identify these systems as well as factors that may influence the relationship between malnutrition and physical performance. There is clinical need to expand on these findings and further characterize the interrelation between the nutritional and physical state in older adults.This study shows that physical performance measures have individual value in relation to malnutrition and should not be used on their own interchangeably to represent physical performance; the present results suggest that focusing on measures of dynamic physical performance and muscle power would be most informative in terms of reviewing the physical impact of nutritional status. Furthermore, future studies should distinguish and examine the association between malnutrition different outcomes related to muscle function, including muscle power, muscle strength, and muscle mass.


Background/Purpose: Malnutrition is a significant global public health burden with greater concern among children under five years in Sub-Saharan Africa (SSA). To effectively address the problem of malnutrition, especially in resource-scarce communities, knowing the prevalence, causes and risk factors associated with it are essential steps. This scoping review aimed to identify the existing literature that uses classical regression analysis on nationally representative health survey data sets to find the individual socioeconomic, demographic and contextual risk factors associated with malnutrition among children under five years of age in Sub-Sahara Africa (SSA). Methods: The electronic databases searched include EMBASE (OVID platform), PubMed (MEDLINE), Cumulative Index to Nursing and Allied Health Literature (CINAHL), Scopus, Web of Science (WoS) and Cochrane Library. Only papers written in the English language, and for which the publication date was between 1 January 1990 and 31 July 2020, were included. Results: A total of 229 papers were identified, of which 26 were studies that have been included in the review. The risk factors for malnutrition identified were classified as child-related, parental/household-related and community or area-related. Conclusions: Study-interest bias toward stunting over other anthropometric indicators of malnutrition could be addressed with a holistic research approach to equally address the various dimension of the anthropometric indicators of malnutrition in a population. Malnutrition is the intake of an insufficient, surplus or disproportionate amount of energy and/or nutrients . MalnutrIn recent times, Sub-Saharan Africa (SSA) has had so much to grapple with in terms of the malnutrition burden. In 2015, SSA accounted for more than 30% of global underweight children . AdditioBeside the SSA region, malnutrition has posed some serious public health challenges in other regions of the world. For instance, in Asia, though considerable steps have made towards the global target, there are lapses in achieving the targets that still exist. The region has experienced a prevalence of overweight among children under five years, which is less than the global average, but it also experienced more than the global average in stunting and wasting, which stood at 22.7% and 9.4%, respectively . SimilarGenerally, malnutrition can lead to cognitive and physical impairment in children, especially those under five years old, with a high rate of morbidity and mortality ,16. A chThis scoping review aimed to identify existing literature that used classical regression analysis, , on nationally representative health survey data sets to find the individual socioeconomic, demographic and contextual risk factors associated with malnutrition among children under five years of age in Sub-Sahara Africa (SSA).The methodological pattern used in this scoping review followed Arkey and O’Malley , Lecac eThere was no review protocol and registration done for this scoping review.The research question was stated having been guided by PICOTS  framework of Agency for Healthcare Research and Quality (AHRQ) . The primary research question for this scoping review is what risk factors are associated with the malnutrition status of children less than five years of age in Sub-Saharan Africa countries that used classical regression methods to analyze a nationally representative survey data set?What are the existing examples of evidence of individual and contextual risk factors associated with the malnutrition status of children under five years in Sub-Saharan Africa countries?What evidence exists in the use of classical regression analysis methods to determine the risk factors related to the malnutrition of children under five years in Sub-Saharan African countries?Other secondary research questions are:The studies included in the review followed the PICOTS  criteria enumerated and defined in The first author (PEO) of the School of Health and Related Research (ScHARR), the University of Sheffield, United Kingdom, carried out the literature search. The process was done at least twice on each of the databases consulted and we compared the outcomes to ensure that relevant papers were not excluded. The selection of bibliographies for screening was done on the basis of keywords and subject headings. The electronic databases searched include EMBASE (OVID platform), PubMed (MEDLINE), Cumulative Index to Nursing and Allied Health Literature (CINAHL), Scopus, Web of Science (WoS) and Cochrane Library. Only papers written in the English language, and published between 1 January 1990 and 31 July 2020, were included.In this scoping review the search strategy involved searching for key terms or text words individually. The phrases were first searched in EMBASE (OVID platform) using “map terms to subject heading”. The search terms applied were derived from the PICOTS categories and they include the variants of Sub-Saharan Africa, under five years, the determinants or risk factors, malnutrition status, and (with/without) regression techniques. These various terms were used with appropriate Boolean connectors, ‘AND/OR’, and with publication dates and research designs applied as restrictions. The sample of the search strategy in EMBASE is displayed in In the EMBASE search strategy result , the pubThe reviewer, PEO, screened all the selected literature for titles and abstracts using the inclusion and extraction criteria as a benchmark . This prInitially the data extracted from the included articles were deposited into a Microsoft Excel spreadsheet designed by the reviewer specifically for this review. The relevant information obtained includes authors/year of publication, the survey type, the sample size, classical regression type and country of study. Other information includes the study aim, the outcomes , the prevalence, various predictor variables assessed , significant risk factors found for each of the malnutrition-related indicators, the specific conclusion reached, and the statistical software used for computation.The results section reports the profile of the quantitative analysis of risk factors associated with malnutrition in under five children in SSA following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) checklists ,22. Twenty-five studies were duplicated in the search at different times . The duplication led to the removal of 47 titles. Out of a total of 177 studies screened for titles and abstract, 138 studies were removed for not meeting the inclusion criteria. A total of 26 studies were finally selected for this study after excluding 13 papers. The reasons for excluding these papers are listed in the chart above .To answer the questions raised in this scoping review, the relevant information was extracted from the selected papers and is presented in One of the inclusion criteria for this scoping review was that the statistical analytical techniques must be classical statistical regression methods. In Among the child-related risk factors, gender and age (in months categories) were the most frequent significant predictors of stunting (13 studies), wasting (four reports), underweight (4 studies), overweight (no study) and stunting (12 articles), wasting (six reports), underweight (4 studies) and overweight (1 study), respectively. In the parental category, maternal education was the most active predictor in 14, 3, 5 and 1 studies for stunting, wasting, underweight and overweight, respectively. Out of the 28 studies that investigated stunting, 16 reported a significant association of household wealth status with stunting. Place of residence from the community-related category was significant in stunting (five studies), wasting (three studies) and underweight (one study). Significant comorbidity was found for a child having diarrhea in the last two weeks before the survey with stunting (four studies) and underweight (two studies) captured in this review.This scoping review aimed to identify the existing literature that used classical regression analysis on nationally representative health survey data sets to find the individual socioeconomic, demographic and contextual risk factors associated with malnutrition among children under five years of age in Sub-Sahara Africa (SSA). The review identified 26 studies and the risk factors for malnutrition, which were classified as child-related, parental/household-related and community or area-related factors. The risk factors for malnutrition identified included age, gender, comorbidities (such as diarrhoea), maternal education, household wealth and place of residence.This scoping review has demonstrated the importance researchers have attached to studying malnutrition  in order to provide a basis for evidence-based decision-making toward meeting the WHO’s nine targets on malnutrition by 2025. Some of the most common determinants of malnutrition indicators include child’s age, sex, birth size, breastfeeding status, and whether the child had a fever in the last two weeks before the survey. Other indicators are the mother’s age, education level, Body Mass Index, and father’s education level. In the household category, wealth status, number of children under five years in the household, source of information, and improved building materials, and from the community-related category, place and region of residence, and Gross Domestic Product (GDP). However, there are a few issues from these studies that need to be discussed here. Firstly, malnutrition in children is a situation where children are either undernourished (less necessary energy and nutrient intake) or ‘over-nourished’ (too much necessary energy and nutrient intake) . The autThe second issue of concern from some of the studies in the scoping review is in the attention given to stunting over other anthropometric indices of malnutrition. This scoping review identified that for every ten studies on malnutrition, at least nine studies are investigating stunting. This trend in studying stunting may be related to the need to meet the WHO target of 40% reduction in stunting prevalence by 2025 , and mayThe third issue of concern is the multiple overlaps in the malnutrition indicators. Though few studies have focused on two or more anthropometric indicators of malnutrition, they were analyzed individually using classical logistic regression methods. In some populations, there are tendencies for multiple forms of malnutrition indicators in children ,51,54. NFinally, the issue of inconsistencies found in some studies concerns the proper way of categorizing undernutrition indicators  into moderate and severe undernourishment ,32,47.For instance, a stunted child has height-for-age (HAZ < −2SD), on a scale, a severely stunted child has HAZ < −3SD. Since stunted is moderate plus severe, then the moderately stunted child is −3SD ≤ HAZ ≤ −2SD. The same classification holds for other anthropometric indicators for undernutrition as displayed in the chart above .This scoping review has some level of strengths. (i) This study is about the first scoping review on risk factors associated with malnutrition in children under five in SSA countries that used classical statistical regression modeling techniques on nationally representative survey samples. (ii) The identification of some grey areas that urgently need research cover, especially in the field of using appropriate statistical methods that will compositely determine the actual index of malnutrition in a population. However, there are some limitations, which include but are not restricted to the following: (i) Some potential studies may have been excluded due to the search strategies adopted. (ii) The grey literature search to seek for possible papers was not done. (iii) The references of the included publications were not searched through to ascertain more pieces of evidence. (iv) SSA countries include other countries that are not English-speaking, so some potential papers not written in English from these countries may have been lost to our search. (v) The studies included had analytical techniques restricted to classical statistics regression methods ; therefore, potential papers that used Bayesian statistical methods in their analyses were excluded. (vi) Linear regression as an analytic technique was omitted in the search and this may have excluded some potential papers. (vii) There was no assessment of the potential risk or publication bias conducted.Areas not covered in this review, especially to satisfy the limitations highlighted above, are potential work for future studies. More important is a review that will map out a piece of study evidence on malnutrition that used either classical regression analysis or Bayesian analysis methods, or both. In addition, studies that include overweight and/or micronutrient deficiencies as part of the indicators of malnutrition among children under five years are urgently needed. Furthermore, studies that will explore the interrelationship between malnutrition and other childhood diseases using appropriate statistical techniques while recognizing the interdependencies of these diseases are areas of future interest.In this scoping review, we have identified several significant risk factors that predict the probability that a child under five years of age in an SSA country will develop malnutrition status. These factors were classified as child-related (CR), parental/household-related (PHR) and area-related (AR) variables. The CR include child’s age, sex, birth weight, type of birth, birth type, diarrhoeal, and place delivered. Factors related to parental/household include mother’s education, breastfeeding, BMI, birth interval, mother’s health-seeking status, mother’s age, household wealth status, improved sanitation, number of children under 5 years in the household, maternal health insurance, type of toilet facilities and cooking fuel, while among the area-related (AR) variables were forest cover lost, community region, and community illiteracy rate. To prevent the wide spread of malnutrition in developing countries, these significant risk factors must be taken into consideration when developing practice and policy formulation. Central to these controls are the maternal education and health status. Pregnant and nursing mothers should have access to a balanced diet.The review also discovered that there was a study-interest bias toward stunting as an index over other anthropometric indicators of malnutrition. Furthermore, the review also identified some limitations in the current studies reviewed when overweight and/or micronutrient deficiencies were excluded as indices of malnutrition. In the authors’ opinion, the exclusion may be partly related to the methodological complications involved in determining the true status of malnutrition when these indices are included. Some of the nationally representative surveys used in the studies reviewed collected information regarding the overweight and/or micronutrient status of children under five years. Micronutrient deficiencies in children of under five years in developing countries are measured by the levels of iron, iodine and vitamin A intake . Apart fFinally, barely five years to the set date of achieving the WHO’s nine targets of malnutrition in children, in this scoping review we conclude that a holistic research approach to equally address the various dimensions of anthropometric indicators of malnutrition in a population is needed. Evidence from such findings will be valuable documents in the hands of many planners/policymakers for informed decision making.


Mg2+ homeostasis is regulated by Mg2+ transporters including Mitochondrial RNA Splicing Protein 2 (MRS2), Transient Receptor Potential Cation Channel Subfamily M, Member 6/7 (TRPM6/7), Magnesium Transporter 1 (MAGT1), Solute Carrier Family 41 Member 1 (SCL41A1), and Cyclin and CBS Domain Divalent Metal Cation Transport Mediator (CNNM) proteins. Recent data show that Mg2+ transporters may regulate several cancer cell hallmarks. In this review, we describe the expression of Mg2+ transporters in digestive cancers, the most common and deadliest malignancies worldwide. Moreover, Mg2+ transporters’ expression, correlation and impact on patient overall and disease-free survival is analyzed using Genotype Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA) datasets. Finally, we discuss the role of these Mg2+ transporters in the regulation of cancer cell fates and oncogenic signaling pathways.Despite magnesium (Mg According to the International Agency for Research, digestive cancers are the most common and deadliest malignancies worldwide . In thisEsophageal cancer (ESAC) is ranked in the seventh position in terms of incidence and in the sixth in terms of mortality . ESAC isHelicobacter pylori infection is a risk factor for GC development, therefore Helicobacter pylori has been classified as a class I carcinogen by International Agency for Research on Cancer  is increased. These results suggest a functional interaction between TRPV1 Ca2+-channels and CNNM4 in colon. Finally, CNNM4-deficient mice treated with azoxymethane followed by dextran sodium sulfate form more polyps. Importantly, histological analyses of CNNM4-deficient polyps reveal the presence of invading cancer cells. These data clearly show a protective role of CNNM4 expression against colon carcinogenesis.In a colon carcinoma LoVo cell model, cytosolic Mgive ones . NeverthVo cells . Moreoveell line . In an ahe colon . Furtherinvasion . Inhibit in mice . Moreove2+ transporter expression is altered in numerous digestive cancers, leading to dysregulation of cell fates. To date, most of these studies are focused on the TRPM7 channel, but the role of other Mg2+ transporters cannot be excluded. However, it still remains unclear if these cancer cell fates may be regulated by intracellular Mg2+ homeostasis disturbance, since the role of Mg2+ as a second messenger is still being debated.To resume, Mg2+ concentration can cause increased tyrosine-kinases activities. Mg2+ is indeed a crucial divalent cation required for the activity of kinases, including non-receptor tyrosine kinases , Focal Adhesion Kinase (FAK), Suppressor Of Cytokine Signaling (SOCS)) and receptor tyrosine kinases  including VEGFR, EGFR, FGFR, and PDGFR) [In cell-based studies, high Mgd PDGFR) . Followid PDGFR) ,101. Actd PDGFR) .Although there is scarce information regarding activation of signaling pathways in digestive cancer, recent papers show evidence for the involvement of at least two pathways in these cancers cells: the AKT/mTOR pathway on one hand, and the JAK/STAT pathway on the other hand.2+ uptake and extrusion [2+ efflux and suppressed AKT/mTOR activity, which is the upstream regulator of Bax and Bcl-2. An increase in AKT activity and supplementation with Mg2+ abolished SLC41A1-induced tumor suppression [Xie and collaborators were able to show a direct relationship between expression of the SLC41A1 transporter and activation of the AKT/mTOR pathway . SLC41A1xtrusion . SLC41A1pression .2+ transporter was found to be associated with the mTOR pathway: CNNM4. This transporter is highly expressed in the colon epithelia, and also strongly expressed in the intestine [2+ absorption from the tubular lumen across the epithelial sheet, by extruding intracellular Mg2+ to the body inside. CNNM4-deficient mice can grow without severe defects but have moderately lowered levels of magnesium in the blood when compared to control wild-type mice due to malabsorption of magnesium [At least another Mgntestine . In thisagnesium .In ApcΔ14/+ mice, which spontaneously form benign polyps in the intestine, deletion of Cnnm4 promoted malignant progression of intestinal polyps to adenocarcinomas. IHC analyses of tissues from patients with colon cancer demonstrated an inverse relationship between CNNM4 expression and colon cancer malignancy, thereby supporting the notion that CNNM4 suppresses the progression of cancer malignancy in humans .2+ efflux is apparently able to suppress tumor progression by regulating energy metabolism [2+ is able to bind several biomolecules, with ATP being most probably the utmost important. CNNM4 knockdown is able to increase intracellular Mg2+ levels, and to significantly increase ATP levels in HEK293 cells. Moreover, CNNM4 knockdown (or Mg2+ supplementation) is able to selectively abrogate AMPK hyperphosphorylation (AMPK being phosphorylated and activated under energy-deficient conditions [CNNM4-dependent Mgtabolism : Mg2+ isnditions ). mTOR inditions  and has nditions .2+ signaling and promotes cell proliferation in the colon epithelia. These results establish the functional interplay between Mg2+ and Ca2+ in the colon epithelia, which is crucial for maintaining the dynamic homeostasis of the epithelial tissue [Moreover, it has been shown that Cnnm4 deficiency suppresses Cal tissue .The second signaling pathway influenced by magnesium levels is the STAT pathway. This influence occurs through Magnesium-dependent Phosphatase (MDP)-1. This enzyme, belonging to the haloacid dehalogenase family, has a protein–tyrosine phosphatase function and is potentially involved in glycation repair .Stat3), as well as the c-Jun N-terminal kinase (JNK) were found to mediate the biological function of MDP1 [Forced expression of MDP1 in the gastric cancer cell line BGC-823 inhibited cell proliferation, whereas the knockdown of MDP1 protein promoted cell growth. Overexpression of MDP1 in BGC-823 cells also enhanced cell senescence and apoptosis. Signal transducer and activator of transcription 3  disease. However, it must be noted (i) that Mg2+ supplementation has not proven successful [Other transporters have been demonstrated to activate magnesium-dependent signaling pathways, such as MAGT1. Indeed, Li and collaborators have identified mutations in this magnesium transporter gene, in a novel X-linked human immunodeficiency characterized by CD4 lymphopenia, severe chronic viral infections, and defective T lymphocyte activation . MAGT1 dccessful , and , gastric cancer (GC), pancreatic ductal adenocarcinoma (PDAC), and colorectal cancer (CRC). These digestive cancers are the most common and the deadliest malignancies worldwide. Numerous epidemiologic studies strongly suggest that digestive cancer incidence and mortality may be dependent on lifestyle, and particularly diet. Mg2+ content is continuously decreased in alimentation of industrialized countries, leading to nutritional deficiency in Mg2+ for a large part of the population, estimated to ~60% in the USA [2+ is the second most abundant cation in the cell, its role in physiology and pathology is less extensively studied than others such as Ca2+, Na+ or K+. Cellular Mg2+ homeostasis is regulated by membrane transporters. Among these transporters, TRPM7 has been clearly identified as the main Mg2+ gatekeeper for cell intake in both non-cancer and cancer cells. On the other hand, the functional characterization of other candidates such as MAGT1 or CNNM4 as Mg2+ transporter is still under debate. The reviewed literature, as well as the Human Protein Atlas analyses indicate that Mg2+ transporter expression is altered in most digestive cancers. The discrepancies between the data could be explained by the low number of cohorts and/or by the methodology used. In particular, antibodies targeting ion channels and transporters often display a poor specificity, inducing potential cross-reactivity with other molecules [2+ transporter expression in cells and tissues by immunochemistry should be systematically completed by other methods of detection such as functional  and transcriptomic analysis. In this work, we analyzed Mg2+ transporters expression in digestive cancers, correlation and their impact on patient overall and disease-free survival using Genotype Tissue Expression (GTEx) and The Cancer Genome Atlas (TCGA), as previously described [2+ homeostasis and cell viability in some cellular models suggesting a possible transcriptomic regulation [The aim of this work was to make an overview of Mg the USA . While Molecules . Analyseescribed . Interesescribed . To our gulation ,114. Intgulation . Therefo2+ transporters and their associated signaling pathways as promising biomarkers in digestive cancers. The transcriptomic analysis of datasets reveals a Mg2+ transporter signature involving TRPM7/MAGT1/CNNM4 in ESAC and PDAC. In PDAC, the correlation between TRPM7, MAGT1 and CNNM4 expression is associated with a low survival. Further studies are needed to better understand the physiological significance of this complex in cancer cell models.To conclude, this review highlights Mg


Forced alcohol  exposure has been shown to cause significant impairments on reversal learning, a widely-used assay of cognitive flexibility, specifically on fully-predictive, deterministic versions of this task. However, previous studies have not adequately considered voluntary EtOH consumption and sex effects on probabilistic reversal learning. The present study aimed to fill this gap in the literature.Male and female Long-Evans rats underwent either 10 weeks of voluntary intermittent 20% EtOH access or water only (H2O) access. Rats were then pretrained to initiate trials and learn stimulus-reward associations via touchscreen response, and subsequently required to select between two visual stimuli, rewarded with probability 0.70 or 0.30. In the final phase, reinforcement contingencies were reversed.We found significant sex differences on several EtOH-drinking variables, with females reaching a higher maximum EtOH consumption, exhibiting more high-drinking days, and escalating their EtOH at a quicker rate compared to males. During early abstinence, EtOH drinkers  made more initiation omissions and were slower to initiate trials than H2O drinking controls, especially during pretraining. A similar pattern in trial initiations was also observed in discrimination, but not in reversal learning. EtOH drinking rats were unaffected in their reward collection and stimulus response times, indicating intact motivation and motor responding. Although there were sex differences in discrimination and reversal phases, performance improved over time. We also observed sex-independent drinking group differences in win-stay and lose-shift strategies specific to the reversal phase.Females exhibit increased vulnerability to EtOH effects in early learning: there were sex-dependent EtOH effects on attentional measures during pretraining and discrimination phases. We also found sex-independent EtOH effects on exploration strategies during reversal. Future studies should aim to uncover the neural mechanisms for changes in attention and exploration in both acute and prolonged EtOH withdrawal. Individuals with Alcohol Use Disorder (AUD) show cognitive impairments, particularly in the domain of cognitive flexibility, broadly defined as the ability to adjust one’s behavior in response to changes in the environment . PreclinSeveral groups have tested the effect of forced EtOH exposure on reversal learning, a robust measure of cognitive flexibility commonly used in experimental animals  that reqVoluntary alcohol consumption models  have been used to assess effects on deterministic reversal learning ,9,10, reThe present study sought to address these gaps in the literature by probing the effects of a chronic intermittent voluntary alcohol consumption model on PRL in male and female rats. Rats were administered a 2-bottle choice procedure, during which they were given access to either both 20% EtOH and H2O, or H2O only, 3 days per week for a total of 10 weeks. Five days after their last day of EtOH access they underwent pretraining and advanced to PRL after meeting several training criteria. Given recent findings on sex differences in EtOH consumption, we hypothesized that females would be more EtOH-preferring and reach higher EtOH consumption levels than males. Here, we corroborate previous findings of enhanced EtOH consumption and escalation in female rats compared to male rats. Surprisingly, we found sex-dependent treatment group differences in trial initiations, with the most robust effects in pretraining that carried through early discrimination. These results were in contrast to intact committed trials  and reward collection times throughout learning. We found changes in WSLS strategy specific to the reversal phase, with EtOH drinkers displaying more exploration (i.e. shift) strategies than H2O drinkers. These effects were not sex-dependent. Taken together, the present results suggest an enduring effect on attention and exploration-based strategies, but not motivational measures, in stimulus-reward association learning in prolonged abstinence from EtOH.ad libitum, and remained in cages with no investigator interference. Following the 3-day acclimation period, animals were handled for 10 min per animal for 5 consecutive days. During the handling period, the animals were given unlimited food and water access and were tail marked. After the handling period, animals were singly-housed under standard housing conditions (room temperature 22–24°C) with a standard 12 h light/dark cycle (lights on at 6am). This study was conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Chancellor’s Animal Research Committee at the University of California, Los Angeles.A timeline of all procedures is shown in Rat home cages were modified to allow for the placement of 2 bottles. Rats  were given access to both water and 20% alcohol simultaneously, with placement of bottles counterbalanced, for a 24-hour period 3 days per week, and only water on the remaining days. Alcohol access was terminated at 10 weeks (29 days of access) after animals’ EtOH consumption stopped escalating. An age-matched control cohort of water (H2O)-only drinking animals  was placed in modified home cages allowing for the placement of 2 bottles with water-only also for a total of 10 weeks. Weight of bottles was measured before and after alcohol and/or water-only access to measure daily consumption amounts with a control cage placed on the same rack to account for leakage.Immediately following the termination of the consumption period, all animals were placed on food restriction to 14 grams/day  or 18 grams/day  of chow for 5 days prior to behavioral testing. Animals were weighed every other day and monitored closely to not fall below 85% of their maximum, free-feeding weight. Behavioral testing was conducted in operant conditioning chambers outfitted with an LCD touchscreen opposing the sucrose pellet dispenser ,32 durininitiation omission. IMT began in the same way as ITCT, but the disappearance of the white graphic stimulus was now paired with the onset of a target image immediately to the left or right of the stimulus (i.e. forced-choice) that the rat was required to nosepoke to obtain reward. During this stage, a trial could be terminated for one of three reasons. First, if a rat touched the center display (i.e. white graphic stimulus) and touched the image displayed on either side, after which there was a dispensation of one sucrose pellet and illumination of the tray-light. Second, if the rat failed to touch the center white graphic stimulus before the display time ended (40 s), the stimulus disappeared, a black background was displayed, and a 10 s ITI ensued, scored as a initiation omission. Third, if the image display time (60 s) ended, after which the stimulus disappeared, a black background was displayed, and a 10 s ITI ensued, scored as a choice omission. For habituation pretraining, the criterion for advancement was collection of all 5 sucrose pellets. For ITCT, the criterion to the next stage was set to 60 rewards consumed in 45 min. The criterion for IMT was set to 60 rewards consumed in 45 min across two consecutive days.The pretraining protocol, adapted from established procedures described in Stolyarova et al. (2017) , consistR(B) = 0.70 and pR(W) = 0.30, respectively. Assignment of the stimulus to better or worse reinforcement was counterbalanced across conditions. If a trial was not initiated within 40 s, it was scored as an initiation omission. If a stimulus was not chosen, it was scored as a choice omission, and a 10 s ITI ensued. If a trial was not rewarded, a 5 s time-out would follow, subsequently followed by a 10 s ITI. Finally, if a trial was rewarded, a 10 s ITI would follow after the reward was collected , and choose between two visual stimuli presented on the left and right side of the screen (displayed for 60 s) counterbalanced between trials, assigned as the Better or Worse options, rewarded with a sucrose pellet, with probability pollected . The criTo test the study hypotheses, a series of mixed-effects General Linear Models (GLM) and ANOVA analyses were conducted using MATLAB   and SPSSThe EtOH consumption data were analyzed with ANOVAs with sex and drinking-group as between-subject factors, and EtOH consumption days (D1-D29) as a within-subject factor, with EtOH consumption (g/kg) as the primary outcome –37. Indefitglme function; Statistics and Machine Learning Toolbox; MathWorks, Natick, Massachusetts; Version R2017a). Probability correct, number of rewards, and initiation omissions, were analyzed using GLM across repeated days of testing per animal with drinking group (EtOH vs. H2O) and sex  as fixed effects and animal as a random effect. In Figures, we show the first 15–20 days of learning to avoid overweighting performance of increasingly fewer animals at the extremes. Omission (sums) and latency data (medians) included one observation per subject and were also analyzed with a GLM with drinking group and sex as fixed effects and animal as a random effect. All post-hoc tests were Bonferroni-corrected to account for the number of comparisons. Statistical significance was noted when p-values were less than 0.05, p-values between 0.05 and 0.06 are reported as marginally significant.Learning data , number of rewards collected, omission, and latency data were analyzed with GLM in MATLAB . For win-stay events we divided the total number of times an animal chose the same stimulus on the trial following a win, by the total number of wins [sum(win-stay)/sum(win)]. For lose-shift events we divided the total number of times an animal chose the alternative stimulus on a trial following a loss, by total number of lose trials [sum(lose-shift)/sum(lose)]. Win-shifts events were inversely proportional to win-stay events [1-sum(win-stay)]. Likewise, lose-stay events were inversely proportional to lose-shift events [1-sum(lose-shift)]. Finally, we compared the frequency of using advantageous strategies  vs. less advantageous strategies  by generating an adaptive score: (win-stay + lose-shift)—(win-shift + lose-stay). Ultimately, we compared the use of advantageous  with less advantageous  strategies by drinking group and sex.Trial-by-trial analyses were conducted to investigate potential sex and group differences in WSLS strategies used in the PRL task. WSLS are strategies commonly examined in decision-making tasks involving risk and reward that can reveal changes in sensitivity to surprising outcomes and feedback learning. Each trial was classified as a r(16) = -0.67, p = 0.005], and number of high-drinking days . Overall females exhibited heavier EtOH consumption patterns than males.Independent samples t-tests showed that females reached a greater maximum level of EtOH consumption   and exhiLearning data were analyzed separately, subdivided into pretraining, discrimination, and reversal phases. Early vs. late discrimination and reversal learning based on prior studies indicating these may be particularly informative to contrast –44.group = -14.00, p = 0.04; sex = -11.38, p = 0.001; group*sex = 12.00, p = 0.01) with EtOH-drinking females requiring more pretraining sessions than EtOH-drinking males . Both EtOH-drinking females  and EtOH-drinking males  needed more sessions to reach criterion than their H2O-drinking counterparts (As pretraining was a unique phase with slightly different dependent measures (i.e. forced choice omissions and latencies), a separate GLM was used to assess the effect of group  and sex , and group*sex interactions on the number of pretraining sessions required to reach criterion to advance to the discrimination learning phase of the PRL task. A significant effect of group and sex emerged, with the EtOH-experienced animals requiring a greater number of pretraining sessions than the H2O-only group  and males  exhibited more initiation omissions than their H2O-drinking counterparts, and EtOH-drinking females exhibited more initiation omissions than EtOH-drinking males . The EtOH group also displayed longer initiation latencies than the H2O group . Finally, we analyzed data for differences in reward latency, defined as the duration to collect the sucrose reward from the food magazine, and found no effect of group  or sex differences  on total number of sessions to reach criterion for the discrimination phase, but a marginally-significant group*sex interaction on this measure was observed , or sex , and no group*sex , or sex*day  interaction on probability correct. There was however a significant group*day interaction , with the H2O-drinking animals choosing the better option increasingly more across days than the EtOH-drinking animals. There was also a significant group*sex*day interaction . Post-hoc comparisons revealed males learned quicker than females , that H2O-drinking females chose the better option increasingly more across days than their EtOH-drinking counterparts (GLM: βgroup = 0.01 p = 0.002); and that the same pattern was not observed for males.A GLM model was used to test the effect of drinking group , sex , days, and their 2-way and 3-way interactions on probability correct (i.e. choosing the better option), number of rewards (i.e. sucrose pellets), and initiation omissions across 20 testing days of discrimination learning (D1-D20). All animals demonstrated learning by showing an increase in choosing the better option across days, , and sex differences  also emerged, with both H2O-drinking animals and males collecting a greater number of rewards than EtOH-drinking animals and females, respectively , with females displaying a greater increase in rewards collected across days , compared to males  and sex differences , with EtOH-drinking animals and females exhibiting more initiation omissions than H2O-drinking and males, respectively , with EtOH-drinking females displaying more initiation omissions than H2O-drinking females ; the same pattern was not observed in males  revealed that EtOH-drinking females decreased their number of initiation omissions across discrimination learning at a slower rate than EtOH-drinking males (p = 0.03) and H2O-drinking females (p = 0.03). There were no differences between male and female H2O drinkers, or between EtOH- vs. H2O-drinking males.Conversely, all animals decreased the number of initiation omissions across days, , whereas H2O-drinking animals exhibited longer reward collection latencies than EtOH-drinking animals . All other types of omission and latency analyses yielded non-significant results.Finally, we conducted GLM analyses on the sum of initiation and choice omissions and median  latencies in discrimination learning, collapsed across days of testing. Our results indicated EtOH-drinking animals exhibited more initiation omissions than H2O-drinking animals , with H2O-drinking animals exhibiting more choice omissions than EtOH-drinking animals. It should be noted that choice omissions represented a small number of occurrences  and this effect was driven by a single outlier (>2 SD from the mean) with 4 choice omissions, which upon removal yielded a non-significant group effect . In summary, the data for early discrimination learning suggest an enduring effect of EtOH experience on initiating trials, with females most affected.Because interesting latency differences were obtained for the pretraining phase and based on prior studies comparing early vs. late discrimination learning, we conducted further latency analyses for other phases of learning to assess if these trends were maintained. An analysis of initiation latencies and omissions, choice (correct and incorrect) latencies and omissions, and reward latencies was conducted for the first 500 trials of discrimination learning to capture early learning in this phase, with animals averaging 89.19±3.38 committed trials per day. There was a significant group difference on initiation omissions , with H2O-drinking animals displaying longer latencies than EtOH-drinking animals, but no significant effect of sex, or group*sex interaction was found for this measure. Similar to what was found during early discrimination, there was a significant group effect on choice omissions in late discrimination , with the H2O-drinking animals exhibiting more choice omissions than the EtOH-drinking animals, but no effect of sex, or group*sex interaction was found for this measure. However, this effect was largely driven by the same animal as during early discrimination, which upon removal, yielded non-significant results . Finally, our results indicated that H2O-drinking animals displayed longer reward collection latencies than EtOH-drinking animals  latencies and omissions, and reward latencies was also conducted for the last 500 trials of discrimination learning to capture late phase learning, with animals averaging 118.98±3.89 committed trials per day. There was a marginally-significant effect of group , sex differences , or group*sex interaction  on sessions to reach criterion for the reversal learning , or sex , and no group*sex , sex*day , group*day , or group*sex*day  interactions on the probability of choosing the better option.A GLM model was used to test the effect of drinking group , sex , days, and their 2-way and 3-way interactions on probability of choosing the better option, number of rewards, and initiation omissions across 15 days of reversal learning. Two females were excluded because they failed to meet criterion for discrimination learning, and never advanced to reversal learning. All other animals demonstrated learning by exhibiting an increase in choosing the better options across days, , or sex , and no significant group*sex , group*day , sex*day , or group*sex*day  interactions on number of rewards collected. All animals decreased the number of initiation omissions across days, , or sex , and no significant group*sex , group*day , sex*day , or group*sex*day  interactions on initiation omissions.All animals increased the number of rewards collected over days, , sex , or group*sex  interaction for initiation omissions. However, though there was no effect of sex , we found that H2O-drinking animals exhibited more choice omissions than EtOH-drinking animals , with a significant group*sex interaction revealing H2O-drinking females exhibited more choice omissions than EtOH-drinking females and H2O-drinking males . Additionally, there was a marginally-significant effect of group on incorrect choice latencies , with H2O-drinking animals exhibiting longer incorrect choice latencies than EtOH-drinking animals. Analyses on initiation, incorrect, and reward latencies yielded non-significant results. Unlike for the discrimination phases, upon removal of 2 outliers (> 2 SD from the mean), the effect of drinking group remained significant, with H2O-drinking animals exhibiting more choice omissions than EtOH-drinking animals .Finally, we conducted GLM analyses on the sum of initiation and choice omissions and median  latencies in reversal learning, collapsed across all days of testing. Similar to the above measure for across- day learning, we found no significant effect of group , with H2O-drinking animals exhibiting longer latencies when choosing the incorrect stimulus compared to EtOH-drinking animals. There was also a significant group*sex interaction  on incorrect choice latency, with H2O-drinking females displaying longer latencies than both H2O-drinking males (p = 0.05) and their EtOH-drinking counterparts (p = 0.02). In summary, the data for early reversal learning suggests there was no longer any effect of prior EtOH experience on initiating trials, as had been previously observed during early discrimination learning, but it is important to note that EtOH-drinking animals were less tentative than the H2O-drinking animals given their faster incorrect choice latencies.An analysis of initiation latencies and omissions, choice (correct and incorrect) latencies and omissions, and reward latencies was conducted for the first 500 trials of reversal learning, to capture early reversal learning. Animals averaged 119.70±8.32 committed trials per day. There were no significant group , but no group differences or group*sex interaction were found. Upon removal of a single outlier, the effect of sex remained . Males displayed longer correct choice latencies than females , whereas H2O-drinking animals exhibited longer reward latencies than EtOH-drinking animals  latencies and omissions, and reward latencies was also conducted for the last 500 trials of reversal learning, to capture late phase learning. Animals averaged 155.11±4.06 committed trials per day. Our analyses on initiation omissions indicated females exhibited more initiation omissions than males , as well as correct choice latencies .Potential differences in win-stay/lose-shift (WSLS) strategies on stimulus responses employed by each group  and by sex  were tested for the first 500 committed trials and last 500 committed trials of the discrimination phase and reversal phase by calculating the frequency of each strategy individually. We compared the frequency of using advantageous strategies  vs. less advantageous strategies  by generating an adaptive score: (win-stay + lose-shift)—(win-shift + lose-stay).group = -0.10, p = 0.01; group = -0.20, p = 0.0002; group = 0.10, p = 0.03; group = -0.05, p = 0.04; There were no significant effects of group, sex, or a group*sex interaction in stimulus-based WSLS strategies individually, or as an ‘adaptive score’ comparison during early or late discrimination learning . HoweverThe present study used an intermittent access model to study the effect of voluntary EtOH consumption on cognitive flexibility using a probabilistic reversal learning paradigm. We included sex as an a-priori moderator. Although forced-exposure models such as intraperitoneal (i.p.) injections ,46 and EWe observed an escalation of drinking over the course of the twenty-nine days of alcohol access, irrespective of sex, an expected pattern when using intermittent-access models compared to continuous access models. Several studies administering intermittent exposure have shown that alternating brief periods of alcohol access with brief periods of no access can actually escalate alcohol consumption to excessive levels ,50–55 coWe found that females reached a higher EtOH consumption level and exhibited greater high-drinking days than males. These findings are consistent with previous studies showing that female rodents drink more EtOH than males –25 and eWe observed the most pronounced impairment following EtOH on sessions to reach criterion during pretraining; a pattern that was not maintained through discrimination or reversal learning. Prior studies testing the relationship between alcohol exposure and performance on reversal learning tasks have largely been mixed, with some studies demonstrating alcohol produced impairments in both discrimination and reversal learning ,58, otheWe found impairments associated with prior EtOH experience, such that animals previously exposed to EtOH required more sessions to reach criterion and exhibited longer initiation and choice latencies during pretraining. Similar deficits were also found during discrimination learning, with a greater number of initiation omissions and longer initiation latencies in the EtOH-experienced group. These effects were most pronounced in female animals (discussed below). There have been studies of EtOH exposure on attention using the 5-choice serial reaction time task –72, consSome interesting sex differences on reversal learning emerged in our experiment. The most pronounced impairments were observed during late reversal, with females exhibiting greater omissions (initiation and choice) and longer initiation latencies than males, irrespective of prior EtOH exposure. This is in agreement with the human literature, which has shown that males outperform females on reversal learning ,74, and A sex-dependent drinking group difference was observed, with EtOH-exposed females more affected than males on measures of attention: they exhibited more initiation omissions than their H2O-drinking counterparts during both pretraining and early discrimination learning, which is also reflected in a greater number of sessions required to reach criterion in early learning. Although prior research has not provided sufficient evidence supporting an attentional deficit specific to females , there iWe observed sex-independent EtOH effects on WSLS strategies during reversal learning. Rats with prior EtOH experience were more likely to use a “shift” strategy whereas H2O-drinking animals were more likely to “stay” with the previous stimulus choice in the reversal phase. Similarly, animals with prior EtOH experience were generally more flexible in early reversal learning (i.e. they exhibited a greater ‘adaptive’ score) than H2O-drinking animals. This suggests that EtOH-experienced rats had a more tenuous representation of trial-by-trial stimulus-outcome contingencies upon criterion-level performance than the H2O-drinking control rats, and could consequently be more flexible. However, all rats generally increased their choice of the better option and rewards collected over time, while decreasing the number of initiation omissions for both the discrimination and reversal phase. The lack of pronounced EtOH impairments on overall discrimination and reversal phases of learning- as measured by global measures such as the probability choosing the better option over time- may be attributed to plasticity following protracted abstinence in rodents –81, and In summary, we observed pronounced trial initiation omissions following EtOH experience in females during pretraining and discrimination learning. These phases are closest in time to the last EtOH experience and constitute the early abstinence period. Additionally, this attentional decrement, which was most pronounced in female animals, was partnered by an enhanced exploration strategy in all EtOH drinking animals, both males and females, later in reversal learning.Alterations related to attention and processing speed in early EtOH abstinence (during pretraining) may have a domino effect on later learning, leading to the sex by drinking group interaction we observe in discrimination learning, and perhaps contribute to the enhanced exploration phenotype in reversal learning. A true test of this would require animals to undergo pretraining, discrimination learning, and drinking prior to any reversal learning. Ultimately, all rats exhibited intact motivation and motor timing, and were able to increase their probability of choosing the better option and number of rewards, while decreasing their initiation omissions. Although voluntary alcohol consumption models, such as the one employed here, do not model severe alcohol dependence like forced-exposure models, they do however reflect escalating, chronic intermittent drinking that corresponds to the early stages of problematic drinking, before individuals transition to alcohol dependence. Attenuated attentional mechanisms in early abstinence may not contribute to decrements in flexible learning per se, but may instead detract from executive functions important in limiting (over)consumption. Future studies should investigate the brain mechanisms and the role of gonadal hormones on alcohol consumption and attention, and systematically compare these measures as predictors of consumption (i.e. relapse) during acute vs. prolonged abstinence.S1 FigA) All animals regardless of drinking group or sex increased their number of rewards collected over the twenty testing days of discrimination learning. H2O-drinking animals and males collected a greater number of rewards than EtOH-drinking animals and females, respectively. Females displayed a greater increase in rewards collected across days. (B) All animals regardless of drinking group or sex decreased their initiation omissions over the twenty testing days of discrimination learning. EtOH-drinking animals and females had more initiation omissions than H2O-drinking animals and males, respectively. EtOH-drinking females displayed more initiation omissions than H2O-drinking females. (C) All animals regardless of drinking group or sex increased their number of rewards collected over the fifteen testing days of reversal learning. There were no group or sex differences on number of rewards collected. (D) All animals regardless of drinking group or sex decreased the number of initiation omissions over the fifteen testing days of reversal learning. There were no group or sex differences on initiation omissions. Bars indicate ± S. E. M.((DOCX)Click here for additional data file.S2 FigA) No group or sex differences in the use of the win-stay strategy. (B) EtOH-drinking animals used the lose-shift strategy more than H2O-drinking animals. Bars indicate ± S. E. M. **p ≤0.01.((DOCX)Click here for additional data file.S3 FigA) EtOH group exhibited longer initiation latencies than the H2O group. (B) EtOH group exhibited longer forced-choice latencies than the H2O group. (C) No group differences were found for reward latencies. (D) No sex differences were found for initiation latencies. (E) Females exhibited longer forced-choice latencies than males. (F) No sex differences were found for reward latencies. Dashed lines in latency histograms represent group medians. Bars indicate ± S. E. M. n = 16 males, n = 16 females, *p<0.05.((DOCX)Click here for additional data file.S4 FigA) No group differences were found for initiation latencies for the first 500 trials. (B) No group differences were found for reward latencies for the first 500 trials. (C) No group differences were found for initiation latencies for the last 500 trials. (D) No group differences were found for reward latencies for the last 500 trials. (E) No sex differences were found for initiation latencies for the first 500 trials. (F) No sex differences were found for reward latencies for the first 500 trials. (G) No sex differences were found for initiation latencies for the last 500 trials. (H) No sex differences were found for reward latencies for the last 500 trials. Dashed lines in histograms of latencies represent group medians. Bars indicate ± S. E. M. n = 16 males, n = 16 females.((DOCX)Click here for additional data file.S5 FigA) No group differences were found for initiation latencies for the first 500 trials. (B) No group differences were found for reward latencies for the first 500 trials. (C) No group differences were found for initiation latencies for the last 500 trials. (D) H2O group exhibited longer reward latencies compared to the EtOH group for the last 500 trials. (E) No sex differences were found for initiation latencies for the first 500 trials. (F) No sex differences were found for reward latencies for the first 500 trials. (G) Females exhibited longer initiation latencies compared to males for the last 500 trials. (H) No sex differences were found for reward latencies for the last 500 trials. Dashed lines in histograms of latencies represent group medians. Bars indicate ± S. E. M. n = 16 males, n = 14 females, *p ≤0.05, **p ≤0.01.((DOCX)Click here for additional data file.


TCAB1, a.k.a. WRAP53β or WDR79, is an important molecule for the maintenance of Cajal bodies and critically involved in telomere elongation and DNA repair. Upregulation of TCAB1 were discovered in a variety types of cancers. However, the function of TCAB1 in tumor cell senescence remains absent.The TCAB1 knockdown cell lines were constructed. The expression levels of TCAB1, p21, p16 and p53 were detected by qRT-PCR and western blotting. Staining of senescence-associated β-galactosidase was used to detect senescent cells. The ubiquitination of the p21 was analysed by immunoprecipitation and in vivo ubiquitination assay. TCGA databases were employed to perform in silico analyses for the mRNA expression of TCAB1, p21, p16 and p53.Here, we discovered that knockdown of TCAB1 induced rapid progression of cellular senescence in A549, H1299 and HeLa cells. In exploiting the mechanism underlining the role of TCAB1 on senescence, we found a significant increase of p21 at the protein levels upon TCAB1 depletion, whereas the p21 mRNA expression was not altered. We verified that TCAB1 knockdown was able to shunt p21 from proteasomal degradation by regulating the ubiquitination of p21. In rescue assays, it was demonstrated that decreasing the expression of p21 or increasing the expression of TCAB1 were able to attenuate the cellular senescence process induced by TCAB1 silencing.This study revealed the importance of TCAB1 for its biological functions in the regulation of cell senescence. Our results will be helpful to understand the mechanisms of senescence in cancer cells, which could provide clues for designing novel strategies for developing effective treatment regimens. INK4a/pRb and p53/p21 pathways [Cellular senescence is a state of irreversible growth arrest where cells remain viable and metabolically active, but non-proliferative even received mitogenic stimulations . The senskp2, CRL4cdt2, and APC/Ccdc20 [The regulation of cellular senescence often requires the function of p21 as a key mediator molecule for signal transduction. By blocking the kinase activity of CDKs, the CDK2 inhibitor p21 is an important cellular factor to brake G1 progression and induce senescence. The expression of p21 is regulated by complex mechanisms during the process of cellular senescence, including epigenetic silencing, transcriptional or translational controls, and protein stability changes from ubiquitin dependent or independent degradation . As a maC/Ccdc20 –8, whichC/Ccdc20 , 10. IntC/Ccdc20 .TCAB1 is a structural and essential component protein of the Cajal body (CB). Through its WD40 domains and the c-terminal region containing 456 to 533 amino acids, TCAB1 interacted with Coilin and SMN proteins . TCAB1 iIn this study, we find that knocking down TCAB1 in stable cancer cells induced striking cellular senescence phenotype. The effect of TCAB1 silencing was mediated by decreased ubiquitination of p21 and its subsequent proteasome-mediated degradation. Removal of cumulated p21 under TCAB1 deficiency allowed the cells to escape from senescence. Taken together, we have identified a new biological function of TCAB1 for its involvement in cellular senescence. Our findings suggested that reducing TCAB1 could be deployed as a valid strategy to develop anticancer treatment as it appeared to be sufficiently to trigger cellular senescence.2 at 37 °C. The RNA interference target sequences corresponding to TCAB1 (shRNA: TATCTGGGACGCATTCACT) and a scrable sequence (TTCTCCGAACGTGTCACGT) were subcloned into lentiviral vectors of GV152 backbone . The lentiviral of LV-TCAB1 were provided by Hesheng genomic technology. The cells were infected with the produced lentivirus in the presence of polybrene  for 48 h, and then selected by 2 μg/ml puromycin (Sigma) for 7 days. The expression levels of the targeted gene were determined by RT-PCR and western blotting for knockdown efficiencies.A549, H1299 and HeLa cells were obtained from the cell bank at Peking Union Medical University and were detected for STR profiles in china. Cells were grown in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum (HyClone) and 100 units/ml penicillin and streptomycin in a humidified incubator with 5% COTotal RNA was isolated from HeLa stable cells using the RNeasy mini kit (Qiagen) following to the manufacturer’s standard protocol. The RNA concentrations and purity were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). A total of 1 μg of RNA each was subjected to reactions to obtain cDNAs using a Reverse Transcription Kit (Promega). Quantitative Real-Time-PCR was performed by an ABI 7500 Real-Time PCR System (Applied Biosystems) using Maxima SYBR Green/ROX qPCR Master Mix (MBI Fermentas). Thermal cycling conditions were 95 °C for 30 s, followed by 5 s at 95 °C and 1 min at 60 °C for 40 cycles. PCR amplification was performed using specific primers: p21 ; TCAB1 F: ; and GAPDH . Relative quantities of mRNA were calculated using the comparative Ct method for each sample. Analyses were done in triplicate to confirm the data.The cell culture samples were lysed in modified Radioimmune Precipitation Assay (RIPA) Buffer (Applygen) containing a protease inhibitor cocktail (Roche). The protein concentrations were measured using the BCA assay kit (Thermo Fisher Scientific). A total protein of 10–20 μg from each sample were loaded and subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to PVDF filters (Millipore). Immunoprobing of target proteins were performed by incubating overnight at 4 °C with the primary antibodies against p21 , TCAB1 or ubiquitin . After rinses, HRP-conjugated secondary antibodies used for western blotting analysis were as follows: anti-rabbit IgG  and mouse IgG . The even loading of isolated proteins was verified with β-actin monoclonal antibody  or GAPDH monoclonal antibody . The blotted filters were visualized for signals using enhanced chemiluminescence (ECL) reagents  according to the manufacturer’s instructions.2. Eight randomly selected representative fields were quantified for the percentage of SA-β-gal positive cells. Data were presented as mean ± SE.For SA-β-gal staining, cells were seeded into 12-well plates and cultured in a DMEM medium. After fixed for 20 min at room temperature in 4% paraformaldehyde, the cells were washed twice with 1× PBS. A freshly prepared SA-β-gal staining solution was used to incubated with the fixed cells overnight at 37 °C with the absence of COTo detect the levels p21 ubiquitination, A549-shTCAB1, H1299-shTCAB1 and shTCAB1 stable HeLa cells were pretreated with 20 μM MG132 (Merck Millipore) for 6 h. The whole cell lysates were collected from 10 cm dishes and incubated with anti-p21 antibodies overnight at 4 °C. Then, 20 μl beads conjugated with protein A/G were added and incubated for 1 h at room temperature. The beads were washed three times with the washing buffer  prior to immunoprecipitation. The released samples in 20 μl 2× SDS protein loading buffer were subjected to western blotting analyses using an antibody against ubiquitin.The transformed stable cells were treated with 100 μg/ml of cycloheximide for 0, 0.5, 1, 2, 3, 4 and 5 h. The whole cell lysates were prepared. The amount of 10 or 20 μg of total protein from each sample was analyzed by Western blotting with an anti-p21 antibody.To transiently silence p21, siRNA targeting p21 was synthesized (Hesheng genomic technology) and transfected with Lipofectamine™ RNAiMAX (Invitrogen) following the manufacturer’s instructions. The siRNA sequence target p21 was: CGUCAGAACCCAUGCGGCATT. Control insertion sequence was: UUCUCCGAACGUGUCACGUT.From TCGA database, the mRNA expression of TCAB1, p21 and p16 of a total of 514 lung cancer patients were retrieved. For better and fair comparison of genes of different expression levels and abundance, the expression levels of each gene were normalize by quartile ranking scales and classified into 9 group from low to high as “0 to 8”. Using the normalized levels as the x and y scales, TCAB1/p21 or TCAB1/p16 in our case, the averaged level of a third gene of interest (p53 in this study) could be ploted with the original raw values to generate a 3D terrain map. Both p53 wild-type (362) and p53 mutation (152) cases were analyzed and presented as comparative panels.+ cells were observed significantly increased in numbers as compared to cells infected with the control virus  cells, we found that the absence of TCAB1 could induce senescence dramatically. Nearly half of the cells were stained positive by SA-β-gal  to inhibit protein biosynthesis, and protein extracts obtained at the indicated time points were analyzed. We found that knockdown of TCAB1 in cells resulted in a significantly extended half-life of p21  can still be degraded [In mammalian cells, the senescence checkpoint is initiated by one of the two major signaling pathways p21 /p53 and p16/Rb. We demonstrated that TCAB1 reduction-induced senescence was not dependent to p16 expression, rather signatured by an increase at p21 protein levels. Elevated levels of p21 in TCAB1-depleted cells resulted from avoiding the degradation from ubiquitin proteasome system. The mechanism of p21 degradation is currently not fully understood. It is believed that the degradation of p21 could be through either ubiquitin-dependent or independent mechanisms. Newly synthetized p21 is stabilized by WIS39, an adaptor that recruits HSP90 to p21  while p2degraded , 29. At degraded . We hypoAnother important finding from this study was that A549 lung cancer cells appeared to be much more sensitive to TCAB1 suppression for senescence induction. As A549 contains the wild type of p53, it could explain, at least partially, our discovery indicating the p21/p53 pathway was responsible for TCAB1 participated senescence regulation. Surprisingly, in the wild-type p53 patients analyzed in the TCGA database, the expression of TCAB1 and p21 in patients with high p53 expression was negatively correlated. This turned out to be reasonable to explain that why p21/p53 pathway for cell senescence do not activate in tumor cells, even when p53 functions are intact. The interconnections among the state and expression of p53 and TCAB1/p21 could be of great biological significance and remains to be further studied. It should be pointed out that the TCGA results are gene expression measures at the mRNA levels, even in support of our hypothesis on TCAB1 and p21 axis on senescence regulation, more direct evidence from assays on TCAB1 and p21 protein levels from clinical samples will add more convincing values.In summary, we observed that knockdown of TCAB1 in cancer cells sufficiently induced cellular senescence of different extent, possibly depending on the status of p53 gene. TCAB1 knockdown was able to increase the p21 protein level by reducing its proteasomal degradation through ubiquitination dependent fashion. In A549 cells with wild type p53, the phenotype of TCAB1-KD induced senescence was more pronounced with p21 dependency as demonstrated by si-p21 rescue experiments. However, whether TCAB1 deletion can induce tumor senescence in vivo remains to be explored. Molecule TCAB1 interacts directly with to influence the ubiquitination of p21 needs further study.Cellular senescence is a prominent tumor suppression strategy to allow cancer cells escape from malignant expansion and development of treatment resistance. Induction of senescence in cancer cells is emerging as a practical therapeutic concept . DepletiAdditional file 1. Fig.S1 A-C