Melanoidins (MLDs) are formed through the reaction of carbonyl compounds and amino compounds in the Maillard reaction (MR) during the heating or storage of food. In this study, the formation, chemical composition, and structural characteristics of black garlic (BG) MLDs stored at different temperatures  over a period of 6 months were investigated. The initial products of the MR formed more often at 4 °C and 20 °C, while higher temperatures (35 °C) promoted the reaction in the middle and late stages of the MR. The higher temperature promoted an increase in molecular weight and MLD content, which can be attributed to the increase in protein and phenolic content. Elemental analysis confirmed an increase in nitrogen (N) content and the continuous incorporation of nitrogen-rich substances into the skeleton. Amino acids, particularly aspartic acid and threonine, were the primary N-containing compounds involved in MLD formation. Additionally, the infrared analysis revealed that the changes in MLDs during storage were characterized by amide I and amide II groups. The MR enhanced the yields of heterocyclic compounds (from 56.60% to 78.89%), especially that of O-heterocyclic compounds, at the higher temperature according to Py-GC-MS analysis. Furthermore, the higher temperature enhanced the molecular weight, maximum height, and roughness of MLDs compared to the control. The antioxidant ability of MLDs was positively correlated with storage temperatures. In summary, temperature had an impact on the formation, evolution, and antioxidant activity of MLDs. Black garlic (BG) is a processed product of fresh garlic. It has gained attention from customers due to its abundance of bioactive substances , such asMLDs have complex compositions and structures due to different reactants and diverse reactions. A few studies have shown that the skeleton of MLDs is established through the cross-linking of carbohydrate degradation products with proteins and other macromolecular substances ,9. MLD sThe structures of MLDs are affected by a series of factors, especially temperature and time . The actThe experiment was a single-factor experiment (temperature). Three temperatures were set, and six time points were taken at each temperature for the experiment. Our studies confirmed that the alterations in the compositions and structures of MLDs during storage. Then, the changes in antioxidant activity were evaluated via DPPH, ABTS, and a reducing power assay. The present study lays the foundation for future research on the formation mechanism and biological activities of MLDs.BG was produced, based on the enterprise standard (QDPH 0001S-2019), by Dali Pinhong Plateau Agricultural Science and Technology Development . All chemicals were of analytical grade and purchased from Sigma Chemical Co. . BG was stored at 4 °C, 20 °C, and 35 °C for 6 months in PETB bottles.w/w). Then, the mixture was ground for 4 min and left to rest in an ultrasonic disintegrator  for 20 min. The supernatant was degreased twice with dichloromethane at 3:2 (w/w). The solution (10 mL) was dialyzed with an ultrafiltration membrane  of 3 kDa in distilled water (1 L). The water was replaced three times during dialysis. MLDs were freeze-dried  and preserved at −80 °C in a dry environment until analysis.MLD sample preparation was carried out based on the previous method of Zhao et al. (2011)  with somMLDs were measured as in the previous method  using a 0 and A are the absorbance values at time zero and time t, and k is the first-order rate constant (month−1) under the corresponding storage conditions. The change in the absorbance of MLDs during storage can be simulated through first-order reactions.Sample solution (0.5 mg/mL) measuring 0.5 mL was added 1.0 mL of DNS. The sample was heated in a water bath at 100 °C for 5 min, and then water was added to reach a volume of 5 mL after cooling in the ice bath. Finally, the absorbance was measured at 540 nm.The Coomassie brilliant blue method was used to measure the content of protein in MLDs. The MLD solution  and the Coomassie brilliant blue reagents were mixed and left to rest for 10 min. The absorbance was measured at 595 nm.The total phenolic (TPC) content in MLDs was determined using the Folin–Ciocalteu method as described by Babbar et al. 2011) [11 [20]. Elemental analysis was performed on the element analyzer . MLDs (3 mg) were wrapped with tin foil and completely burned at a temperature above 1150 °C. Then, the element signal was measured with a thermal conductivity detector to calculate the content of the samples. https://cheMLData.Nist.gov/ (accessed on 5 May 2023)), and pertinent literature was used to compare results. Pyrolysis–gas chromatography–mass analysis was carried out in a gas chromatograph equipped with a mass spectrometer , as previously described by Rochat et al. (2007) . The GC The molecular weight of MLDs was determined via high-performance gel permeation chromatography  with a UV detector and an automatic sample injector. All separations were carried out using chromatographic columns (TSKGgel G3000SWXL 7.8 × 300). Four standards were used for molecular weight calibration: Thyroglobulin bovine (670 kDa), γ-globulins from bovine blood (150 kDa), albumin chicken egg grade VI (44.3 kDa), and ribonuclease A type I-A (13.7 kDa). −1 for 64 scans [FT-IR analysis was conducted with an FT-IR attenuated total reflection (ATR) spectrometer . Spectra records were kept at a resolution between 400 and 4000 cm64 scans . The morphology of MLDs was determined via AFM . The 50 μL diluted sample (25 μg/mL) was deposited onto a mica slide. Nitrogen was blown over the samples at room temperature for 1 min before analysis. 3[Fe (CN)6] solution  and then heated in a water bath at 50 °C for 20 min. The Cl3CCOOH solution  was added and centrifuged at 4500 r/min for 10 min. The supernatant (2.5 mL) was mixed with 2.5 mL of deionized water and 0.5 mL of FeCl3 . The absorbance at 700 nm was measured after mixing at room temperature for 10 min.s is the MLD absorbance and A0 is the absorbance of the control group.The antioxidant activity was measured according to modified methods of Babbar et al. 2011) [11 [20], p < 0.05 were considered to show significant differences. All experiments were performed in triplicate. Statistical analysis was performed using the SPSS 21.0 software . Statistical data were expressed as mean ± standard deviation. Significant differences among the means were determined via ANOVA corrected for multiple comparisons with Duncan’s multiple range test, and values of 280 nm), 325 nm (A325 nm), and 420 nm (A420 nm) is shown in 280 nm at 4 °C was the most significant with the increase in temperature, while A325 nm and A420 nm increased more at 20 °C and 35 °C. After the same amount of time in storage, A280 nm decreased with the rising temperature, while A325 nm and A420 nm increased. The absorbance at 280 nm, 325 nm, and 420 nm corresponded to the initial, intermediate, and final stages of the MR, respectively. A280 nm also demonstrated that the colorless intermediate was produced via the condensation of glucosamine and Amadori rearrangement in the initial stage of the MR [MLDs are the final product of the MR and appear as brown in aqueous solution. MLDs have a wide UV absorption range, covering almost the entire UV spectrum. In the present study, MLDs in BG were characterized via spectrophotometry to evaluate their properties. The absorbance of MLDs in BG under different storage conditions at 280 nm  were produced due to a variety of reactions, including amino acid degradation (Strecker degradation), sugar dehydration, and sugar fragmentation. Highly colored products were formed through aldol condensation, aldehyde amine condensation, and the formation of heterocyclic nitro compounds in the final stage (420 nm). The rate constant of the first-order reaction equation showed that the reaction rate was the fastest at 4 °C, which means the rate of MR product formation in the initial stage was fastest at 4 °C. The reaction rate in the middle and late stages of the MR increased with the increase in temperature. The reaction rate of MLDs stored at 35 °C was 1.55 times faster than the rate of those stored at 20 °C in the middle stage of the MR, while it was 3.66 times as fast as the rate of those stored at 20 °C in the later stage of the MR. Therefore, more initial-stage products formed at lower temperatures, while more MLDs were formed at the higher temperature over the same amount of storage time. Overall, the temperature effectively promoted the MR, thereby accelerating the generation of high-molecular-weight (HMW) MLDs. The results are similar to those found in a previous report by Yang et al. (2023), which showed that the content of MLDs in distillers’ grains increased with an increase in heating temperature . They foThe specific chemical compositions of MLDs are unclear, but sugar and protein are considered to form the skeleton of MLDs. As shown in It could be seen that proteins were continuously added to the MLD structure as the MR progressed . This result is in line with the observation made by Kang, who reported that N content increased during MLD formation from BG processing [In summary, we identified that the increase in temperature and time caused the continuous construction of the MLD skeleton, which was caused by the continuous incorporation of carbon-rich and nitrogen-rich substances. The increasing integration of carbon-rich substances, such as reducing sugar and total phenol, led to the final polymeric MLD skeleton. The content of N increased during storage at high temperatures with a significant difference  proved that there was a ring-opening fracture of the furan ring . In summAfter the initial analysis, PCA was used for multivariate analysis to further identify differences A,D,G. OnFurthermore, the heat map was applied to explore differences among different groups of samples. The components with significant differences among the 4 °C MLD pyrolysis components were 3-methyl-2-cyclopentenone, 2,5-dimethylfuran-3,4  -Dione, and 2,5-furan carbaldehyde. Significant differences in the 20 °C MLD pyrolysis components were reflected in 5-hydroxymethylfurfural, 2-formyl-5-methylfuran, and furfural. At 35 °C, the main molecules among MLD components showing significant differences were glacial acetic acid, 3-methyl-1,2-cyclopentanedione, and 2-methyl-2-cyclopentenone. Overall, the change in heterocyclic compounds and ketones was the most significant feature during storage. Ketones come from the degradation of sugars. Glacial acetic acid might come from the degradation of saccharides at high temperatures  or from MLDs are high polymers with different MWs, and the molecules are closely connected. The weight-average MW of MLDs under different storage conditions is shown in Our results verify that the MW of MLDs increased during storage at 20 °C and 35 °C, which means more HMW MLDs were produced in the middle and late stages. The decrease in MLDs’ MW after storage at 4 °C was due to the massive initial-stage products of the MR, resulting in a decrease in the weight-average MW, which could explain why MLDs stored at 4 °C had higher absorption at 280 nm. The proportion of MLDs with a HMW stored at 20 °C and 35 °C was greater, which is consistent with previous results showing absorption at 420 nm . In brie−1 and 450 cm−1 , but the peak moved to 1604 cm−1 at 4 °C, 1601 cm−1 at 20 °C, and 1600 cm−1 at 35 °C after 6 months of storage. The MLDs that were not stored peaked at 1254 cm−1 (the amide III group), but the peak moved to 1241 cm−1 at 4 °C, 1239 cm−1 at 20 °C, and 1240 cm−1 at 35 °C after 6 months of storage. The main differences in the infrared spectra were mainly concentrated in the amide I and amide III groups as the storage time and temperature increased, but the difference in storage time was more significant. The MLDs that were not stored peaked at 1622 cm2) may be lost, but they can be increased by the presence of reactions such as Amadori compounds (C=O), Schiff bases (C=N), and pyrazine (C–N) [In summary, we identified that the chemical changes in the MR resulted in a change in the infrared spectra of the MLDs due to the alteration of several functional groups. Functional groups (including -NHne (C–N) . It has ne (C–N) . These rne (C–N) . AdditioThe surface morphology of MLDs stored for different periods was obtained via AFM . SurfaceOur results suggest that the changes in surface morphology were consistent with the MW, which could explain the decrease in Ra at 4 °C and the increase in Ra at 20 °C and 35 °C. Low-MW MLDs were more likely to aggregate to form more HMW MLDs ,35, thusThe DPPH, ABTS radical scavenging activities, and total reducing power were evaluated based on the antioxidant properties of natural botanicals. MLDs under different storage conditions possessed excellent antioxidant activity  tended to promote the formation of HMW compounds. Ketones and heterocycles were derived from precursor substances, which played significant roles in MLD formation. Therefore, the smoothest MLD surfaces were observed at 4 °C, while rougher MLD surfaces were formed at higher temperatures. In addition, the antioxidant capacity was positively correlated with storage temperatures. In summary, temperature had an impact on the composition, structure, and antioxidant activity of MLDs. Further research is needed to investigate the potential changes in their functional characteristics and food safety as a result.


The pooled prevalence of the risk of malnutrition in those with dementia was 57.43% . Malnutrition is widespread in those with dementia living in long-term care. Further research exploring malnutrition in other industrialized countries using validated assessment tools is required.Dementia is a common syndrome in older people. Dementia alters eating behaviors, hunger and thirst cues, swallow function, ability to self-feed, and recognition and interest in food. There is significant variation in the reported prevalence of malnutrition among older people who live in long-term care. The aim was to conduct a systematic literature review and meta-analysis of the prevalence of malnutrition in those with dementia living in long-term care using a validated nutrition assessment tool. Scopus, Web of Science, CINAHL, and Medline were searched. A random effects model was used to determine the prevalence and risk of malnutrition. Data were retrieved from 24 studies. Most of the studies were from Europe or South Asia. The prevalence of malnutrition ranged from 6.8 to 75.6%, and the risk of malnutrition was 36.5–90.4%. The pooled prevalence of malnutrition in those with dementia in long-term care was 26.98% (95% CI 22.0–32.26,  Dementia is a syndrome associated with chronic progressive degeneration of the brain . DementiIrrespective of the type of dementia or cause, food intake can be significantly altered due to the complex nature involving many physiological, pathological and psychological factors ,6. In thAs dementia advances, so does the requirement for assisted living due to their behavioral and psychological changes, including wandering, progressing cognitive impairment, and difficulties partaking independently in activities of daily living (ADL) . AccordiMalnutrition is ‘a subacute or chronic state of nutrition, in which a combination of varying degrees of under- or overnutrition and inflammatory activity has led to changes in body composition and diminished function’ . Studies2, which underscores the necessity for using a validated assessment tool to diagnose malnutrition [Unfortunately, malnutrition is often undetected and neglected in long-term care facilities due to a lack of malnutrition screening and inadequate dietetic staffing. Biomarkers such as serum albumin and transferrin are unreliable assessment methods as they are influenced by other confounding factors such as inflammation, infection and the presence of co-morbidities . Body mautrition . Severalutrition . Studiesutrition ,16,17,18The question of interest was “in people diagnosed with dementia (population) who are living in residential long-term care (context), what is the prevalence of malnutrition  AND  AND  as Medical Subject Heading terms and keywords as per the database. The search terms had been identified by analyzing keywords in similar studies. The electronic databases Scopus, Web of Science, CINAHL, and Medline were searched on 24 April 2022 with no restrictions. The search terms were developed by one author (E.P.) in conjunction with a specialist librarian and revised by two authors of the research team (K.W. and K.L.). The reference lists of relevant studies on similar topics of dementia, malnutrition and long-term care facilities were also searched and a search for unpublished reviews on PROSPERO was conducted. The search strategies for each database are listed in The search was conducted independently by one author (E.P.) and uploaded to Covidence software  where duTo be eligible for inclusion, studies were required to be primary research studies where malnutrition was assessed using a validated nutrition assessment tool, were conducted in long-term care or equivalent, and residents must have been diagnosed with dementia or cognitive impairment using relevant assessment tools or with a confirmed diagnosis reported in their medical record. Studies were excluded if they assessed malnutrition using non validated measures , reported for those without a probable diagnosis of dementia using a validated tool, including those with acute delirium, and studies not in English. Studies were also excluded if the outcome of interest, specifically the number of individuals with malnutrition and dementia, was unable to be determined or was not reported.Data extraction was completed independently by one author (E.P.) in Microsoft Excel   and a seThe Academy of Nutrition and Dietetics Criteria Checklist for Primary Research was used to assess the quality of each study . The toop < 0.05. The I2 statistic was used to evaluate and indicate degree of variance between studies and study heterogeneity, with a higher score closer to 100% indicating higher heterogeneity between the studies. A random effects model was used due to the diversity of studies globally and was determined to be better able to capture the true prevalence of malnutrition. Publication bias was assessed using a funnel plot and Egger’s test and the DerSimonian and Laird method was used for between study heterogeneity for rates of malnutrition.Data on the total number of participants with dementia, and the prevalence of malnutrition and/or those at risk of malnutrition were calculated and inserted into Microsoft Excel . Data were then exported into MedCalc (version 20.11)  to conduThe initial search yielded 2946 studies. After the removal of 1494 duplicates, 1452 studies were screened via their title and abstract. This resulted in 365 full-text articles assessed for eligibility, with 24 studies included for analysis. Studies that did not specifically report on the number of those with dementia ,29 were A total of 8775 participants with dementia were included in the review ranging from 2 to 2379 participants per study  Table 1Table 1. MNA  and MNS Ten studies reported the mean age of those with dementia, with the lowest mean age being 74.5 ± 7.68 years  and highSix studies used medical records to obtain the dementia diagnosis of the patients ,43,47,50A wide range of tools were used for the diagnosis of dementia . These iSeveral validated nutrition assessment or screening tools were identified as in use. This included the Mini Nutritional Assessment (MNA) in fifteen studies ,49,51,52Nine studies did not state who conducted the nutrition assessment ,46,48,50p < 0.0001, I2 = 94.12%)   (p = 0.48), risk of malnutrition (p = 0.52), and combined prevalence and risk of malnutrition (p = 0.41).Twenty-four studies reported on the prevalence of malnutrition . Prevale 94.12%) . The poo 97.38%) . The poo 98.42%)  [29,30,3Most of the 24 studies were rated as positive (18 studies) ,49,50,52To our knowledge, this is the first systematic literature review reporting the prevalence of malnutrition in those with dementia living in long-term care using established, validated nutrition screening or assessment tools. Analysis of the 24 eligible studies suggest that the prevalence and risk of malnutrition in this population and setting is high, with a pooled prevalence of malnutrition of 26.98%, risk of malnutrition at 57.43%, and the combined pooled prevalence was 79.66%.These high rates of malnutrition in those with dementia are concerning due to the life expectancy increasing worldwide, thus an increased likelihood of individuals developing dementia as they age . Ten stuOur results are similar to a systematic review using only the MNA . Cereda Of interest in this review was the gender distribution in the included studies. Nine studies reported on gender distribution, with the lowest percentage of females at 59%  and highp < 0.05) [Many factors contribute to the high prevalence of malnutrition in those with dementia. This includes knowledge that those with AD often have an increased resting energy expenditure (REE) and feeding difficulties ,68. An a < 0.05) . It is i < 0.05) . Further < 0.05) . One stu < 0.05) . Similar < 0.05) .Screening and diagnosis of malnutrition can be conducted using a range of validated tools. The MUST and MNA-SF can be used for screening and the MNA and PG-SGA for assessment. The MNA-SF and MNA are validated for older adults only ,72. NineInterestingly, no study in our review compared malnutrition rates in those with dementia in long-term care to those in the community; thus, we cannot compare nutritional status between locations. One review found the prevalence of malnutrition in long-term care to be the highest at 21.6% compared to only 9.2% in the community . This isAn unexpected finding of this study was the limited number of studies and limited distribution of studies globally. Three quarters of the studies were conducted in Europe and 25% of the studies were conducted in South Asia. This review is therefore not able to provide insight into the issue of malnutrition in the United Kingdom, North America, South America, Africa, or Australasia. The World Alzheimer’s Report in 2015 found that the highest prevalence of AD globally was in East Asia and Western Europe and determined that the distribution of new dementia cases was 49% in Asia , 25% in Europe, 18% in the Americas and 8% in Africa . These hIt is important to note that dietitians are essential staff in a long-term care facility, but unfortunately, only two studies used dietitians to conduct the nutrition assessment, and nine studies did not state who conducted the assessment. Nurses play a crucial role in long-term care facilities; however, they are likely to need more training in the prevention, detection and management of malnutrition in residents with dementia. One study examining nurses’ ability to detect malnutrition found an alarming discrepancy between malnutrition prevalence according to nurses (11% of residents) compared to 32.9% using objective criteria for malnutrition . SimilarThere are several limitations to this systematic literature review. Studies were restricted to articles in English, which meant that eligible studies in other languages may have been excluded. The restriction to scientific databases meant some relevant studies may not have been found, and we did not contact the authors for further information; therefore, potential data may have been excluded. The high level of heterogeneity between studies may be the result of differing tools used to assess the outcome of malnutrition. While a random effects model was used, this heterogeneity indicates there is some uncertainty in the results. Dementia diagnoses are known to be under recorded in low- and middle-income countries , which pTo our knowledge, this is the first synthesis of this topic and can be used to inform future interventions and advocacy efforts for people living in long-term care. Overall, this review suggests that there is a high prevalence and risk of malnutrition in those with dementia living in long-term care facilities. Given the high prevalence, the use of validated nutrition assessment tools to assess malnutrition is strongly recommended and suggests that these tools are ideally administered by dietitians to enable rapid access to appropriate medical nutrition therapy. Future research should explore the prevalence of malnutrition in other settings including long-term care institutions in the United Kingdom, North America, and Australasia, as well as in other subgroups with dementia, such as those with dysphagia and neurological conditions.


Here, this cross-species analysis focused on KCNN3 DNA methylation, gene expression, and single nucleotide polymorphisms including alternative promoters in KCNN3 that could influence surface trafficking and function of KCa2 channels. Bisulfite sequencing analysis of the nucleus accumbens tissue from alcohol-drinking monkeys and alcohol dependent mice revealed a differentially methylated region in exon 1A of KCNN3 that overlaps with a predicted promoter sequence. The hypermethylation of KCNN3 in the accumbens paralleled an increase in expression of alternative transcripts that encode apamin-insensitive and dominant-negative KCa2 channel isoforms. A polymorphic repeat in macaque KCNN3 encoded by exon 1 did not correlate with alcohol drinking. At the protein level, KCa2.3 channel expression in the accumbens was significantly reduced in very heavy drinking monkeys. Together, our cross-species findings on epigenetic dysregulation of KCNN3 represent a complex mechanism that utilizes alternative promoters to impact firing of accumbens neurons. Thus, these results provide support for hypermethylation of KCNN3 as a possible key molecular mechanism underlying harmful alcohol intake and alcohol use disorder.The underlying genetic and epigenetic mechanisms driving functional adaptations in neuronal excitability and excessive alcohol intake are poorly understood. Small-conductance Ca In addition, prolonged excessive ethanol consumption produces neuroadaptations in projection neurons and neural circuits that are proposed to sustain heavy drinking12. Although there are three FDA-approved pharmacotherapies for treating AUD, only one  targets genetic variation in individuals with AUD. While pharmacologically targeting known single nucleotide polymorphisms (SNPs) can reduce relapse rates in a subpopulation of individuals with AUD13, mixed results suggest a critical need for further investigation of the (epi)genetic factors and neuroadaptations that contribute to the excessive ethanol drinking in AUD.Alcohol (ethanol) use disorder (AUD) is a devastating brain disease driven by complex interactions between genetic, epigenetic, and environmental factors. Genetic factors increase the propensity for risky drinking2+-activated K+ (KCa2 or SK) channels in cortical-striatal brain circuitry involved in motivational processing have emerged from preclinical studies as a target for treating AUD19. In this circuitry, neuronal firing patterns20 of nucleus accumbens core (NAcC) medium spiny neurons (MSNs) and substantia nigra dopamine neurons are controlled by KCa2.3 channels encoded by the KCNN3. Previous functional mouse genomics studies, found quantitative trait loci (QTL) containing Kcnn3, particularly NAcC Kcnn3 transcript levels, were negatively correlated with voluntary drinking in genetically diverse BXD strains18. Further, the promoter region of Kcnn3 was associated with ethanol preference in selectively bred rat lines21. In addition, intrinsic excitability and reduced KCa2 channel function and KCa2.3 channel protein expression were increased in the NAcC of excessive ethanol drinking and dependent rodents22. Functionally, blocking KCa2 channels in the NAcC with apamin increased voluntary ethanol drinking in mice17, whereas positive modulators of KCa2 channel function reduced home cage drinking and operant self-administration22. Importantly, the ability of apamin to inhibit KCa2 channel function was completely lost in NAcC MSNs from ethanol dependent mice, but not in rats that had access to 7-weeks of operant self-administration of moderate amounts of ethanol15. Together, these studies identified KCNN3 in general, and NAcC KCa2 channel function in particular, as a potential regulator of excessive ethanol consumption and dependence in rodent models of chronic ethanol exposure.Small-conductance CaKCNN3 encoded by exon 123. Higher numbers of CAG repeats reduced KCa2 channel function in transfected HEK293 cells24, and this polymorphism has been associated with neuropsychiatric conditions, such as schizophrenia and anorexia nervosa28. While this polymorphism did not confer risk for developing the disease, longer CAG repeat length is associated with higher cognitive performance in individuals with schizophrenia24. This finding is consistent with a known role for KCa2.3 channel regulation of cognitive function and plasticity of intrinsic excitability that is an important mechanism for forming new learned associations 30. Because the CAG trinucleotide repeat is conserved in nonhuman primates31 and macaques exhibit a range of ethanol drinking that mimics human consumption34, the present study explored the relationship between CAG repeat length and ethanol drinking in rhesus macaques with low- and heavy-drinking phenotypes.There are two polymorphic CAG repeats in the N-terminus of human 36, and a recent study demonstrated that knockdown of DNA methyltransferases reduced Kcnn3 expression and increased intrinsic excitability of cultured cortical neurons37. Thus, we measured DNAm levels at a differentially methylated region in exon 1 (MR-ex1) that coincides with a cross-species regulatory region within the KCNN3 promoter. In humans, the KCNN3 gene encodes four known transcripts by making use of alternative first exons and alternative splicing. Similar to the CAG trinucleotide repeat, alternative KCNN3 transcripts influence function of KCa2 channels. KCNN3 transcript SK3_1B encodes a truncated channel that functions as a dominant-negative to suppress endogenous KCa2 channel currents38, whereas transcript hSK3_ex4 encodes a protein with an additional 15 amino acid insertion within the S5-PHelix loop that renders the channel insensitive to apamin block39. In chronic ethanol drinking mice and monkeys, changes in DNAm of exon 1 altered the expression of KCNN3 transcripts in the NAcC. Here, we report a complex cross-species relationship between NAcC KCNN3 and excessive ethanol drinking that ultimately leads to reduced KCa2.3 channel protein expression in mice and monkeys.DNA methylation (DNAm) is an epigenetic mark that contributes to modulation of gene expression by modifying the accessibility of transcription factors to chromatin. Alterations in DNAm are reported in heavy drinking humans, monkeys and rodentsn = 66, Macaca mulatta) from seven different cohorts  were included in this study  and described in detail in Supplemental Materials. Monkeys were individually housed and ethanol self-administration was induced using schedule-induced polydipsia, as previously described34. For all cohorts, monkeys had open access to 4% ethanol  and water (ethanol subjects) or water only (control subjects) for 22 h/day, every day, for over 12 months (see33 for further details on these seven cohorts). The 49 monkeys with access to ethanol were classified into three different age categories based on their age of first ethanol access and four different drinking categories based on previously described criteria32. All of the animal procedures used in this study were approved by the Oregon National Primate Research Center IACUC and were performed in accordance with the NIH and the National Resource Council’s Guide for the Care and Use of Laboratory Animals.Male and female rhesus macaques  at ~ 7 weeks of age. Baseline ethanol drinking (22 h day/15% ethanol v/v) was established prior to treatment with 4 repeated weekly cycles of chronic intermittent ethanol (CIE) exposure in vapor inhalation chambers, alternated with weekly home cage drinking sessions, as previously described40. Genomic DNA and RNA were extracted from male and female monkey and male mouse NAcC samples using the All Prep DNA/RNA/miRNA Universal kit  following the manufacturer’s recommendations. Blood samples drawn from macaques prior to ethanol self-administration were used for CAG repeat analysis. Briefly, blood was collected in EDTA tubes and DNA was isolated using QIAamp DNA mini kit following manufacturer’s instructions (QIAGEN Sciences Inc).After the 12 month open access period, a detailed necropsy protocol was used to systematically collect tissues from all macaquesKCNN3 CAG repeat region as previously described41. The primers and methods are described in Supplemental Materials. We found that the first exon 1 CAG repeat was not variable in rhesus macaques; thus, these studies focused on the second exon 1 CAG repeat length.Blood DNA was used to analyze the number of CAG repeats within the second KCNN3 promoter region using NAcC tissue from macaques and mice following published methods31 and as described in Supplemental Materials. Primers were designed to amplify a 646 bp region of the KCNN3 within exon 1A and intron 1 in human, rhesus macaque, and mice. Because of the length of the region, two sets of primers were designed to cover the whole region .Bisulfite amplicon sequencing was used to measure the DNAm rates of a DMR within the Supplemental Materials. The primer sequences are described in Supplemental Table 3. Since most of the alternative transcripts are not annotated in the Rhesus or mouse genome, we used the human annotations to design the primers, then identified the homologous sequence in the rhesus macaque (MacaM)42 and mouse (GRCm38.p3) genome. The mRNA expression levels were normalized using the phosphoglycerate kinase (PGK1) gene. This gene was demonstrated to be a reliable control for brain gene expression43. We also previously confirmed that different levels of ethanol use did not affect its expression4.The NAcC RNA quantity and quality was evaluated and qPCR was performed in triplicates assays, as described in 44, as fully described in Supplemental Materials.After extraction, tissue samples containing the NAcC extracted from female control and long-term drinking rhesus macaques were prepared for western blot analysis following our previously published methods in monkey brain tissueCa2.3 protein expression level per comparison group. All variables analyzed followed a normal distribution. Welch’s one-way ANOVA was used to compare the difference in average methylation between controls and ethanol drinkers with Games-Howell post-hoc tests. One-way ANOVA was used to compare mRNA relative expression levels between groups. Prior to applying one-way ANOVA, Levene’s test was used to test homogeneous variance assumption for parametric methods. When heterogeneous variance was detected, we used the nonparametric Kruskal-Wallis test. Bonferroni or Tukey correction for the multiple comparisons were used to correct the overall type I error rate. Two-tailed independent t-test was used to compare the difference in average methylation rate between controls and dependent mice. Based on the Levene’s test for homogeneous variance, we used the appropriate p-value (homogeneous or heterogeneous variance). The allele frequency distribution of CAG trinucleotide repeats between controls and drinking monkeys was compared using the Kruskal-Wallis test . Normalized western blot data were analyzed by a two-tailed t-test in Prism. Ethanol drinking data in mice were analyzed by a repeated-measures mixed linear model with a Tukey post-hoc test .Data from heavy and very heavy drinking monkeys were combined due to small sample sizes in the transcript analysis and bisulfite sequencing studies. All statistical analyses were carried out using IBM SPSS Statistics  except where noted, with values α < 0.05. The Shapiro-Wilk test  was used to assess the normality of the average methylation rate, mRNA expression rate, and K33.Sixty-six, male and female rhesus macaques, enrolled based on no common parents, were used in this study. Previously, daily ethanol drinking was mathematically modeled based and determined 4 categorical levels of intake: low, binge, heavy and very heavy. In the subjects of this study, there were 16 low drinking (LD), 9 binge drinking (BD), 9 heavy drinking (HD), and 15 very heavy drinking (VHD) monkeys. The average daily (22 h) ethanol intake (range: 0.47 to 5.15 g/kg) across the 12 months of open access for each of the drinking monkeys along with their age at drinking onset and percentage of drinking days over 3 g/kg are shown in 46, mice exposed to CIE significantly increased their voluntary ethanol intake  = 2.47, p = 0.0491). Post hoc analysis indicated that the two treatment groups were not different at baseline (p = 0.6685), but differed during weekly test drinking sessions 1 (p = 0.0074), 2 (p = 0.0468), and 3 (p = 0.0034). In the ethanol dependent mice, drinking levels in all four test sessions were significantly higher than their intake during baseline (p = < 0.0001).To more closely match the monkey drinking paradigm, the standard two-bottle choice, limited-access mouse model of dependence-induced escalation of drinking was modified to allow mice open access to ethanol (15% v/v) for 22 h/day. Ethanol drinking prior to and following each weekly exposure to CIE is shown in KCNN3 contains two polymorphic sites composed of a variable number of CAG repeats that have been associated with KCa2 channel activity. It has been reported that higher numbers of CAG repeats reduces KCa2 channel currents24. Thus, we investigated the variability of these polymorphisms in ethanol-drinking rhesus macaques and their influence on KCNN3 regulation and transcript levels. In our studies, the first CAG repeat array that was not polymorphic, however the second CAG repeat copy number was highly variable across rhesus monkeys (H(4) = 2.354, p = 0.5023; p = 0.6946; p ≥ 0.1631; p = 0.3563; Supplemental Fig. 1). Data for expression of KCNN3 transcripts and MR-ex1 DNAm rates were obtained from the following sets of studies.The promoter region of MR-ex1 of  monkeys  and rangKCNN3 gene and promoter across human, rhesus macaque, and mouse, we identified ten regions with potential conserved regulatory region, based on sharing over 95% sequence homology and ≥ 75% CpG identity. We then analyzed the DNAm patterns in these ten candidate regions between ethanol-naïve, LD, and HD/VHD rhesus macaques. Because of the small sample size, BD were not included in this analysis, while HD and VHD were combined based on their similar drinking behavior. Among the different regions, only a region of 646 bp overlapping with exon 1 and intron 1 of KCNN3 . While the overall CpG conservation was lower in the mouse as compared to both human and rhesus macaques , the high sequence and CpG similarity of this region across species suggests functional relevance and underscores the potential translational value of the DNAm signal identified in this study. Overall, the CpGs within MR-ex1 showed generally low DNAm in controls, with methylation levels ranging from 7–24% in males . Furthermore, and in agreement with a potential role of this region as a promoter48, several transcription factors relevant to neuronal regulation and known to have a role in mediating the effects of ethanol on gene regulation are predicted to bind to it, including GR (glucocorticoid receptor49), ER-α (estrogen receptor50), CREM (cAMP responsive element modulator51), CREB (cAMP responsive element binding protein52), Sp153, GATA-354, NeuroD155, C/EBP (CCAAT-enhancer-binding proteins56) and AP-2α57 . In addition to the fact that this differentially methylated region is located in exon 1 (605 bp downstream of the transcription start site of exon 1A), it is upstream of exons 1B and 1C , and could act as a regulatory region contributing to differential expression of KCNN3 transcripts.Comparison of the MR-ex1 region to human ENCODE dataKcnn3 methylation levels ranged from 0.1–17%, with average methylation rates for 19 out of 21 CpGs below 5% in the controls. Interestingly, three CpGs showed significantly higher methylation rates in CIE-exposed drinking mice as compared to rates in controls  and two were located in the binding sites for AP-2α .We next investigated the DNAm profile of the MR-ex1 region in mice that were drinking ethanol for 22 h in the CIE dependence model. Similar to ethanol-naïve rhesus macaques, DNAm in this region was relatively low in air-exposed control mice. controls . These CKCNN3 mRNA expression. The mouse (GRCm38.p3) and rhesus macaque (MacaM or Mmul10) genomes are not annotated with as much detail as the human. Thus, in order to investigate the effects of ethanol drinking on the expression of the different KCNN3 transcripts, we designed primers to amplify two exons common to all reported transcripts (SK3_ex7/8), as well as primers to specifically amplify transcript SK3_ex4, SK3_ex1B, and SK3_ex1C in human. Next, each amplicon’s orthologous sequence was identified in mouse and rhesus macaque, and species-specific primers for the different transcripts were designed. It should be noted that transcripts SK3_ex1B and SK3_ex4 encode dominant-negative and apamin-insensitive isoforms of KCa2.3 channels, respectively, and transcript SK3_ex1C was not detected in the NAcC, in agreement with previous studies indicating this transcript is not expressed in the brain59.We next evaluated the potential relevance of MR-ex1 hypermethylation in regulating SK3_ex7/8) showed no differences among the different ethanol-drinking male monkey groups  = 2.721, p = 0.0870, n = 8–9/group; SK3_ex1B  = 3.547, p = 0.0462, n = 7–10/group) and SK3_ex4  = 9.89, p = 0.0009, n = 8–9/group; Supplemental Table 4). In addition, transcript SK3_ex4 was significantly increased in the accumbens of LD male monkeys  = 1.802, p = 0.2134; SK3_ex1B: two-tailed t-test; t(2) = 0.1494, p = 0.8950; SK3_ex4: two-tailed t-test; t(2) = 0.6137, p = 0.6019); thus, samples from these two age groups were pooled for further analysis. When collapsed across age, 12 months of open access to ethanol regardless of drinking phenotype significantly increased KCNN3 transcript SK3_ex4 expression  = 10.88, p = 0.0009; SK3_ex7/8: one-way ANOVA: F = 1.798, p = 0.1941; SK3_ex1B: one-way ANOVA: F = 2.935; p = 0.0803; The expression of the exons common to all transcripts  = 0.036, p = 0.972, n = 4–6/group; SK3_ex1B (two-tailed t-test: t(10) = 2.658, p = 0.024, n = 5–7/group; SK3_ex4 (two-tailed t-test: t(9) = 2.477, p = 0.037, n = 5–6/group; KCNN3 and the shifts in transcript expression affected KCa2.3 channel protein expression, we performed western blot analysis in accumbal tissue from the same female rhesus macaques. Characterization of KCa2.3 channel immunoreactivity in ethanol-naïve monkey NAcC samples revealed a linear dynamic range across twofold dilutions between 1.25 and 40 μg of protein  = 2.585, p = 0.0324, n = 5/group; Ca2.3 channel, such as the dominant-negative isoform 3 with a predicted molecular weight of 47 kDa, were unsuccessful with commercially available antibodies.Because Ca2 currents and expression in the nucleus accumbens of rats and mice22, we next determined if Kcnn3 transcript expression is altered in ethanol dependent mice. Similar to the monkey data, ethanol drinking and/or dependence did not affect Kcnn3 expression of SK3_ex7/8  = 3.931, p = 0.0529; SK3_ex1B expression  = 7.228, p = 0.0114; SK3_ex4 was significantly increased in drinking mice regardless of their history of CIE exposure  = 32.29, p < 0.0001; Given that chronic ethanol drinking and dependence reduced KKCNN3 and changes in gene expression in heavy drinking macaques and ethanol dependent mice. In both monkeys and mice, ethanol drinking and dependence was associated with hypermethylation of conserved CpGs at a predicted regulatory region in exon 1A of KCNN3. In parallel with the hypermethylation, excessive drinking increased expression of a dominant-negative transcript of KCNN3 that is transcribed using an alternative exon downstream of exon 1A. Consistent with chronic ethanol-induced loss of apamin-sensitive currents in accumbens and orbitofrontal neurons61, ethanol drinking increased expression of the transcript that encodes apamin-insensitive KCa2 channels. We also found a reduction in KCa2.3 channel protein in heavy drinking female macaques that is congruent with decreased expression reported in rodent models of chronic ethanol exposure. These results suggest that ethanol-induced regulation of KCNN3 transcripts is a conserved mechanism that underlies functional changes in KCa2 channels reported in rodent models.The results from these studies provide cross-species evidence for alcohol-associated alterations in DNAm signals mapping to 62. Our analysis identified a DMR that spans exon 1 and part of intron 1 of monkey and mouse KCNN3 in a region containing CpGs that are highly conserved across species. Across male and female monkeys, more than half of the CpGs in this DMR were hypermethylated in HD/VHD, but not in LD or ethanol-naïve monkeys, an effect that is also conserved in ethanol dependent mice that show escalated drinking. Importantly, the DMR in exon 1 of KCNN3 coincides with a predicted promoter region with binding sites for neural relevant transcription factors. Furthermore, this DMR is upstream of two alternative exons 1 (1B and 1C). Together, these findings suggest that the DMR is strategically located to potentially regulate alternative transcript expression of KCNN3. A previous characterization of the promoter region upstream of the exon 1A transcription start site (TSS) identified consensus sequence binding sites for CREB, AP-1, and AP-248. Additional binding sites for these same transcription factors are predicted to bind to MR-ex1, specifically to significantly differentially methylated CpGs. Numerous signaling transduction pathways that are altered by ethanol consumption lead to CREB activation63, a key mediator in the development of addiction. Upon activation, CREB can exert its influence upon target gene transcription and interact with promoter-bound cofactors. Previous evidence showed that CREB modulates BK channel expression64, and our results suggest that it may also modulate KCNN3 expression. AP-1 complexes containing FosB, which accumulates in the NAc after drug intake, modulates promoters of genes relevant to addiction, such as GluA2 and dynorphin65. The MR-ex1 region also contains binding sites for glucocorticoids, which have been extensively associated with AUD67. Others have shown that glucocorticoids and stress exert profound effects on intrinsic excitability in neurons through regulation of ion channel activity, including KCa2 channels71. Thus, it is possible that ethanol, by modulating transcription factor levels as well as availability of binding sites by DNAm, modulates KCNN3 expression.In the current study, we identified a DMR that maps to an ion channel gene previously implicated in excessive drinking, ethanol-seeking behaviors, and ethanol-induced plasticity of intrinsic excitability4. In a recent whole-genome analysis of the NAcC methylome in additional ethanol-naïve and HD/VHD macaques5, several KCNN3 DMCs  with alcohol intake levels following prolonged alcohol drinking and repeated cycles of abstinence and relapse (unpublished data). These results suggest that pre-existing DNAm signatures of KCNN3 and other genes could be a risk factor for future heavy alcohol drinking. In mice, induction of ethanol dependence increased evoked firing in NAcC MSNs17 and induced synaptic proteome adaptations in the NAcC72. Because alterations in neuronal firing underlie synaptic integration, learning processes and may facilitate drug-associated synaptic remodeling74, our findings suggest that a change in the methylation status of key CpGs is a critical cross-species mechanism that might regulate coordinated neuronal excitability and synaptic adaptations that lead to uncontrolled drinking.Using a genome-wide approach, we previously reported DMRs associated with the modulation of genes that regulate synaptic plasticity in the NAcC of heavy drinking monkeysKCNN3 transcripts that encode KCa2.3 channels that reduce surface trafficking and apamin sensitivity. A combination of transcripts and protein expression data suggest that with heavy ethanol consumption there is a decrease in transcript SK3_ex1A expression while there is an upregulation in transcripts SK3_ex1B and SK3_ex4. While expression of the dominant-negative transcript was only elevated in heavy drinking monkey, and expression of the apamin-insensitive transcript was increased by ethanol intake regardless of the drinking phenotype or the age of drinking onset. These results are in agreement with our functional and behavioral data on reduced apamin sensitivity in the nucleus accumbens and orbitofrontal cortex of ethanol dependent mice and self-administering rats61. We previously reported that apamin microinfusion into the NAcC increased drinking and bath application of apamin reduced KCa2-mediated currents in NAcC MSNs in non-dependent C57BL/6J mice. However, the ability of apamin to influence drinking and KCa2 currents was completely lost when mice were exposed to CIE. Moreover, ethanol intake and evoked firing in NAcC MSNs were increased and there was a reduction in KCa2 channel currents and protein levels in the ethanol dependent mice. Although it is unknown if heavy ethanol drinking in macaques alters firing properties of NAcC MSNs, the shift in transcripts and the reduction in total KCa2 channel protein suggests that prolonged ethanol intake increases intrinsic excitability similar to results from rodent models of ethanol self-administration15 and dependence75. In addition to reduced Kcnn3 gene expression and increased intrinsic excitability, a recent study reported a loss of apamin’s ability to increase evoked firing in cultured cortical neurons treated with DNA methyltransferase inhibitors37. Thus, these data provide support that the increase in SK3_ex1B and SK3_ex4 transcript expression through hypermethylation of KCNN3 exon 1A is an underlying mechanism driving these functional and behavioral adaptations across species and brain regions.In monkeys and mice, heavy ethanol drinking and dependence was associated with increased expression of Kcnn3 as a candidate signature gene that is associated with binge-like and excessive ethanol drinking21. In contrast to mounting evidence and our hypothesis, high numbers of polymorphic trinucleotide repeats encoded by exon 1 of KCNN3 did not segregate with a heavy drinking phenotype in this population of rhesus macaques. There are a number of possibilities that could explain these negative findings. Although long CAG repeats in KCa2.3 channels reduced apamin-sensitive currents24, function of this polymorphism was characterized in transfected HEK293 cells and not mammalian central neurons. In the current study, CAG repeat number was measured from blood samples taken after chronic ethanol consumption. While traditionally considered stable, there is some evidence that CAG repeats can vary between types of tissue  and can expand across time to accelerate disease progression77. Thus, future longitudinal studies are necessary to track CAG repeat number in the NAcC of LD and HD/VHD monkeys.Previous studies using functional genomics and rodents with divergent drinking phenotypes have identified KCNN3 by excessive alcohol consumption represent a complex mechanism through the use of alternative promoters that likely impact intrinsic excitability of NAcC MSNs, and, ultimately, ethanol-seeking behaviors. We propose a model in which MR-ex1 functions as a regulatory region to modulate the expression of the alternative transcripts SK3_ex1B and SK3_ex4. Our findings provide the first evidence that hypermethylation of the MR-ex1 region of KCNN3 by heavy alcohol drinking is a key cross-species mechanism that may be important for the maintenance of excessive drinking and the development of AUD.In summary, these cross-species findings of genetic and epigenetic adaptations in


Objective YouTube , one of the most accessed sites on the internet, has become a widespread source of healthcare information for patients. Videos about coronary artery bypass grafts (CABG) have accrued tens of millions of views on the platform, yet their educational quality is unknown. This study investigates the educational landscape of videos regarding CABG procedures on YouTube.MethodsYouTube was queried for “Coronary Artery Bypass Graft Surgery” and “Coronary Artery Bypass Graft Procedure”. After applying exclusion criteria, 73 videos were assessed. Two independent reviewers rated the material with the Global Quality Scale (GQS)  to judge educational value. A ratio of view count to days since upload was applied to assess video popularity. Source, modality, and date of upload were recorded for each video as well.ResultsAn average GQS score of 2.94 was found, indicating poor educational quality of the 73 YouTube videos on CABG procedures. Videos uploaded by physicians  had a significantly higher average GQS score than those uploaded by non-physicians (p<0.001). When content was grouped by delivery method, physician-led presentations (24/73 or 32.9%) produced the highest average GQS score of 3.35; conversely, patient-friendly delivery methods (18/73 or 24.7%) yielded the lowest average GQS score of 2.36 (p<0.001). Neither the view ratio nor the days since upload significantly correlated with the educational quality of the video.ConclusionAlthough CABG videos are readily available on YouTube, they often contain considerable biases and misleading information. With online sources for healthcare education now commonplace, physicians must be aware of the vast quantities of low-quality videos patients often encounter when weighing different treatment options. Further analysis of CABG videos on YouTube may allow physicians to ameliorate this gap by producing videos that are not only high quality but highly viewed on the platform. Internet use has increased in prevalence around the world as countries continue to expand their online infrastructure . RecentlWith over two billion monthly viewers, YouTube  is the largest online video platform, and a popular source for medical information ,6. HowevDespite this disparity between content quality and quantity, many patients highly value the healthcare information they find online. A previous survey found that the Internet was the second most popular source of healthcare information with 11% of individuals deferring to online information instead of consulting their doctor . CliniciCoronary artery bypass grafting (CABG) continues to be the most ubiquitous cardiac surgery in the world . ApproxiSearch strategywww.youtube.com) account was created, and Google Chrome  was set to "incognito mode" to eliminate any personalized tailoring of the YouTube algorithm to the relevant search. Two distinct phrases were entered into YouTube’s search engine to identify relevant videos on May 18, 2020: “Coronary Artery Bypass Graft Surgery” and “Coronary Artery Bypass Graft Procedure”. The filter option on YouTube was then set to “filter by view count” which organizes the videos in descending order from highest to lowest view count at the time of the search. Videos with less than 25,000 total views were excluded as these were not popular enough to warrant analysis. This left a total of 118 videos for evaluation, before applying additional exclusionary criteria. Videos that were not in English (n=11), not relevant to CABG procedures (n=16), less than one minute in duration (n=4), and duplicates (n=14) were also excluded based on previous studies [Our methodology was based on previous research studies that conducted similar analyses of health-related YouTube content ,19,20. A studies . Only En studies . Videos Video assessmentThe selected videos were scored in a similar process implemented by previous studies ,20. All Descriptive statistics including video title, hyperlink, duration, total likes and dislikes, total comments, days since upload, view count, content, source, and delivery method were recorded. Videos were then organized into three categories based on the delivery method of the video. These categories included videos that were patient-friendly modalities, live procedures, or presentations. The rationale behind these categorizations was to better characterize the current catalog of content regarding CABG procedures on YouTube as well as determine if variations in the content’s educational quality existed because of the chosen modality. Patient-friendly modalities included animations, news reports, and patient accounts, as this information was often straightforward and easiest to interpret without any prior medical knowledge. Live procedures were considered the least consumer-friendly modality as they often required a medical background to understand the video content. This is because videos of live procedures often contained little to no description of the content being presented and required a complex understanding of the surgery being performed and knowledge of key anatomical landmarks to comprehend the video. Presentations fell in the middle of comprehensibility with some including advanced analyses of CABG procedures, and others containing foundational knowledge for a broad audience. Content was also divided into two groups based on whether a physician was present in the video to substantiate any claims made about the procedure.The like-to-dislike ratio  was calculated to judge community approval as seen in previous studies . The vieStatistical analysisStatistical analysis was performed with IBM SPSS Statistics for Windows, Version 25.0 . The normality of the data was tested with the Shapiro-Wilks test. Continuous variables were reported as mean, median, and standard deviation, while categorical variables were reported as frequencies and percentages. All numerical results were rounded to two decimal places. An intraclass correlation coefficient (ICC) was calculated between reviewers to evaluate the agreement of the independent scores. The interpretation of the ICC was based on previously established criteria, which suggested a value above 0.8 was considered “excellent”, between 0.6 and 0.8 was considered “substantial”, between 0.4 and 0.6 was considered “moderate”, and below 0.4 was considered “poor” . PairwisEthics statementAll information was taken from existing data that was publicly available and is therefore exempt from institutional review board approval. A total of 73 videos that contained medical information pertinent to patients considering the procedure were included for final analysis after excluding 45 based on preset criteria to ensure adequate relevance. The average GQS score of all 73 videos analyzed was 2.94, indicating an overall poor educational quality (standard deviation (SD): 0.8). The GQS scores awarded to each video ranged from 0 to 4 with no video obtaining a perfect score of 5 from either independent reviewer.Table Patient-friendly modalities  accounted for 24.7% (18/73) of videos, live surgeries accounted for 42.5% (31/73) of videos, and 32.9% (24/73) of videos were presentations ; conversely, patient-friendly delivery methods yielded the lowest average GQS score of 2.36 (SD: 0.82) (p<0.0001). Live surgeries had an intermediate average GQS score of 2.95 (SD: 0.49) which was significantly higher than patient friendly videos (p<0.01) and significantly lower than presentations (p<0.01)  included a physician substantiating the claims made in the video, while the remaining 23.3% (17/73) did not. Content with at least one physician present in the video had an average GQS score of 3.16 (SD: 0.26). This was significantly higher than videos that did not contain a physician, which had an average GQS score of 2.21 (SD 0.53) (p<0.0001) Figure .The average view ratio of the 73 videos analyzed was 247.8 views per day (SD: 761.3), ranging from 7.2 to 6,041 views per day. There was no significant difference in the average GQS score between videos with view ratios greater than 50 views per day (37/73) and videos with view ratios less than 50 views per day (36/73) (p=0.57).After excluding videos with 0 likes and dislikes (n=4), the average like-to-dislike ratio was 16.9 likes per dislike (SD: 15.7), with a range of 3.8-77.8 likes per dislike. No significant difference in the average GQS score was found between the videos with like-to-dislike ratios above 10 likes per dislike (43/69) and videos with like-to-dislike ratios below 10 likes per dislike (26/69) (p=0.54). No significant correlation was found between the days since upload and the average GQS score of the videos. Similarly, video duration and average GQS scores were not significantly correlated. The ICC for GQS scores was 0.52, which indicated moderate agreeability between the independent reviewers.YouTube is currently the most popular video platform on the internet, and patients are increasingly relying on it as a source of healthcare information ,13,27. RAlthough an average GQS score of 2.94 exposes the low reliability of YouTube CABG content overall, different results are shown when video modalities are analyzed separately. Patient-friendly delivery methods had the highest levels of bias and misinformation with an average GQS score of 2.36. This is because these videos sacrificed medical accuracy for high comprehensibility. Animations represented 50% (9/18) of this category and were often rife with misleading descriptions of the CABG procedure. This was not surprising as previous studies have revealed the low educational quality of medical animations ,30. SimiVideos that contained a physician verifying the presented content had a higher average GQS score than those that did not contain a physician. This finding was expected, as it agreed with previous studies on this topic ,30. DoctWhen videos were stratified by those with lower versus higher engagement and popularity measures , no significant differences were found in average GQS scores. This reveals a clear disconnect between community approval and the educational quality of YouTube CABG videos. Numerous previous studies with conflicting evidence regarding engagement parameters and YouTube content quality are present in the literature ,31,34-36Analysis of video duration yielded no significant correlation with average GQS scores, as seen in previous studies ,39. HoweDespite the current paucity of high-quality CABG content on YouTube, the platform has potential as a reputable educational source. Research has shown that increased patient volumes, physician shortages, and fee-for-service payment models have decreased doctor visit durations; leaving many patients inadequately educated on important healthcare issues -42. YouTLimitationsRegarding the limitations of this study, YouTube is a dynamic platform that is growing every day. Thus, video metrics taken at one point in time will not accurately represent those video parameters at a later point. This limitation would be present in any study of a similar design and was minimized by having both independent reviewers record video metrics on the same day. Furthermore, videos below 25,000 views were not evaluated as these were less likely to be watched by patients; however, this may have excluded popular content that was uploaded recently. Another limitation was that videos included in the study were only in English which may reduce its generalizability to other countries. However, English is considered the universal language across the world . AdditioAlthough CABG videos are readily available on YouTube, this study revealed that they contain considerable biases and misleading information. This is troublesome as patients are increasingly utilizing the internet to make informed medical decisions. However, clinicians who understand the educational landscape of CABG content on YouTube can effectively guide their patients toward reliable videos. Important interventions such as increased physician authorship of videos and more guidance for patients in the online space could potentially pave the way for online videos to effectively offer appropriate patient education in the future.


Older patients are at an increased risk of malnutrition due to many factors related to poor clinical outcomes.This study aims to develop an assisted diagnosis model using machine learning (ML) for identifying older patients with malnutrition and providing the focus of individualized treatment.We reanalyzed a multicenter, observational cohort study including 2660 older patients. Baseline malnutrition was defined using the global leadership initiative on malnutrition (GLIM) criteria, and the study population was randomly divided into a derivation group  and a validation group . We applied 5 ML algorithms and further explored the relationship between features and the risk of malnutrition by using the Shapley additive explanations visualization method.The proposed ML models were capable to identify older patients with malnutrition. In the external validation cohort, the top 3 models by the area under the receiver operating characteristic curve were light gradient boosting machine (92.1%), extreme gradient boosting (91.9%), and the random forest model (91.5%). Additionally, the analysis of the importance of features revealed that BMI, weight loss, and calf circumference were the strongest predictors to affect GLIM. A BMI of below 21 kg/m2 was associated with a higher risk of GLIM in older people.We developed ML models for assisting diagnosis of malnutrition based on the GLIM criteria. The cutoff values of laboratory tests generated by Shapley additive explanations could provide references for the identification of malnutrition.Chinese Clinical Trial Registry ChiCTR-EPC-14005253; https://www.chictr.org.cn/showproj.aspx?proj=9542 Epidemiological studies show that the proportion of aging people is expected to exceed 20% of the world's population by 2050, with 80% living in low- and middle-income countries . As the Despite the high incidence and increased risk, a consensus diagnosis of malnutrition is not reached. One of the most important reasons is that the multidimensions need to be incorporated when assessing the nutritional status of inpatients. The global leadership initiative on malnutrition (GLIM) criteria advocate a “two-step” approach to diagnosing malnutrition, as follows: (1) screening the patients for nutrition risk by using validated nutrition screening tools, such as the nutritional risk screening 2002 (NRS-2002) and the Mini Nutritional Assessment–Short Form, and (2) for those who are at risk, diagnosing malnutrition if one of three phenotypic criteria  and one of two etiologic criteria (reduced food intake or assimilation and inflammation or disease burden) are met . ResearcGiven the above background, this study was performed to explore the distribution of the core nutritional features in older patients to establish a decision-assisted system for assessing malnutrition and providing directions for individual intervention using ML approaches. Furthermore, we investigated the optimal cutoff value for important features, which could help aid the evaluation of nutritional diseases in the elderly.This study was led by the Geriatric Nutrition Study Group of the Chinese Society for Parenteral and Enteral Nutrition, Chinese Medical Association. From June to September 2014, a clinical nutrition survey was conducted to assess the changes in nutritional status during hospitalization in 7122 Chinese patients from 34 large hospitals in 18 cities. The patients enrolled in this study were from a variety of clinical departments, including gastroenterology, respiratory medicine, neurology, oncology, general surgery, thoracic surgery, orthopedics, and geriatrics. The inclusion criteria were as follows: aged≥18 years; hospital stay of 7-30 days; no urgent surgery performed before 8 AM the day following admission; patients had to be conscious, willing to accept the multiple nutrition assessment, and have signed written informed consent. The patients belonging to the following categories were excluded from the examination: those who were younger than 18 years; with an expected hospital stay of <7 days or >30 days; unconscious; refused participation in the nutritional assessment or did not sign the written informed consent.The study protocol was approved by the Ethics Committee of Beijing Hospital  and registered in the Chinese Clinical Trial Registry (No. ChiCTR-EPC-14005253). This study included only older adults . The flowchart of patients is shown in For participants at malnutrition risk (NRS2002≥3 in this study), at least one phenotypic criterion and one etiologic criterion should be met to establish the GLIM diagnosis: only if NRS2002 score is ≥3, GLIM score would be calculated. At least 1 phenotypic criterion and 1 etiologic criterion were assigned GLIM (+); the participants could be diagnosed with malnutrition; all phenotypic criteria and etiologic criteria were GLIM (-), and the diagnosis was negative. In addition to the above, the diagnosis can be “Not Known.” Participants who could not develop GLIM diagnosis were deleted. Please find the original scale of NRS2002 and GLIM in In this study, the GLIM diagnosis was retrospectively determined according to the information collected in the study. A low BMI was diagnosed according to the Asian standards described in the GLIM criteria, and reference values for severe malnutrition were defined according to a previous study conducted among an Asian population ,16. CalfP=.65) and Creatinine (P=.66), were used to construct the prediction model [In this study, feature selection was carried out in 2 steps. Firstly, based on recommendations and a review of literature, we chose 25 variables for the ML models, which included demographics , medical history , physical examination , and laboratory test . According to the statistical differences between groups and expert opinions, 23 variables, except blood urea nitrogen  in the sub–data sets and data sets. Preprocessing was based on the features of the training set to avoid data leakage. Ordinal values for categorical variables were assigned to represent duration and converted into categorical values; categorical values were one-hot encoded. All other quantitative variables were standardized. After preprocessing, of 2660 patients diagnosed with malnutrition, 855 (32%) were divided as 684 (80%) into the training set, and 171 (20%) into the test set, respectively.For training models, we applied 5 widely used ML algorithms in assisted diagnosis prediction , including logistic regression , tree-baAfter development, prediction performance was assessed using the area under the receiver operating characteristic curve (AUROC), sensitivity, specificity, and accuracy. To enhance the clinical applicability, we combined the diagnostic prediction results with clinical feature importance explanations to identify a patient with malnutrition. In this study, we used Shapley additive explanation (SHAP) values, to interpret feature contributions and assess the clinical significance of predictive models ,28.P value is the measurement of the marginal contribution of each feature in different combinations (Equation 1).The SHA0ϕ is the average predicted value of all the samples, known as the base value, jϕ is the SHAP value of the feature, and M is the total number of features. When jϕ is greater than zero, this feature improves the predicted value and has a positive effect. According to the SHAP value, the effect direction and intensity of each feature can be obtained. Where P values to explore individual-based decision-making processes in the view of local explanation.For the ML model interpretation, we used both globally explainable and locally explainable methods . The gloData preprocessing, model development, and external validation were performed in Python . Model algorithms and hyperparameter tuning tools were based on Python scikit-learn (v0.24.2).t test. The normality of continuous data was tested using a Shapiro-Wilk test. Categorical data were expressed as n (%) and were compared using a chi-squared test. All tests were 2-tailed, and P<.05 was considered statistically significant. Statistical analyses were performed using SPSS Statistics .Continuous data were expressed as means (SD) and were compared using a The GLIM criteria diagnosed 855 patients with malnutrition. GLIM diagnosed that malnutrition was significantly associated with age, BMI, hand grip strength, albumin, total protein, hemoglobin, lymphocyte count, total bilirubin, alanine transaminase, and platelet count .The performance of predictive models was evaluated in terms of sensitivity, specificity, accuracy, and AUROC. In a 10-fold cross-validation performed on the training set, the XGBoost performed best in AUROC (92.5%), and logistic regression had the worst AUROC and second-worst sensitivity . In the To gain a thorough understanding of the general impact of features, the importance of features included in the XGBoost was quantified. Features and their subtypes were sorted by the magnitude of impact.P value magnitude over all samples. SHAP summary plots succinctly display the magnitude, prevalence, and direction of a feature's effect. As each dot corresponds to a single sample, the swarm plot avoids conflating the magnitude and prevalence of the feature effect into a single number. Therefore, apart from the magnitude of the features' global importance, it is informative concerning the exact impact on specific individuals.Categorical predictor variables were presented separately. The importance of features was sorted by the sum of the mean absolute value of the SHAP value plot by combining feature importance with feature effects. The y-axis is defined by the feature, and the x-axis is defined by the SHAP value. The plot describes the features’ overall influence on the model prediction. Each point in each feature represents an individual case, with colors ranging from blue  to red . The data points further to the right represent the features that contribute to the higher risk of malnutrition using GLIM for a given individual case. The data points to the left represent the features that contribute to the lower risk of GLIM. The vertical line in the middle represents no change in risk. We found that the data points  with lower BMI values had a higher risk of GLIM.2 was associated with a higher risk of GLIM. Patients had a high risk of GLIM when their platelet count value was below approximately 280×109/L. As shown in Furthermore, the lab tests and BMI were used as the continuous variable in our prediction model. This study examined the marginal effect of those features on the predicted outcome of an ML model using a SHAP dependence plot. As shown in At the same time, we use the explainable method of ML to explain the constructed ML model. As shown in P value, we identified the most important features and their recommended cutoff values in evaluating malnutrition in older people. We also interpreted and visualized the model in individuals. Our model provided insights to the diagnosis of malnutrition and possesses the characteristic of high interpretability.In this study, GLIM was retrospectively used to evaluate the nutritional status of older inpatients, and an assisted diagnosis model of malnutrition with a quite high performance was established by the ML algorithm. Using ML, we validated and visualized a tool that incorporated medical history and laboratory tests for identifying malnutrition in older inpatients based on the GLIM criteria. Through the SHATo the best of our knowledge, this is the first study using ML to validate nutritional status in older patients. The etiology of malnutrition in older individuals is multifactorial and consists of physiological, social, and economic parameters, often different from the young. Thus, it is important to develop a practical malnutrition tool and optimal cutoff values targeted to older people. The AUROC of the ML model achieved 0.921, which is comparable to findings by previous studies performed in different populations ,18,30. T2, they are exposed to a higher risk for malnutrition. This coincides with the result from the current national BMI data set [According to the analysis of the dependence plot, we could observe the cutoff values of the features with a higher risk of malnutrition. The cutoff point of BMI was approximately 21, which suggests that when BMI of older Chinese people is below 21 kg/mdata set . The resThis study has some limitations. First, the GLIM diagnosis was retrospectively determined. Although the original study collected a wide range of nutritional factors, more information was needed for accurate diagnosis and grading. In this study, GLIM-recommended muscle measurements such as dual-energy absorptiometry or bioelectrical impedance are not available; calf circumference was used as an alternative measure. In addition, we established the GLIM diagnosis based on Asia BMI thresholds, so the applicability of the model in a non-Asian population should be reevaluated. Furthermore, the training and test sets were derived from the same study, which means the model needs to be validated by prospective research. Lastly, the data were slightly imbalanced, which means the precision of the model training was relatively low because of the lower number of malnutrition cases.In conclusion, using ML, we could get an accurate and dynamic diagnosis of malnutrition based on the GLIM criteria. This model could be used as a decision tool to assist clinicians. The cutoff values of laboratory tests also provide references for the identification of malnutrition in Chinese older inpatients. Further studies are needed to simplify and validate the model in clinical conditions.


Video games are a popular sedentary activity among people with impaired mobility; however, active video game hardware typically lacks accessibility and customization options for individuals with mobility impairments. A touchpad video game system can elicit moderate physical activity in healthy adults; however, it is unclear if this system is usable by adults with impaired mobility.The purpose of this study was to assess the usability of a touchpad video game controller system adapted for adults with impaired mobility. Additional outcomes explored were enjoyment, perceived exertion, self-efficacy, participant feedback, and researcher observations of gameplay.Participants played several video game titles for 20 minutes with a touchpad video game controller as they stood or sat in a chair or their wheelchair. Usability was assessed with the System Usability Scale (SUS) and the Health Information Technology Usability Evaluation Scale  surveys after gameplay. After each video game, participants reported enjoyment using a visual analog scale (0 to 100 mm) and a rating of perceived exertion using the OMNI 0 to 10 scale. Self-efficacy was measured before and after gameplay. Participants provided feedback at the end of their session.In total, 21 adults  with a mean age of 48.8 (SD 13.8) years with various mobility impairments participated in this study. The touchpads received mean usability scores on the SUS 80.1 (SD 18.5) and Health-ITUES 4.23 (SD 0.67).The SUS scores reported suggest the touchpad system is “usable”; however, the Health-ITUES scores were slightly below a suggested benchmark. Participants reported moderate to high enjoyment but perceived the exertion as “somewhat easy.” Self-efficacy was moderate to high and did not differ pre- to postgame play. The participants regarded the touchpads as novel, fun, and entertaining. The generalizability of our results is limited due to the heterogenous sample; however, our participants identified several areas of improvement for future iteration. Habitual physical activity improves health and quality of life; however, half of the people with a disability in the United States are categorized as physically inactive -4. WhileWhile half of adults in the United States engage in sedentary video game play , active Given that AVGs can foster feelings of autonomy, competency, and relatedness, these games may fulfill basic psychological needs and augment the enjoyment derived from participation in physical activity ,24. FurtThe research and development team with the Rehabilitation Engineering Research Center on Interactive Exercise Technologies and Exercise Physiology for People with Disabilities previously showed that AVG play can be adapted for wheelchair users , be enjoThe TPS is an internally developed AVG controller designed to add physical activity to sedentary video games. This prototype translates physical contact into video game commands. Because the TPS is recognized as a USB controller, this system can provide commands to any video game title that features controller support. The current proof-of-concept has been adapted for use by people with impaired mobility.We constructed 6 touchpads using a particle board base, conductive aluminum tape, electrical wire, and duct tape. The touchpads were wired to a MAKEY-MAKEY circuit board  that was connected to the video game computer as a controller. All but 1 touchpad measured 20 cm × 20 cm, and the final touchpad was larger (61 cm × 31 cm). Each touchpad was placed on an adjustable stand that enabled varying height, distance, and orientation . The smaller touchpads were placed in front or to the side of the player. The larger touchpad was placed behind the player.To ensure safety, adjustable parallel bars were located on both sides of the player during gameplay. The participants played video games approximately 1.8 m away from a flat-screen television. Touchpads were placed so they did not block the player’s view of the screen. All wires were secured away from the player to prevent any trip hazard during gameplay.This was a cross-sectional usability testing study. We assessed usability and embedded participant feedback to help explain our results. All written consent and data collection took place at the University of Alabama at Birmingham within the RERC RecTech Exercise Science and Technology Laboratory. Participants attended a single 60- to 90-minute session to test the usability of the system.The procedures of this study were approved by the University of Alabama at Birmingham’s institutional review board (IRB 300003265).We recruited participants from the local community of Birmingham, AL, using flyers and word of mouth. Sample size estimates were based on identifying common usability barriers for the system, and issues specific to the 3 modes of play . According to Cazañas et al , a samplInterested individuals were included if they were an adult 18 to 75 years of age, had a self-reported mobility impairment, and possessed the ability to exercise with their upper extremities. Individuals were excluded if they were unable to converse in English, weighed greater than 181.4 kg (400 lbs), had significant visual impairment that prevented them from seeing a large flat-screen television, had cardiovascular disease within the previous 6 months, had severe pulmonary disease or renal failure, currently pregnant, ongoing exacerbation of a health condition, or any other condition that would interfere with testing procedures. Participants received a US $50 gift card at the end of their visit.Participant usability of the TPS was assessed using the System Usability Scale (SUS) and the Health-Information Technology Usability Evaluation Scale . The SUS is comprised of 10 statements, and participants rate their agreement of each statement with a 5-point Likert scale from “strongly disagree” to “strongly agree.” Across 206 usability tests and 2324 responses, the SUS was found to be a robust and reliable (α=.91) tool to measure usability ,37. The Similar to the SUS, the Health-ITUES is made up of 20 statements that participants rate on a 5-point Likert scale from “strongly disagree” to “strongly agree” . The subParticipants reported their enjoyment with a visual analog scale (VAS), which is a 10-cm scale with anchor phrases at each end . The ancParticipants rated their perceived exertion using the OMNI 10-point ratings of perceived exertion (RPE) scale . The sca3,2 (intraclass correlation coefficient) 0.83 (95% CI 0.62-0.91)  How How13] HSimilar to previous studies using AVGs among individuals with impaired mobility, participants moderately enjoyed using the TPS  20] Sim Sim20] SThese data suggest that our participants perceived their exertion as somewhat easy to somewhat hard during video game play using the TPS , which iThe participants regarded the touchpads as novel, fun, and entertaining but they did encounter some accessibility barriers. Modifications such as repositioning the touchpads, adding conductive tape to prosthetics, and providing moisturizer for dry skin were not anticipated because they were not encountered in previous testing of the TPS  or with For future iterations, the stands used to mount the touchpads need to resist movement when a player exerts considerable force to activate. Due to the large body movements and quick reactions required to play video games using the TPS, the touchpads need to stay fixed in position during gameplay. Even though each touchpad was made large to mitigate the need for movement precision, touchpad movement during gameplay may cause the player to lose screen focus and thus introduce frustration. The TPS needs to remain robust to a varying degree of force from multiple directions to better accommodate individuals with impaired mobility. The TPS is currently being refined based on usability, participant feedback, and researcher observations. This system can be refined to work with adaptive video game equipment such as the Microsoft Adaptive Controller. Because the TPS is not limited to a single user, future research should examine the use of this system for multiple players simultaneously.First, the participants in this sample represented various mobility impairments; therefore, the number of players within each mode of play is few, which makes mode comparisons difficult. Second, our participants reported a wide range of weekly physical activity minutes , limitinThe TPS enabled people with impaired mobility to participate in AVG play. The TPS allows typical sedentary video games to be played by adults with impaired mobility while sitting, standing, and with their own mobility aids. Participants found the TPS to be usable, experienced moderate enjoyment, and achieved moderate intensity physical activity through gameplay. Key areas of improvement to the system were identified based on our measures, participant feedback, and observation.