Paenibacillus polymyxa has been accepted as the candidate of the cell factory for many secondary metabolites. However, as the regulatory expression elements in P. polymyxa have not been systematically investigated, genetic modification on account of a specific metabolism pathway for the strain is limited. In this study, a xylose-inducible operon in the xylan-utilizing bacterium ATCC842 was identified, and the relative operon transcription was increased to 186-fold in the presence of xylose, while the relative enhanced green fluorescent protein (eGFP) fluorescence intensity was promoted by over four-fold. By contrast, glucose downregulated the operon to 0.5-fold that of the control. The binding site of the operon was “ACTTAGTTTAAGCAATAGACAAAGT”, and this can be degenerated to “ACTTWGTTTAWSSNATAVACAAAGT” in Paenibacillus spp., which differs from that in the Bacillus spp. xylose operon. The xylose operon binding site was transplanted to the constitutive promoter Pshuttle-09. The eGFP fluorescence intensity assay indicated that both the modified and original Pshuttle-09 had similar expression levels after induction, and the expression level of the modified promoter was decreased to 19.8% without induction. This research indicates that the operon has great potential as an ideal synthetic biology tool in Paenibacillus spp. that can dynamically regulate its gene circuit strength through xylose.With numerous industrial applications,  Paenibacillus polymyxa is a biological control and plant-growth-promoting bacterium that produces numerous primary and secondary metabolites [P. polymyxa have been characterized and broadly applied in the agriculture, food, and pharmaceutical industries. Various types of exopolysaccharides produced by P. polymyxa using different carbons [abolites ,2,3,4. Tabolites  could heabolites , and alkabolites  produced carbons  could beP. polymyxa by optimizing the culture medium. Zhang et al. [P. polymyxa [P. polymyxa. To improve the expression efficiency, using a free-expression system with relatively higher copy numbers might be a promising strategy.To obtain these valuable products more efficiently, technologies involving fermentation engineering and genetic and metabolic engineering have been introduced in recent decades. For instance, Polymyxin E is an antibiotic with therapeutic efficacy against Gram-negative bacterial pathogens, and Yu et al. ,10 improg et al.  improvedpolymyxa . HoweverP. polymyxa successfully [P. polymyxa if an appliable inducible expression system were constructed for this strain.Certain promoters derived from other bacterial species have been integrated into essfully ,13. Neveessfully , while nBacillus subtilis, Bacillus licheniformis, Bacillus megaterium, and Bacillus coagulans [Bacillus spp. requires xylose isomerase and xylulose kinase, which are encoded by xylA and xylB, respectively. Both genes are regulated at the transcription level by a xylose-responsive repressor ROK family protein encoded by xylR. When xylose is deficient, XylR binds the operator, prevents RNA polymerase transcription, and regulates PxylR and PxylAB. The XylR binding site contains GTTT N7 AAAC [Via the unique regulatory mode, inducible expression systems can be activated with high expression levels by chemical or environmental induction or by autoinduction ,16,17. Coagulans ,30,31,32 N7 AAAC ,34, whic N7 AAAC ,36. The  N7 AAAC .P. polymyxa, although this bacterium can grow with xylan and produces 2,3-butanediol acid using xylose as the carbon source, the pathway and operator associated with xylose metabolism in this strain have not been identified. To construct valid inducible expression systems in P. polymyxa, in the present study, we elucidated the sequences of xylA, xylB, and xylR, and verified the relationships between their transcriptional intensity and the presence of xylose or glucose as a carbon source. A novel inducible promoter was generated and verified herein through the verification of promoter expression intensity by green fluorescent protein (eGFP), analyzation of the xylose operon structure for the confirmation of the XylR binding site, and transplantation of the bases in the repressor protein binding site to the constitutive promoter after identification and localization. With the introduction of the xylose-inducible promoter in P. polymyxa, the current study provides a promising expression element with the intention to extend the application potential of this certified biosafety microorganism.For P. polymyxa ATCC842 has been published in the National Center for Biotechnology Information (NCBI) database (GenBank: CP049783.1). The analysis revealed that the presence of the predicted xylose operon encoded three putative open reading frames. The encoded proteins were annotated as ROK family transcriptional regulator , xylose isomerase , and xylulokinase . The gene direction of the hypothetical transcriptional regulator differed from those of the other two genes, and the latter were xylose metabolism proteins. Hence, there was a bidirectional promoter between these two reverse genes. This bidirectional starting structure resembled those of the xylose operons from B. subtilis, B. licheniformis, and B. coagulans. ROK family proteins are associated with sugar metabolism and include a sugar kinase and a sugar-responsive repressor. To identify the function of this ROK family protein, we aligned its protein sequence with that of XylR from B. subtilis  and B. licheniformis , which regulate xylose utilization, and with that of Lmo0178  from Listeria monocytogenes, which regulates α-glucan decomposition  after 4 h of culture with xylose. The transcription of all three genes continued to be upregulated  after 8 h of culture with xylose  after 4 h of culture, and more so after 8 h of culture . After 12 h, the transcription intensity levels of these genes were only 0.06-fold, 0.04-fold, and 0.09-fold, respectively.The whole-genome sequence of position . The ali results a show thh xylose b. After h xylose c, the trP. polymyxa cultured at 30 °C and 37 °C, respectively  experiment, the ROK family protein was identified as a candidate for regulating the bidirectional promoter, which was confirmed by a shifted band in the lane with the biotinylated promoter as a probe. As shown in P. polymyxa. Moreover, xylose was found to promote the operon whereas glucose inhibited it.As shown in this section, the ROK family transcriptional regulator may in fact bind the xylose operon. The predicted ROK family protein and the bidirectional promoter comprised the xylose operon in P. polymyxa with the plasmid pHY300PLK harboring the xylose operon from B. subtilis, B. licheniformis, and P. polymyxa was induced with xylose, and its fluorescence intensity was measured. The corresponding plasmid construction scheme is shown in xyl, Blxyl, and BSxyl after an induction of 4 h were 1132, 1019, and 128, respectively, and these levels were 1748, 1639, and 577, respectively, after 8 h. For the biomass, the OD656 was measured as 0.54, 0.42, and 0.58, respectively, after 4 h of induction, while it was 0.55, 0.53, and 0.69, respectively, after 8 h of induction. The growth rates of P. polymyxa containing plasmids harboring PPxyl and BLxyl were lower than those of the cells carrying plasmids harboring BSxyl. The fluorescence intensity levels of the cells harboring PPxyl or BLxyl were significantly higher than those of the cells harboring BSxyl  and damaged site (DTE) were introduced in an EMSA experiment  were found to be limited by XylR based on the observation of a decrease in fluorescence intensities . In comparison, the fluorescence intensity of cells containing Psr23 was greater than the control group, and a high expression efficiency was identified after an induction for 8h, whereas the expression efficiency of cells with Psa24 was still low even with the inducer.In this study, the possible available types of inducible promoters in as Psr23 a. In anoas Psa24 b. As shoP. polymyxa by identifying promising expression elements, a xylose-inducible expression system was constructed and verified in this study. On the basis of the previously reported xylose operon in other Bacillus spp., the xylose operon candidate in P. polymyxa was compared against those of other bacterial taxa. The results reveal that those strains shared a similar basic bidirectional start promoter and regulatory mechanism. The construction of a xylose operon is shown in P. polymyxa xylose operon indicated a difference in the length (135 bp) of the bidirectional promoters, which were 241 bp and 224 bp long in B. subtilis 168 and B. licheniformis ATCC14580 xylose operons, respectively [B. subtilis, B. licheniformis, and B. coagulans. In P. polymyxa, however, the XylR binding site spanned both −35 regions from PxylAB to PxylR. In comparison, the regulator binds the −35 box more efficiently than the UTR region [Bacillus spp., the XylR protein controls PxylAB transcription via a roadblock mechanism, whilst it sterically hinders RNA polymerase access to the PxylAB promoter in P. polymyxa [xylR by preventing RNA polymerase from binding to the promoter. In this manner, the entire operon system is muted to avoid extra energy costs. Another issue that requires attention is the positions of the cre site in the promoter. Bacillus spp. has a typical cre sequence of TGWNANCGNTNWCA [P. polymyxa xylose operon. In the B. subtilis XylA operon, a cre was located at +36 bp [B. licheniformis, a cre was localized at the core of the xylose operon. In plasmid-based systems, CCR also reduces expression. A mutation in the cre site may inhibit transcription regulation by CcpA [P. polymyxa, the putative cre site TGTAAACGTTTACT at the position between the −10 region and the RBS was detected herein, which differed from those of the other bacterial xylose operon systems. The difference gives the promoter the potential to be regulated by glucose and the possibility of relieving inhibition by modification.To improve the possible application potential of ectively . This obR region . In addipolymyxa . The XylCGNTNWCA , while dt +36 bp , and the by CcpA . For P. P. polymyxa was then developed in this study after the above structural analysis. The corresponding functionality of the Pxyl promoter was further verified through the eGFP assay. In terms of the effect of temperature, the P. polymyxa growth rate was higher at 30 °C than at 37 °C, whereas the cellular fluorescence intensity was lower at 30 °C than at 37 °C. The optimum growth temperature for P. polymyxa was 30 °C, while the optimum PPxyl expression temperature was 37 °C. For optimum P. polymyxa fermentation, incubation should be performed at 30 °C. Subsequently, the induction was conducted at 37 °C after the appropriate biomass was attained. All xylose operons from other bacterial species used in this study were verified to work in P. polymyxa, although they showed different expression efficiencies. All xylose operons from other bacterial species used in the present study were verified to work in P. polymyxa, although they showed different expression efficiencies. Brito [B. methanolicus functioned in both P. larvae and P. polymyxa. However, as expression elements may not be adapted to a foreign host, operons from other bacterial species may have different promoter activities. In this study, the expression efficiency of the xylose operon from B. subtilis was extremely low in P. polymyxa, despite the fact that it is widely used as an expression system owing to its high expression level in B. subtilis. Thus, xylose operon from B. subtilis seems not to be a suitable expression system in P. polymyxa.A xylose-inducible promoter that functions in s. Brito  reportedP. polymyxa, the XylR protein from P. polymyxa was expressed and aligned with other ROK family proteins for the prediction of the XylR critical domain sequence. After alignment, the corresponding sequence was suggested to be ACTTAGTTT-AAGCAAT-AGACAAAGT, and the degenerate sequence ACTTWGTTT-AWSSNAT-AVACAAAGT was applied for all Paenibacillus spp. Due to the important roles played by the bases ACTTNG-CNAAGT at the leading and trailing ends in the XylR protein binding process, any alteration in these sequences was proposed to impede the binding. Overall, changes to the fifth A/A bases of the binding site were found to have less impact on the binding than changes to the other bases. Without entirely matching, the consensus sequence TTNGTTT-AAACNAA was determined for B. subtilis and applied to B. licheniformis and B. megaterium [N-terminal effector subdomain position and super wing in the transcriptional regulator [Paenibacillus spp. were also observed, and C and G showed high affinity for this binding site [mtlA, PmanA, PgutA, and ParaB, which have similar binding site sequences but are controlled by their corresponding transcriptional regulators [To further confirm the XylR protein binding site of gaterium . The secegulator . Similaring site . The preing site  may helpgulators .luxS sequence to stop RNA polymerase transcription via the roadblock effect of the XylR protein, the modified Pshuttle-09 promoters Psr23 and Psa24 were inducible and had low leakage and high efficiency in response to inducers. However, the expression efficiency of Psa24 was significantly weaker than that of Psr23. A similar phenomenon was also reported in a previous study [Considering the high expression efficiency of Pshuttle-09 in both B. subtilis and E. coli , the Xylus study , in whicP. polymyxa is highly efficient and could serve to construct a xylose-inducible system. This study is the first to present a sugar-inducible promoter from P. polymyxa that compensates for the regulatory expression element deficiency in this bacterium. With the construction of a free-expression system using the foregoing xylose operon, the endogenous xylose-inducible promoter was empirically demonstrated to be more efficient than the exogenous promoter from B. subtilis. A modified xylose-inducible Pshuttle-09 promoter was finally generated by transplanting the XylR binding site into a constitutive promoter after the identification and localization of the key bases of the regulatory site within the promoter via the comprehensive elucidation of the corresponding structure through EMSA.In summary, the xylose operon of E. coli was used in plasmid construction, while E. coli BL21(DE3) served to express the proteins. The E. coli transformants harboring plasmids modified by pHY300PLK were selected on Luria–Bertani (LB) agar plates supplemented with ampicillin (100 μg/mL). The E. coli harboring XylR expression vector was selected on LB agar plates fortified with kanamycin (30 μg/mL). The P. polymyxa transformants were selected on tryptic soy agar plates supplemented with tetracycline (20 μg/mL). The media used for the culture and activation of E. coli and P. polymyxa were LB medium  and tryptic soy broth (TSB) medium , respectively shaken at 200 rpm, and the E. coli was cultured at 37 °C while the incubation temperature of P. polymyxa was 30 °C.The strains and plasmids used in the present study are listed in eGFP as a reporter gene was constructed to verify the validity and efficiency of the selected promoter. After the characterization of the promoter function for the constructed plasmid, the predict promoter PPxyl from P. polymyxa was inserted into the plasmid to test the corresponding efficiency within the bacterium. The function of PPxyl was determined, and xylose operons from B. subtilis and B. licheniformis were amplified and inserted into the plasmid to compare relative expression efficiency. The primers used are listed in eGFP gene was amplified with the eGFP-f/r primer pair and inserted into pHY300PLK plasmid at HindIII/SmaI, thereby generating the pHY-e plasmid. The upstream transcriptional regulator in the hypothetical xylose operon from P. polymyxa containing the promoter PPxyl  was obtained from P. polymyxa ATCC842 using the PPPxyl-f/r primer pair. The operons BSxyl  and BLxyl  were amplified from the genomes of B. subtilis 168 and B. licheniformis ATCC14580 using the BSPxyl-f/r and BLPxyl-f/r primer pairs, respectively. The operons were cloned into pHY-e by homologous recombination and the pHY-PPxyl, pHY-BSxyl, and pHY-BLxyl plasmids were generated from these clones. To construct plasmid pHY300-PS09, the HapaII promoter was obtained from pMA5 using HapaII-f/r, Pshuttle-09 was obtained using P09-f/r, and xylR  was obtained using 09xylR-f/r. All fragments are linked to pHY-e at HindIII by homologous recombination. The Psr23 and Psa24 fragments were obtained using Psr23-f/r and Psa24-f/r from pHY300-PS09, and they were further introduced into pHY300-Psr23 and pHY300-Psa24, respectively. All plasmids were transferred into P. polymyxa to verify the corresponding expression efficiency.A plasmid with a pHY300PLK backbone and harboring P. polymyxa was activated in TSB medium at 30 °C with shaking at 200 rpm for 10 h. Then, 0.5 mL of medium containing activated P. polymyxa cells was transferred to 50 mL of solution A (TSB with 0.5 M sorbitol) and cultured  for 8 h until OD656 = 0.8–0.9. The bacterial suspension was centrifuged at 6000× g at 4 °C for 5 min, kept in an ice bath for 30 min, and resuspended four times in solution B (10% (v/v) glycerol with 0.5 M sorbitol and 0.5 M mannitol. The cells were then resuspended in 1200 μL of solution B, divided into 20 equal parts, and stored at −80 °C until the subsequent experiments.P. polymyxa. Then, 4–8 μg of plasmid was mixed with 60 μL of P. polymyxa competent cells. The suspension was kept in an ice bath for 5 min, and the competent cells were transferred to a precooled 0.1 cm Gene Pulser cuvette and kept on ice for another 5 min. The electroporation conditions were 2000 V and 5 ms. Then, 1 mL of recovery medium (LB medium with 0.5 M sorbitol and 0.38 M mannitol) was added to the Gene Pulser cuvette immediately after the shock was terminated. After 8 h of recovery at 30 °C under 200 rpm shaking, the cells were applied to plates containing the corresponding antibiotics.Plasmids with xylose operons were introduced by electrotransformation into P. polymyxa strains harboring the plasmids were cultured in TSB medium at 30 °C and incubated at 200 rpm for 12 h. Then, a 5% (v/v) of the culture solution was inoculated into fresh TSB medium. After 12 h of fermentation, xylose inducer was added to a concentration of 20 g/L, and the fluorescence intensity of the culture was measured every 2 h thereafter. The samples were centrifuged to obtain the cells, and the latter were washed twice with phosphate-buffered saline (pH 7.4). The samples were diluted to OD656 = 0.5. Then, 200 μL aliquots were placed in each well of a 96-well microtiter plate. The sample plate was checked with a TECAN-SparK Plate Reader . The scanning program and fluorescence intensity calculation were derived from a previously published report [The recombinant d report .E. coli. BL21(DE3). The xylR (GenBank No. CP049783.1) was copied from the P. polymyxa genome using the xylR-f/r primer pair and cloned at the NheI-HindIII sites of pET28a. A 134-bp promoter was amplified by the PE-f/r primer pair and ligated into pMD19-T to construct a plasmid template and obtain DNA probes for the polymerase chain reaction (PCR)-based EMSA. The 5′-biotin-labeled primer and others were used to amplify the biotinylated fragments. All marked fragments were purified by 2% agarose gel electrophoresis. The remaining steps were implemented following the instructions of the Chemiluminescent EMSA Kit .The ROK family proteins predicted to be XylR transcriptional regulators were expressed by P. polymyxa was activated overnight in TSB medium to determine the effects of xylose on the predicted operon. The latter had the overlapping promoters PxylR and PxylAB to regulate the transcription and the expression of XylA and XylB. The cells were incubated for another 12 h after 5% cultures were transferred to TSB medium, following which 2% xylose, glucose, or water was added. Each culture was sampled every 4 h. The mRNA was extracted with a Simply P Total RNA Extraction Kit , and the cDNA was generated and amplified with a Prime Script RT Kit . The genes xylR, xylA, and xylB were amplified with xR-F/xR-R, xA-F/xA-R, and xB-F/xB-r, respectively. The 16S housekeeping/internal reference gene was amplified with 16sF/R. The reaction conditions of the RT-qPCR were determined using a Universal YSBR qPCR Master Mix . The RT-qPCR was performed in a qTOWER2.0 thermal cycler .


Purpose: To evaluate the educational content, quality, and reliability of YouTube videos regarding the Achilles tendon and Achilles tendon injuries.Methods: The first 50 videos found on YouTube after searching “Achilles tendon” were evaluated and classified by content type and uploader source. Reliability and accuracy were assessed using the Journal of the American Medical Association (JAMA) benchmark criteria, nonspecific educational content was assessed via the Global Quality Score (GQS), and specific educational content was assessed using the Achilles Tendon Specific Score (ATSS). ANOVA was performed to determine differences in video reliability and educational content quality by video content type and source. Multivariate stepwise regressions were used to evaluate the effects of specific video characteristics on JAMA benchmark criteria, GQS, and ATSS.Results: The 50 videos evaluated had a cumulative view total of 53,323,307, a mean of 1,066,466, and a range of 1,009 to 42,663,665 views per video. Most videos focused on disease-specific information (38%) and exercise training (22%). Most videos were uploaded by non-physicians (34%) or medical sources  (32%). A higher view ratio was an independent predictor of lower JAMA scores (lower reliability and accuracy)  and increased video duration was an independent predictor of greater GQS  and ATSS scores  .Conclusion: Videos on YouTube regarding the Achilles tendon were viewed numerous times, but their educational content and reliability were poor. Providers treating patients for Achilles tendon-related pathologies should initiate a dialogue with patients about their use of internet sources and should educate them on their optimal usage. They should warn them of the low quality of YouTube-derived information and provide them with reliable sources that may better give them control over their own care. The internet is increasingly prevalent in the lives of Americans with an estimated 85% using it daily, 48% using it multiple times a day, and 31% using it almost constantly as of January 2021 [YouTube is one of the most popular sites with 71% of Americans in a study reporting use, and it is being increasingly used by people seeking opinions and facts . With thStudies have shown that the use of internet sources to obtain healthcare-related information increases with a decreased perceived quality of care and quality of patient-physician relationship . StudiesDespite the prevalent use of online resources, YouTube has no peer-reviewed process or quality control methods in place for most health-related videos, which may present patients with low quality, misleading, and/or incomplete information ,8,9. PreThis study aims to evaluate the educational content of YouTube videos regarding the Achilles tendon. The Achilles tendon is the largest tendon in the human body, functions in plantar flexion of the foot, and is most commonly injured during athletic activities such as basketball . A recenThe authors of this study searched the term “Achilles Tendon” on YouTube on May 24, 2021. A Google Chrome  incognito tab was used to eliminate any confounding factors influencing the results. The videos were queued by the default filtering method of “relevance” and the first 50 videos were recorded for evaluation (Appendix). This is a reasonable approach as previous studies on orthopedic-related topics have been similarly conducted and accepted in peer-reviewed journals . ExclusiVideo characteristicsVideo characteristics were recorded for each of the following variables: title, video duration (in minutes), number of views, video source, content type, days since upload, view ratio (views per day), number of likes, number of dislikes, like ratio ((likes * 100) ÷ (likes + dislikes)), and video power index (VPI). The VPI uses the following formula: like ratio * view ratio ÷ 100; it has been utilized in prior studies to measure video popularity by views and likes .Video sourcesVideo sources categorized were academic (research group or colleges/universities), physician (independent or physician groups without research or college/university ties), non-physician , athletic trainers, medical sources , or commercial.Video contentContent categories included the following: exercise training (Achilles tendon therapy and rehabilitation), disease-specific information, patient experiences/anecdotes, surgical approaches and/or techniques, non-surgical intervention or therapy, or advertisements.Video reliability and educational content assessmentThe Journal of the American Medical Association (JAMA) benchmark criteria were utilized to assess the reliability and accuracy of each video. The JAMA benchmark criteria are four nonspecific and objective criteria that may be evaluated in online resources. For each of the four criteria Table  present Assessment of nonspecific educational content quality was done via the Global Quality Score (GQS). GQS is a non-validated but commonly used metric used to assess the quality of online resources . GQS utiAssessment of Achilles tendon-specific educational content was done via the Achilles Tendon Specific Score (ATSS). ATSS is composed of 19 criteria derived from the American Academy of Orthopaedic Surgeons (AAOS) guidelines and adapted from similar metrics in prior studies ,17,18. FStatistical analysisAll statistical analyses were performed in SPSS version 27.1 . Descriptive statistics such as means, standard deviations, minimums, maximums, frequencies, and percentages were used to quantify characteristics, reliability, and quality scores  for each video. Each video was independently rated by four authors for JAMA benchmark criteria, GQS scores, and ATSS scores. None of the authors were aware of the scores given by other authors as all authors recorded their evaluations on separate spreadsheets. After all four authors had evaluated all 50 videos for JAMA benchmark criteria, GQS scores, and ATSS scores, data were collated in SPSS and analyzed for mean scores. Intra-observer reliability scores for JAMA benchmark criteria, GQS scores, and ATSS scores were then calculated using reliability analysis.Distributive tests for the normalcy of data were performed with Kolmogorov-Smirnov and Shapiro-Wilk tests. One-way analysis of variance (ANOVA)  and Kruskal-Wallis  tests were used to determine whether video reliability and quality varied by video sources and content.Multivariate linear stepwise regression was performed to evaluate the effects of specific video characteristics, content type, or video source on JAMA benchmark criteria score (reliability), GQS , and ATTS . P-values < 0.05 were considered statistically significant.The first 50 YouTube videos obtained from the search were evaluated as none of them met exclusion criteria. These videos had a cumulative view total of 53,323,307, and the other video characteristics are listed in Table The most common information presented in videos was disease-specific information (38%) followed by exercise training 22%), surgical techniques 16%), and nonsurgical management (14%)  for JAMA benchmark criteria, 0.851 (0.764-0.910) for GQS, and 0.925 (0.860-0.959) for ATSS.Analysis of variance Table  showed nMultivariate stepwise linear regression analysis was run to assess if there were independent associations between video characteristics, content, or source with JAMA benchmark criteria, GQS, or ATSS scores. Analysis showed that videos with increased view ratios (more views per day) had lower overall JAMA benchmark criteria scores . Videos with longer duration had increased GQS scores  and ATSS scores . No independent associations were found between uploader source or content and JAMA benchmark criteria, GQS, or ATSS scores.The first 50 videos queued in this study had a very large number of views with a cumulative total of 53,323,307, a mean of 1,066,466, and a range of 1,009 to 42,663,665 views per video. These results are like previous studies on the quality of YouTube-derived content related to orthopedic topics such as kyphosis, posterior cruciate ligament (PCL), or disc herniation . In a siOverall, the authors of this study found the reliability and accuracy, non-specific educational content, and specific educational content of individual videos to be of low quality. The mean JAMA benchmark criteria score of 2.69 out of 4.0 suggests that the reliability and accuracy of individual YouTube video content are moderate to low on average. The mean GQS score of 2.64 out of 5.0 suggests that the nonspecific educational quality of individual YouTube video content is low and that the ability of any single video to sufficiently inform consumers is suboptimal to poor. The mean ATSS score of 4.66 out of 19 suggests that the specific educational content in individual videos is not comprehensive and that they give patients only a fraction of all the information they would need to have a good understanding of the Achilles tendon and its pathologies. These findings suggesting low quality were like studies such as Kunze et al. (2019), which reported a mean JAMA benchmark criteria score of 2.02 out of 4, GQS score of 2.3 out of 5, and posterior cruciate ligament score (PCLS) of 2.9 out of 18 [mayoclinic.org or orthoinfo.aaos.org), and then consult with their physician, they could form a loose basis for understanding their condition and be better able to convey their goals and expectations. Though the content of YouTube videos is lacking in reliability and overall content, it is not generally created to be used as a comprehensive guide. Instead, if implemented correctly, usage of these videos may act as a primer to help further educate patients and provide them with some sense of control in their treatment, plan, and care.Though individual YouTube videos do not provide viewers with comprehensive educational content on the Achilles tendon, a person searching for this information online is unlikely to only obtain information from one video or one source. Thus, if a patient were to view multiple videos, reference other sources (such as Notably, no statistically significant associations were found between video uploader source or video content and the JAMA benchmark criteria, GQS, or ATSS scores of Achilles tendon-related videos. This suggests that video quality and reliability are not related to the source or content type. However, this study found that a higher view ratio related to a lower JAMA benchmark criteria score indicates that videos with greater popularity are less explicit with authorship, attribution, currency, and/or disclosure of conflicts of interest. GQS and ATSS were only increased with increased video duration, which is reasonable as a longer video would allow the uploader more time to provide a better and more comprehensive understanding of that specific topic.If the educational content of these videos is to be accurate and thus maximize its benefit to the patient, it should follow the peer-reviewed reliable principles and guidelines established by the AAOS, which were adapted into the ATSS of this study. As stated above, the content of individual YouTube videos is of low quality and non-comprehensive but could act as a primer to help patients better understand their condition. When used in conjunction with high-quality resources such as the AAOS website, these videos could help ease patients into a position where they are more comfortable with their condition and more able to accurately and effectively communicate with their attending provider.LimitationsThis study was conducted on and analysis was limited to the first 50 YouTube videos queried using the term “Achilles tendon.” This limits the generalizability of this study but may mirror the actual search patterns of many users as they rarely search beyond the first or first few pages of results when searching a topic . This stThe internet is becoming an increasingly powerful tool to obtain many forms of information, and as people become more used to using this tool, it is important that healthcare providers be aware of its use and potential for misuse. There is largely no peer-reviewed or regulatory system in place for YouTube videos or many online sources. Thus, providers should engage with their patients by asking them if they have used online resources to understand their condition and should help patients better utilize these resources. YouTube is a massive and growing free platform with millions of users and videos that are easily accessible and often used even though the information provided there is of low quality and lacking in content. Other resources such as the AAOS or Mayo Clinic websites are of higher quality but are far less popular, less utilized, and/or mostly provide information as text and images, which is often less engaging and more intimidating than the dynamic nature of videos where the source is walking you through a topic. The internet and the use thereof will only continue to grow in the coming years. If providers are able to properly address these resources with patients, participate in resource creation, and inform patients about their use, they may be able to help patients find a sense of control in their care. Subsequently, patients may find grounds to better discuss their knowledge with their provider instead of simply being educated by them. Through this cooperation, patients can seek out proper care at the appropriate time, thus reducing the risk of exacerbating injuries and future tendon ruptures.


While alcohol-induced psychotic disorder (AIPD) is a well-recognised clinical disorder, relativery little is known about aspects such as epidemiology, course and treatment of the condition. Current evidence suggests AIPD can be clinically distinguised from alcohol-withdrawal delirium and schizophrenia. AIPD is associated with high comorbidity with other psychiatric disorders, high re-hospitalization and mortality rate, namely suicidal behaviour.The objetive of the study was to examine the correlates, clinical features, psycopathology, and short-term response in an inpatient sample with alcohol-induced psychotic disorder, predominant hallucinations (ICD-10 F10.52) admitted to Centro Hospitalar Psiquiátrico de Lisboa.We collected retrospectively data from all admitted patients to our Alcohol Unit between January 2010 and January 2020 with the diagnosis of AIPD. The exclusion criteria were: presence of preexisting psychotic disorder, delirium, or other substance use disorders. We characterized our sample in Demographic categories, Clinical categories, Treatment and Short-term course.A total of 113 subjects were included in the study. The prevalance of alcoholic hallucinosis was found to be 1.3% of all patients who received inpatient treatment. Most individuals reported auditory hallucinations, that iniciated when they decrease their alcohol intake, and 1 in 4 had past episodes of AIPD.There are specific challenges in studiyng AIPD, such as the relatively rarity of the disorder, its often transient nature and high levels of comorbidity. A high degree of recurrence and morbidity indicates a need to prevent, and intervene early with an abstinent-oriented management goal.No significant relationships.


The aim of this study was to investigate the personality traits, and P300 component in the offspring of parents with alcohol dependence (OPAD) currently engaged in risky drinking and those not engaged in risky drinking, and to further explore the correlates of problematic alcohol use.n = 35) and those currently engaged in risky drinking . P300 was measured to test the possible electrophysiological differences. The correlates of alcohol use were analyzed.A case-control study was conducted according to the cutoff of the Alcohol Use Disorder Identification Test (AUDIT). The frequency of the TaqIA polymorphism of the dopamine receptor D2 gene associated with alcohol dependence was compared between the two OPAD groups. Tridimensional Personality Questionnaire (TPQ), The Alcohol, Smoking, and Substance Involvement Screening Test (ASSIST), and the MINI-International Neuropsychiatric Interview (M.I.N.I.) were measured or interviewed in OPAD not engaged in risky drinking  and harm avoidance subscale scores  than resilient OPAD, while the total scores of each dimension showed no significant difference. OPAD engaged in risky drinking showed more tobacco use than OPAD resistant to risky drinking. OPAD with risky drinking showed a shorter P300 latency than resilient OPAD on Fz electrodes. AUDIT scores of OPAD were correlated with P300 latency.Vulnerable OPAD showed higher novelty seeking subscale scores (NS4; 4.45 ± 2.012 vs. 3.31 ± 1.728, P300 differed between OPAD with and without risky drinking and alcohol use was associated with P300 latency, indicating that P300 may be used in the early detection of vulnerable OPAD and early intervention in the future. Alcohol dependence is a chronic mental disease that has both genetic and environmental determinants . Large nGenetic studies have shown that people with TaqIA of the dopamine receptor D2 gene (DRD2A1) is significantly associated with alcohol dependence . Many inTridimensional personality, especially novelty seeking, differs between people with alcohol dependence and without alcohol dependence . Some stP300 event-related potential (ERP) has been shown to reflect an objective physiological basis of cognitive functions , such asTo better understand the biological factors that may be associated with the outcomes for OPAD, this study aimed to investigate the genetic, psychological and P300 characteristics in the OPAD currently engaged in risky drinking and those not engaged in risky drinking, and further explore the correlates of problematic alcohol use.Fifty-five young adults were enrolled through advertisements on Wechat at Peking University Sixth Hospital. Participants completed the first screening to ensure whether they met the criteria for an offspring of a parent with alcohol dependence (OPAD) by asking “Did your father drink continuously for more than 1 year in your childhood and were there negative impacts on his physical and/or mental health, or an impact on his work or that of others?” according to the International Classification of Diseases, tenth edition (ICD-10) alcohol dependence criteria. Affirmations of all these three screening questions that indicated the participants were more than likely to be the offspring of parents with alcohol dependence. We only recruited offspring of fathers with ICD-10 alcohol dependence, in order to avoid the confounding effect of maternal alcohol abuse during pregnancy. All participants were right-handed and aged 18–45 years. They were excluded if they had a history of severe physical or neurological disease, conscious-loss or learning disability, or maternal alcohol use, and excluded if either parent had a history of schizophrenia, bipolar disorder or dementia.All experimental procedures received approval from the Institutional Review Board of Peking University Sixth Hospital (No. 202046). All the participants provided written informed consent in writing at the beginning of the initial screening and participants were compensated for 100 RMB as their transportation allowance.All participants were instructed to refrain from using any psychoactive substances, including alcohol, tobacco, caffeine, and sedative medications 24 h before testing. Each of them was interviewed and measured during three sessions, including gene sampling, psychological assessment, and EEG acquisition, which were all conducted by two well trained psychiatrists.+ or A1−) was designated according to the genotype carried by participants. The A1+ group included eight A1A1 genotypes and 25 A1/A2 genotypes.The 55 participants were instructed to clean their mouths using pure water and then provide oral swabs; DNA was extracted using standard techniques. DNA was used in the polymerase chain reaction as a template for the determination of DRD2 TaqI A alleles. DRD2 TaqI A genotyping was performed as described in another study . Psychian = 35) and those in the risky drinking group had score of 7 or greater (n = 20) according to the cutoff of 7 (The Alcohol Use Disorder Identification Test (AUDIT) was used to divide all the participants into the risky drinking and no risky drinking groups. Participants who met the criteria for the no risky drinking group had a score below 7 . The construct validity was 0.48–0.76 (P < 0.01), and the retest validity was 0.97 (P < 0.01)  and 12 subscales . TPQ is a normed and validated 100 item true-false questionnaire , and it .48–0.76 . The MIN < 0.01) .The paradigm consisted of frequently presented standard tones (90%), and infrequent target tones requiring a button press (10%). Standards (50 ms) and targets (100 ms) were 1,000 Hz, 75 dB pure tones. The task comprised a fixed pseudorandom sequence of 750 stimuli, with an interstimulus interval of 475–525 ms, divided into three blocks. Participants were instructed to press a response key to target tones only, using their right hand.Participants sat in front of a computer monitor and wore the headset for EEG recording during the oddball task . The EEGhttp://sccn.ucsd.edu/eeglab/). All recorded artifact-free EEG data were resampled to 500 Hz, referenced to an average of channels, and bandpass filtered in the range of 1–45 Hz to avoid the interference of 50 Hz signals. The time series were segmented into epochs with time- locked to the target auditory stimulus onsets (−200 to 500 ms) and baseline corrected (−200 to 0 ms). Epochs with excessive artifacts were removed. The data were then decomposed to perform an independent component analysis (ICA) via the runica algorithm. ICA components associated with vertical eye movements, heartbeats and other obvious artifacts were visually identified and removed according to their spatial, spectral, and temporal properties.Signals were analyzed offline with the MATLAB R2014a -based EEGLAB toolbox  than the no risky drinking group. There was no significant difference in the genotype ratio between the two groups. No other psychoactive substances were used as measured by ASSIST .There were no significant differences in the total scores of novelty seeking, harm avoiding and reward dependence between the two groups. The risky drinking group of OPAD showed higher scores on NS4 and HA4 . There were no significant differences in the amplitude of P300  with the latency of P300 at Fz electrode  scores and shorter latency of P300. Furthermore, the AUDIT scores were negatively correlated with the latency of P300 and positively correlated with NS4 and HA4 scores.In this study, the total scores of novelty seeking, harm avoidance and reward dependence personality did not differ between OPAD with and without risky drinking, while OPAD with risky drinking showed more significant disorderliness (NS4) and fatigability and asthenia (HA4) than OPAD without risky drinking. The division of participants into groups based on their current risky drinking is justified by the observation that OPAD who do not have risky drinking at a young adult age are likely to represent resilient individuals, and OPAD with risky drinking are considered to be vulnerable individuals . PreviouThe risky drinking participants in the current study showed not only risky drinking but also significantly more tobacco use than the no risky drinking group. Prior studies also showed that both alcohol and other substances were used in OPAD, which may result from the need to reduce people's negative feelings , 37. TheThis study showed the alteration in latency, not the amplitude of P300 in OPAD with risky drinking than in OPAD without risky drinking. Some previous studies found lower  or higheIn our study there were significant negative correlations between AUDIT scores and P300 latency, while other studies didn't find such correlation , 22, posThere are scarce studies focusing on vulnerability for offspring of parents with alcohol dependence and exploring possible psychophysiological mechanisms, especially controlling the genetic and other confounders. The results may provide a special and specific research direction for future studies in determining psychological and psychophysiological endophenotypes that can help with early warning and intervention. The current study further detected the difference between two relatively homogeneous groups of OPAD, and this should be more difficult in that both groups are already at risk and they are likely to be similar across many features. There are several possible limitations as follows. First, the probands were diagnosed according to the self-report of the OPAD, which may result in recalling bias, although we thoroughly checked the symptoms and criteria items to lessen the bias. Second, we do not include comparison groups with risky and no risky drinking whose parents have no history of alcohol use disorders, which may distinguish the impact from current risky drinking or the risk conferred from family. Selection bias may exist in that the participants were mostly had a high education level .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Malnourished individuals were older, had lower estimated protein intake , higher C reactive protein  and tumor necrosis factor α . Malnourished patients had higher sRAGE  and esRAGE (648 [408–1049] vs. 476 [355–680] vs. 545 [380–730] p = 0.033). In the multivariate analysis, only sRAGE maintained its association with malnutrition (p = 0.02) independently of aging and inflammation. Conclusions: in CKD patients, RAGEs isoforms, but not AGEs, are associated with malnutrition, irrespective of systemic inflammation, aging, and renal function.Background: in patients with chronic kidney disease (CKD), the inflammatory and pro-oxidant milieu may contribute to malnutrition development. In this study, we investigated the relationship between inflammation, advanced glycation end-products (AGEs), and their receptors (RAGEs) with malnutrition in CKD patients. Methods: we evaluated 117 patients. AGEs were quantified by fluorescence intensity using a fluorescence spectrophotometer, soluble RAGEs isoforms, and inflammatory interleukins by ELISA. Malnutrition was assessed by a malnutrition inflammation score. Results: mean age was 80 ± +11 years, eGFR was 25 ± +11 mL/min/1.73 m The etiology of malnutrition in chronic kidney disease is complex and multifactorial: reduced food intake because of anorexia, changes in taste, uremic gastritis, and the high number of medicines prescribed may all contribute to reducing energy and protein intake ,2,3. CKDAGEs, throughout the interaction with their receptors , contribute to increased systemic inflammation. The accumulation of AGEs can increase circulating monocytic cell levels  and AGEsIn end-stage renal disease patients’, levels of circulating AGEs have been associated with the prevalence of malnutrition ,14. This2 of variation). GFR was estimated according to the CKD-EPI formula (eGFR). We excluded patients with cancer, advanced cirrhosis and/or ascites, severe heart failure (NYHA class III–IV), nephrotic syndrome, thyroid diseases, bowel inflammatory diseases, and inability to cooperate. We also excluded patients treated with immunosuppressive drugs or who had been hospitalized in the last three months. Twenty-four hours urinary collection and biochemical parameters were collected the day of the visit after overnight fasting of at least 12 h. The study was conducted according to the ICP Good Clinical Practices Guidelines, and it was approved by the Ethics Committee of our Institution . All patients signed informed consent.We evaluated cross-sectionally 117 prevalent patients between 9/2016 and 3/2018 that were enrolled according to the following criteria: age ≥ 65 years, CKD stages 3a to 5 in conservative therapy, and with a relatively stable glomerular filtration rate (GFR) over the previous 6 months .Quantification of total isoforms of RAGEs was performed as previously indicated . Briefly®, Promega). Fluorescence intensity was expressed in arbitrary units (AU). AGEs were then normalized for total protein content.AGEs levels were dosed as previously reported ,19. BrieSerum cytokines concentration were dosed in duplicate using the following ELISA kits according to the manufacturer’s instructions:Quantikine ELISA Human CCL2/MCP-1 Immunoassay DCP00, Human TNF-alpha ELISA Kit  and Quantikine HS ELISA Human IL-6 Immunoassay HS600B . Zero was included, as the last standard value, in each resulting curve. Results were validated by using Quantikine Immunoassay Control Group 1–4 or 10 . Absorbance readings were measured at 450 nm by spectrophotometer .Nutritional status was assessed with malnutrition inflammation score (MIS). MIS consists of ten domains, each of which with a score scale 0–3 according to the severity level . A totalProtein intake was determined by normalized protein catabolic rate (nPCR) on 24 h urinary urea excretion .Energy intake was assessed with dietary diaries that were compiled the two days preceding the visit. We calculated nutrient intake using the nutritional software Winfood .2)).We also measure the following anthropometric measurements: body weight, height, body mass index , in parametric distributions, or median with interquartile range (IQR), in non-parametric data. Categorical variables were summarized as percentages. We performed Student’s t-test and ANOVA to compare parametric variables, while we performed, when appropriate, the Mann–Whitney “U” test or Kruskal–Wallis for the comparison of not parametric ones. Multivariate linear regression was used to test for the correlation between inflammatory interleukins, AGEs, and RAGEs isoforms; correcting for the influence of eGFR.CRP levels were considered as Log-values, in this case, to compensate for nonlinear distribution. We also evaluated the correlation of malnutrition, as a dependent variable, with sRAGE and esRAGE in two multivariable logistic regression models that included all the principal variables that were associated or had an interaction with AGEs and malnutrition.Statistical Analysis was conducted using IBM SPSS software .2. Median malnutrition inflammation score was 4 [3–8], while eGFR ranged from 60 to 15 mL/min/1.73 m2, with a mean value of 25 ± 11 mL/min/1.73 m2 (stage IV CKD).Our study enrolled 117 patients whose general characteristics are depicted in Nutritional metabolic markers were averagely normal .Regarding inflammatory markers, CRP levels were generally increased in our cohort (0.4 ± 0.7 mg/dL). Mean TNFα, IL6 and MCP-1 levels were .p = 0.002) and had an individual protein intake (estimated by nPCR) that was lower than non-malnourished and at-risk for malnutrition patients . Conversely, energy intake was comparable in the three groups As evident in p = 0.002) and prealbumin . No difference concerning eGFR, lipid profile, and BMI was evident between the different groups.Concerning serum parameters, malnourished patients had lower concentrations of serum albumin  and TNFα  levels were significantly higher in malnourished patients compared to the other groups.Concerning inflammatory markers , both CRp = 0.035 and p = 0.033, respectively). AGE/sRAGE ratio and cRAGE/esRAGE ratio did not show any statistical difference between different nutritional statuses. In The correlations between AGEs and RAGEs with the nutritional status are summarized in p < 0.0001, p = 0.003, p = 0.001 respectively). Secondarily, lower AGEs/sRAGE values were significantly correlated with lower albumin and nPCR values (p = 0.005 and p = 0.01 respectively).We also performed additional analyses to test for eventual correlation between AGEs, sRAGE, and relative isoforms and the main biochemical parameters evaluated in our cohort. These results are shown in detail in p = 0.05), while sRAGE and cRAGE were negatively correlated with it . Secondarily, higher values of AGEs/sRAGE ratio significantly correlated with higher CRP values (p < 0.0001). We performed a series of multivariate linear regression analyses to evaluate whether there was any relationship between AGEs, RAGEs, and their ratio with inflammatory markers that were mainly associated with malnutrition . Since CRP had a skewed distribution, it was log-transformed for the analysis. Moreover, considering that AGEs, RAGEs, CRP, and TNFα levels are influenced by eGFR, all models were corrected for patients’ eGFR .We also performed additional analyses to test for eventual correlation between AGEs, sRAGE, and relative isoforms and pro-inflammatory markers. First of all, AGEs levels were directly and significantly correlated with CRP levels . The same interactions were observed for esRAGE . Then, we created two different models, including separately sRAGE or esRAGE (in order to avoid redundancy in the model) with the principal variables that were associated or showed any interaction with overt malnutrition . However, CRP and age maintained the strongest association with malnutrition .sRAGE  maintainp = 0.055). Furthermore, in this case, CRP and age were the variables with the strongest association with malnutrition .In In our study, we confirmed that in patients with advanced CKD, chronic moderate inflammation is associated with malnutrition. Alongside that, we found that sRAGE concentration and AGEs/sRAGE ratio are associated with inflammatory markers, suggesting that AGEs/RAGEs interaction might modulate inflammation. Above all, we found that in CKD patients, sRAGE levels are associated with malnutrition independently of other well-known risk factors. Previous studies investigating the relationships between AGEs, sRAGE, and malnutrition in CKD were focused on end-stage renal disease patients undergoing chronic dialysis. To our knowledge, this is the first study that evaluated the correlation between inflammation, AGEs, RAGEs isoforms, and malnutrition in CKD patients not yet in dialysis.Among patients with CKD, the coexistence of malnutrition and inflammation is a well-recognized condition ,23,24,25In uremic patients, AGEs are correlated with CRP and TNFα . First op-cresyl sulfate) can increase ROS production, enhancing oxidative stress in CKD patients. This leads to proteins, lipids, and DNA modifications, many of which are composed of protein carbonyl compounds, which are precursors of pentosidine, which derives from protein and lipids oxidation and glycation [In a recent study conducted by Suliman et al. in patients with end-stage renal disease, plasma pentosidine (a subtype of AGE) was associated with inflammation and with the development of malnutrition . Some urlycation ,44,45. Ulycation .Luketin M et al. described a negative correlation between AGEs, LDL-cholesterol, triglycerides, and BMI in hemodialysis patients and a positive association between AGEs and malnutrition . sRAGE wConcerning the therapeutic approach against AGEs, in patients with diabetes, AGE formation can be prevented by hypoglycemic medicines, vitamins, and smoking cessation . MoreoveDifferently from previous studies, we did not find any significant difference in serum AGEs concentrations in well-nourished patients with respect to those that were at risk of malnutrition or malnourished. Instead, we observed an association between malnutrition and increased levels of sRAGE and esRAGE. The lack of a direct correlation between AGEs and malnutrition may depend on several discrepancies between our study and the previous ones. First of all, our study evaluated patients with advanced CKD but not yet in dialysis; therefore, we did not find such pronounced AGEs accumulation as it is described in dialyzed patients. Secondarily, eGFR, which is the main determinant of AGEs accumulation in CKD, was the same in the three groups of patients classified according to their nutritional status, and this may have nullified the possible effect of different degrees of renal dysfunction on AGEs accumulation. However, despite the fact that AGEs were not associated with malnutrition, we found that malnourished patients had higher levels of CRP that were directly associated with AGEs and AGEs/sRAGE ratio . This suWe acknowledge that our study has some limitations. First of all, the cross-sectional design does not allow for attributing any causal relationship to the association between sRAGE isoforms levels and the onset of malnutrition in CKD patients. Secondly, our study is monocentric, and our population is relatively small. However, the monocentric nature of our study allowed us to reduce the possible sources of bias by using a highly standardized protocol for patients’ selection, biochemical analyses, and clinical observations. In particular, we applied strict inclusion and exclusion criteria that let us exclude patients that may have developed malnutrition because of specific clinical conditions.A significant point of strength of our study is that the relationship between inflammation, AGEs, RAGEs, and malnutrition was thoroughly investigated. Furthermore, we studied in depth the association of AGEs and malnutrition by addressing not only AGEs but also RAGEs isoforms and the modulation of AGEs/RAGEs interaction played by inflammation. This is, to the best of our knowledge, the first time that a comprehensive evaluation of the association between the AGEs–RAGE system and malnutrition has been conducted in CKD patients not undergoing dialysis.Our study shows that RAGEs levels are independently associated with malnutrition in patients with advanced CKD. Furthermore, we found that in these patients, inflammation can modulate AGEs/RAGEs interaction, indicating a possible correlation between inflammation, oxidative stress, and malnutrition. Whether these findings were confirmed, we could hypothesize that the modulation of AGEs/RAGEs interaction may contribute to preventing the development of malnutrition in this high-risk population.


The behavioral immune system (BIS) comprises manifold mechanisms, that may assist the physiological immune system (PIS) in counteracting infection and can even reduce the risk of contagion. Previous studies have found initial evidence for possible interactions between the two systems. However, most of these findings were correlative and have not been replicated. Further, none of these studies examined whether disease stimuli that indicate an enhanced airborne transmission risk may trigger a different immune response in comparison to stimuli that predominantly evoke core disgust. In the present study, we employed a video-priming approach to get further insight in the influence of the perception of disgust- and disease-related stimuli on the rapid physiological immune response, as indicated by changes of secretory immunoglobulin A (S-IgA) in saliva. We created three video primers that represented different categories of disgust- and/or disease-associated content. Two of the videos showed disease-related situations that were associated with contagious respiratory virus infections, varying in concealment of aerosols. The third video incurred no heightened airborne contagion risk, but comprised situations that are known to elicit core disgust, such as rotten foods, decaying animal carcasses, or cockroaches. A fourth video acted as control showing landscape impressions. The different video primers varied in their contagion risk and disgust-evoking potential. Given the role of S-IgA in the mucosal immune defense, we expected differences in the S-IgA response between the two videos indicating a heightened airborne contagion risk and the core disgust video, with the highest S-IgA to occur after the aerosol video. For this, we used the data of 107 healthy participants in a between-subjects design with the four video primers. We found a significant increase of S-IgA in response to both the disease- and the disgust-related videos, which correlated positively with the perceived contagion risk of the displayed situations. Nevertheless, there was no significant difference in the increase between the three disease- and disgust-related videos. We also found that people with a high contamination disgust produced less S-IgA in such situations, which is a hint for a compensating relationship between the BIS and PIS. Our observations suggest that the mere visual perception of videos showing realistic situations of an increased contagion risk can elicit a heightened release of salivary antibodies. •Realistic videos of respiratory diseases are efficient to trigger immune responses.•Secretory Immunoglobulin A (S-IgA) in saliva increases after video-priming.•S-IgA increase is positively correlated with perceived contagion risk.•S-IgA increase is inversely correlated with the trait contamination disgust. While both the unspecific, innate and the specific, adaptive immune system are highly effective, most functions of the PIS are very resource consuming and can have negative consequences when misdirected . This leAs a complex system, the BIS may detect potential pathogens, and trigger defensive responses like disgust, avoidance behavior, social exclusion and sickness behavior . The ongPrevious studies found initial evidence for possible interactions between the BIS and the PIS: (1.) avoidance behavior increased after recent activation of the PIS , (2.) beA more direct measure to investigate the relationship between the PIS and BIS is to experimentally confront participants with disgust evoking stimuli, in order to provoke behavioral and physiological immune responses. Disgust may have evolved as a response to objects that represent a potential threat of (infectious) diseases . The emoSetting the focus on the mucosal immune response In the present study, we took a similar approach as Since the pathogens associated with respiratory diseases enter the body through mucosal tissues of the respiratory tract, we focused on the mucosal immune system of the oral cavity, specifically the change of salivary S-IgA. S-IgA is the main mucosal immunoglobulin and plays an important role in the first-line-immune-defense against pathogens that enter the body through mouth or nose . It is cIn our study, young healthy participants watched one of the four videos and answered an online survey to assess inter-individual state and trait differences in the BIS (see details below). Saliva samples were collected at baseline and twice after the video, from which changes in S-IgA were assessed. Given the well-established role of S-IgA in the mucosal immune defense , we expeTo shine more light on inter-individual differences in the behavioral immune system we also utilized established questionnaires that capture trait as well as state disgust  and se22.1https://osf.io/9hxpt/) between-subjects design we confronted the participants with three different disease- and/or disgust-related stimulus sets. In addition to that, we also included a control group, who watched a video with neutral content (landscape impressions and city panoramas). A power analysis with G*power . These videos differed in their disgust-evoking potential and their disease-related content. The aim was to compare three disgust- and/or disease-associated primers, which triggered different degrees of disgust and fear of contagion. All videos showed a combination of short video clips and pictures that were assembled in a video of 1:20 min length. In order to achieve a sufficient priming effect, each video was repeated once . The videos consisted of royalty free material from pages like Database . Most ofAerosol Disease Video (A): This video was intended to trigger a high level of disgust and high fear of contagion. It comprised video clips and pictures of people, who were sneezing unconcealed, either directly into the camera or in the vicinity, whereby some visibly emitted aerosols.Concealed Contagion Disease Video (CC): This video showed people sneezing without emitting aerosols . Other people in the video showed visible signs of sickness, such as looking feverishly or laying sick in bed. We predicted this video to elicit less disgust and a medium to high fear of contagion, compared to the Aerosol Disease Video.Core Disgust Video (CD): Presuming that non-airborne disease threats trigger a different immune response, we created a video showing rotten food, dead animals with maggots, rats, and other disgusting items. Through this video, we tried to elicit a similar disgust response as in the Aerosol Disease Video, yet with a significantly lowered fear of contagion.Control Video (C): In this video, we combined video clips and photos of buildings, skylines, traffic intersections and other landscape views. This video was intended to trigger no disgust- or disease-related responses.2.3Our participants underwent online surveys (see 2.5 Procedure below) that were programmed with the software LimeSurvey and Inquisit 6 . The Zoo2.3.1Mood Scale: Participants rated how they feel, answering 24 questions adapted from the German MDBF  mood-scale from 1 to 5 . For example, tired, satisfied, happy, nervous, etc. : Participants evaluated their trait disgust sensitivity on the modified version of the Disgust Scale established by I might be willing to try eating monkey meat, under some circumstances.“, for which participants indicated their agreement on a 5-point Likert-scale (from 0 = ‘Strongly disagree’ to 4 = ‘Strongly agree’). The rest of the items had the participant rate situations from 0 = ‘Not disgusting at all’ to 4 = ‘Extremely disgusting’, for example, “While you are walking through a tunnel under a railroad track, you smell urine.”. The resulting score were then categorized into the Core-Disgust-Score (12 items), and the Contamination-Disgust-Score (5 items).Perceived Vulnerability to Disease (pVtD): Participants evaluated their perceived Vulnerability to Disease, by using a 15-item self-report instrument designed by In general, I am very susceptible to colds, flu and other infectious diseases.” and “I prefer to wash my hands pretty soon after shaking someone's hand.”. The participants had to evaluate each item with a 7-point Likert-scale (1 = ‘Strongly disagree’ to 7 = ‘Strongly agree’). The resulting score was further categorized into the sub-scores Germ-Aversion (8 items) and the Perceived-Infectability- (7 items).2.3.2Video-Questionnaire: Participants had to answer questions about the video content. We first asked three questions that required a recall of details, such as “How many elderly men were portrayed in the video?”. The participants had a choice between five options, such as “None”, or “Only 1 elderly man”. Furthermore, participants were shown 15 pictures of which 10 were screenshots from the video previously shown, while 5 were not shown in the video. The participants were asked, “Was this person/situation portrayed in the video?” and had to choose between the options “Yes” and “No”. These questions allowed us to implicitly evaluate whether participants had payed attention to the details of the video, participants that answered less than 50% correct would be excluded.I was distracted during the video.” and “If this was a real situation I would have become sick.” (Contagion risk question), which they answered on a 5-point Likert scale form −2 ’Completely incorrect’ to +2 ’Completely correct’. For analysis this scale was converted to a span from 0 to 4.Lastly participants answered explicit questions on how much attention they had paid during the video and how realistic they would rate them. For this we used nine statements, such as “Modified Differential Emotions Scale (mDES): Participants were further asked to recall the explicit feelings they experienced while watching the video. For this “absolute recall task”, we asked them how they felt, while watching the video using 6 statements, such as “How strong was your feeling of disgust, antipathy and revulsion while watching the video?”. The statements about feelings had to be rated on a 9-point Likert-scale from 0 “not at all” to 8 “completely” and the relative recall of feelings depicted an identical number of negative  and positive feelings  and reflected emotional state after the video.Evaluation of Stimuli: Participants evaluated the amount of disgust in relation to the scenes and picture in the videos. For this, we used 37 screenshots from all four videos independent of whether they had been shown to the respective participant . The participants had to decide on how they felt, when watching the situation or person on a given screenshot. For this, they used a 7-point Likert-scale from 1 “no disgust at all” to 7 “extremely strong disgust”. Through these questions, we were able to evaluate the actual impact of the stimuli in terms of their disgust potential, and were also able to assess the influence of the video prime on subsequent stimulus evaluation. For results of every single stimuli, see 2.4lkmann, Karlsruhe, Germany. There, an immuno-nephelometric analysis to determine the concentration of S-IgA in saliva was performed on the Atellica® NEPH 630 System S-IgA.The participants received a kit for saliva collection at home. It was sent by mail, since the restrictions related to the COVID-19-pandemic precluded tests in our computer lab at the university. The kit included three 2 ml Eppendorf Tubes and an instruction of proper saliva sample collection. Once the experiment was completed, the envelope with the samples was immediately sent back to the institute. During the test session, the participants were asked to take their saliva samples at pre-defined time points during the surveys. The experimenter, who was blind to the participant's condition, instructed and monitored the saliva sample collection via Zoom calls. The experimenter also answered any study-related questions prior and after the test session via Zoom. However, the subject was left in private while he/she watched the video prime and filled in the online-survey (questionnaires and demographic data). Upon arrival at the institute, the saliva samples were frozen at −20 °C. For analysis, the frozen saliva samples were sent on dry ice to the MVZ Laboratory Vo2.5duration = 5.5 min), participants were asked to give the third and last saliva sample. Then, participants completed the evaluation of the stimuli . Previous evidence suggests that S-IgA shows its daily peak in the morning and then drops to a stable level in the afternoon . In the also see .2.6For data analysis, we calculated S-IgA concentration against the time it took the subjects to fill the tube with 1.5 ml of saliva [(mg/dl)/min]. All data was tested for deviation from a normal distribution using the Kolmogorov-Smirnov test with a statistical threshold of p = .050. Since all tests were significant, we used non-parametric post-hoc tests.Firstly, we used Kruskal-Wallis-tests, with Primer as independent and post-video evaluation scores as dependent variables to assess how primer category affected the evaluation of the video content. Again, we utilized Mann-Whitney-U tests as post-hocs.a).Secondly, we assessed whether the increase in S-IgA was affected by the category of the video primers. For this, we utilized a general linear model for repeated measures (GLM) with Sample  as within-subject factor and Primer  as between-subjects factor. We used the Mann-Whitney-U test as post-hoc test. All post-hoc analyses included Bonferroni adjusted p-values (pFor the assessment of correlations between the video-evoked increase in S-IgA and (1.) state disgust, (2.) trait disgust, (3.) trait VtD, and (4.) and fear of contagion, we employed the spearman rank correlation. All statistical analyses were performed in IBM SPSS Statistics 27. Figures were created with R Studio and MS-Excel.3We excluded data of 9 participants, who were exposed to more than three of the stressors listed in the initial survey in the 48 h before the test. The final analysis was therefore based on data of 107 participants, with an average age of 24.72 years (σ = 3.60 years). Of these, 27 participants received the Concealed Contagion Disease Video (16 f/11 m), another 27 watched the Core Disgust Video (18 f/9 m) or the Control Video (16f/11 m), and the remaining 26 participants received the Aerosol Disease Video (14f/12m).3.1How strong was your feeling of disgust, antipathy and revulsion while watching the video?”. The Kruskal-Wallis-Test showed that the four video primers differed significantly in disgustingness , the Concealed Contagion Disease Video elicited less disgust than these two . Furthermore, all three disgust- or disease-related primers triggered higher disgust ratings than the Control Video .In order to test, whether participants indeed differentially perceived the three videos in terms of their disgustingness and contagion risk we analyzed two survey questions, which were asked after the video. The first question was part of the mDES and asked: “If this was a real situation I would have become sick.”. The Kruskal-Wallis-Test showed that the four primers differed significantly in the perceived contagion risk . However, there was no significant difference in the rating between the three primers themselves .Regarding the contagion risk associated with the stimuli in the video, we asked participants to rate the following statement in the Video-Questionnaire: “4p2 = 0.133) as well as an interaction of Sample and Primer  = 3.09, p = .030, ηp2 = 0.083) on S-IgA. To further investigate these effects, we split the data by Primer and performed Wilcoxon signed-rank tests between the Baseline sample and Sample 2, which was collected directly after the video. We found that watching any of the three disgust- and/or disease-related primers led to a significant increase of S-IgA after the video, while the control primer did not (see Using a 4 (Primer) x 2 (Sample) GLM, we found a significant main effect of Sample  = 15.75, p < .001, η not see , Fig. 4.a = 1, η < 0.001; A vs. CD: U = 317,5,p = .551, pa = 1, η = 0.007; A vs. C: U = 256,0, p = .091, pa = .546, η = 0.054; CC vs. C: U = 239.0, p = .030, pa = .180, η = 0.087; CC vs. CD: U = 313.0, p = .373, pa = 1, η = 0.015; CD vs. C: U = 279.5, p = .141, pa = .846, η = 0.040; also see Supplement To further compare these increases between video primers, we calculated the difference between Sample 2 and the Baseline sample (ΔS-IgA). Yet, we found that none of the four primers showed a significantly larger increase than any of the others after the Bonferroni correction.  with the ΔS-IgA. This was done to investigate if state disgust, evoked by the presented videos, was related to the increase in S-IgA concentration. We found no significant correlations between the two state disgust measures and the ΔS-IgA .Furthermore, we correlated the above-mentioned rating of the perceived contagion risk with the increase of S-IgA. Here, we found a significant correlation , which is displayed in We correlated the rating of how disgust evoking presented stimuli were from (1.) the Evaluation of Stimuli, and (2.) the mDES . Running the same GLM as before, we still found the significant main effect of Sample  = 11.32, p = .001, ηp2 = 0.130) and the interaction between Sample and Primer  = 6.64, p = .002, ηp2 = 0.149, In fact, only a subgroup of the participants, who watched the two disease-related primers perceived the video content as a realistic contagion risk, hinting that the video material might not have been realistic enough for each observer. This finding led us to conduct a secondary analysis, in which we excluded participants from the analysis, who did not rate the disease video primers as a potential contagion risk (n = 25). In addition, we also excluded participants, who in turn perceived the control primer as a contagion risk (n = 3). After excluding these 28 participants, we combined the two disease-related primers  to one Disease Primer (n = 28), in order to keep the sample size comparable to that of the other two primer groups. The correlation between contagion risk and ΔS-IgA stayed significant after exclusion of these cases , which yet did not differ from the Control Primer .Using the same post-hoc analysis we found a significant increase in the disease- and disgust-related primer  or the Core Disgust subscale , we found a significant negative correlation with the Contamination Disgust subscale  trait disgust, and (2.) VtD, we correlated the ΔS-IgA to the DS-R and pVtD scales and subscales. Since the control group showed no significant increase in S-IgA, we excluded these participants from the analyses. While we found no significant correlation between the ΔS-IgA and the total DS-R . Furthermore, we did not find a significant correlation between the increase of S-IgA and the pVtD sum scale  or its subscales Germ Aversion  and Perceived Infectability .As the core disgust subscale mainly targets the items that are represented in the core disgust video, we also correlated the core disgust subscale separately to each primer. We found a significant correlation between the ΔS-IgA and core disgust in participants that were presented with the core disgust video  induced by the two disease primers did not differ significantly from the control group, yet in the disease primer groups a significant rise from Baseline to Sample 2 could be documented which was not visible in the control primer . MoreoveFurthermore, we found that the perceived contagion risk associated with the videos correlated positively to the increase of S-IgA, implying that perceived contagion risk is an important factor in defensive immune responses. This assumption led us to perform a secondary analysis, from which we excluded participants that did not see the disease-related primers as a realistic contagion risk. Here we found a significant difference in ΔS-IgA between the disease-related and control primers, while the core disgust primer still did not significantly differ from neither the control nor the disease-related primer . Lastly,Furthermore, we found no significant correlation between our measures of state disgust  and the S-IgA increase. This is coherent with findings of previous studies measuring S-IgA , IL-6 S, TNF-a/a7Our data show activation of the PIS  following visual exposure to disease- and disgust-related stimuli. While one may speculate that the increase in S-IgA reflects a proactive immune response, that may prepare the organism for the upcoming pathogens associated with the sneezes and coughs, our study does not provide direct evidence for a heightened immunity in individuals who responded with increased S-IgA to the respective videos. However, such a proactive defense mechanism seems likely, since S-IgA in saliva plays an important role in immune exclusion and intracellular neutralization . Adding 8Our findings are indicative of an enhanced activation of salivary S-IgA secretion through visual perception of potentially contagious objects and people. We also found that people with a high contamination disgust secreted less S-IgA in such situations, which might be a hint for a compensating relationship between BIS and PIS. To get a further insight into the association of the BIS and PIS more research is needed. For future studies we suggest a better baseline control to reduce variance in data. This might be done by testing the participants in the controlled environment of a laboratory, by letting them relax for some minutes before the baseline sample and putting stricter restrictions on participants before testing . We also suggest the implementation of additional implicit behavioral measures  to get a better understanding of the behavioral response to the presented disease stimuli, since this aspect of the BIS may be insufficiently be represented by self-report questionnaires alone.None.10.13039/501100005711Universität Hamburg.This research was funded by the regular research budget of the Neuroendocrinology and Human Biology Unit, at the The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


EDA1 , EDAR , EDARADD , and WNT10A  genes are responsible for most EDs. Bi-allelic pathogenic variants of WNT10A have been associated with autosomal recessive forms of ED, as well as non-syndromic tooth agenesis (NSTA). The potential phenotypic impact of associated modifier mutations in other ectodysplasin pathway genes has also been pointed out. We present on an 11-year-old Chinese boy with oligodontia, with conical-shaped teeth as the main phenotype, and other very mild ED signs. The genetic study identified the pathogenic variants WNT10A (NM_025216.3): c.310C > T; p. (Arg104Cys) and c.742C > T; p. (Arg248Ter) in compound heterozygosis, confirmed by parental segregation. In addition, the patient had the polymorphism EDAR (NM_022336.4): c.1109T > C, p.  in homozygosis, named EDAR370. A prominent dental phenotype with minor ectodermal symptoms is very suggestive of WNT10A mutations. In this case, the EDAR370A allele might also attenuate the severity of other ED signs.Ectodermal dysplasias (EDs) represent a heterogeneous group of genetic disorders characterized by the abnormal development of ectodermal-derived tissues. They include the involvement of the hair, nails, skin, sweat glands, and teeth. Pathogenic variants in  The ectoderm is one of the three germ layers of the embryo. Around the third week of development, it differentiates to form the central and peripheral nervous system, skin, oral mucosa, tooth enamel, mucosa of the nostrils, sweat glands, hair, and nails, among other structures.The specific differentiation of cells of ectodermal origin is regulated by very specific signaling pathways, such as WNT, BMP “bone morphogenic protein”, and FGF “fibroblast growth factor” pathways . StructuEDA1, EDAR, EDARADD, and WNT10A genes are responsible for the majority of EDs  oWNT10A: c.310C > T; p. (Arg104Cys), generates a less impactful amino acid change in the protein it encodes; its validated frequency is 0.065% in East Asian populations and it has not been identified in South Asia.The father comes from a region with a high potential for geographic isolation in eastern China, Daxue, a mountainous area of Tibet. The maternally inherited pathogenic variant, WNT10A gene. Neither of them presented with oligodontia or a complete ED phenotype, except dry skin (HP:0000958), keratosis pilaris (HP:0032152) in the father, and plantar hyperkeratosis (HP:0007556) in the mother. It should be noted that the paternal WNT10A pathogenic variant p. (Arg248Ter) is a nonsense mutation that leads to the end of protein synthesis, resulting in a smaller nonfunctional protein.Both parents, as carriers, confirm the recessive inheritance pattern attributed in this case to the WNT10A gene have been associated with variable phenotypes, ranging from asymptomatic to a severe ED phenotype. Heterozygous pathogenic variants can lead to tooth agenesis. Homozygous or compound heterozygous WNT10A variants, as in the case of our patient, may lead to a wide phenotypic spectrum, from STHAG4 to odonto–onycho–dermal dysplasia and Schöpf–Schulz–Passarge syndrome [Pathogenic variants in the syndrome . The latsyndrome , as wellsyndrome .WNT10A gene. It is very likely that the phenotype depends on the characteristics and location of the genetic changes, and, consequently, on the deficiency generated in the Wnt10 protein. In general, patients carrying biallelic nonsense pathogenic variants in WNT10A have a much more severe dental agenesis, whereas heterozygous individuals carrying a nonsense or a missense pathogenic variant are often unaffected or have a mild phenotype. Heterozygous compound patients were missing up to 6 permanent teeth, whereas homozygotes were usually missing 6 to 26 teeth, mostly around 16 [WNT10A gene and showed severe dental involvement, while the parents did not present any dental involvement. Recently, WNT10A-linked oligo/hypodontia phenotypes have been described to be related with minor ectodermal manifestations, such as mild hair and nail anomalies, as described in our patient [It has been observed that the number of missing teeth in the permanent dentition strongly depends on whether the affected individual is a homozygous or heterozygous carrier of mutations in the round 16 . This is patient .WNT10A gene and its resulting phenotype that we report have not been previously described. Our patient could share a clinical condition with STHAG4 or odonto–onico–dermal dysplasia. A broad clinical spectrum has been defined among homozygous carriers of these variants separately, as a WNT10A homozygote genotype for c.310C > T; p. (Arg104Cys) delineated in ED-affected Turkish children [WNT10A: c. 742C > T; p. (Arg248Ter) homozygous variant associated with NSTA [According to the literature, the compound heterozygous genotype in the children  or an adith NSTA , OODD [3ith NSTA , or SSPSith NSTA . PotentiT; p. Arg8Ter homoith NSTA . Table 2EDAR gene: c. 1109T > C; p. , it has been suggested that it could act as a modifying variant of the ED phenotype in patients carrying causal variants in the EDA gene, one of the main susceptibility genes for ED [WNT10A gene mutation carriers. However, we cannot exclude it, given the relationship among the different molecular pathways involved in ectodermal derivatives development. It is difficult to attribute to the fact that the patient is a carrier of such a homozygous polymorphism, the few symptoms that he presents at the dermatological level or in other structures, since there are no series analyzed to establish such an association with mutation carriers in the WNT10A gene.Regarding the variant in the s for ED . It is dEdar protein that it encodes.According to the functional prediction software for genetic variants Alamut Software, version 1.5.1, the physical–chemical characteristics of the T/C nucleotide change that generates the polymorphism could be important for the The fact that both parents were homozygous for the EDAR370A allele, and therefore the patient, is not surprising. The frequency of this variant in the population from which the patient’s ancestors came from has reversed to become the consensus allele, with 92.1% described in East Asian populations. The allelic frequencies described for the C allele in European, African, and Caucasian populations have remained between 1 and 2%. Its genetic drift shows great contrasts between different populations and ethnic groups. Some authors have suggested that its homozygous genotype could confer some advantage. Its evolutionary conservation is evident in the population of origin of the patient’s family [WNT10A pathogenic variants in compound heterozygosis. This supports previous reports and highlights the importance of searching skin and nail anomalies in patients consulting for apparently isolated dental anomalies to establish an accurate diagnosis during childhood. The association of the EDAR370A homozygous allele might also attenuate the severity of ED signs in this patient, although more related data are needed.Our patient presented a prominent dental phenotype and minor ectodermal signs carrying biallelic


To determine whether adverse childhood experiences (ACEs) of children of alcoholics (COA) in male were associated with their current “risky drinking”.This case–control study used the Alcohol Use Disorder Identification Test  to divide the participants into two groups, a “risky drinking” group (N = 53) and a "non-risky drinking” group (N = 97). Demographic data, Adverse Childhood Experiences-International Questionnaire (ACE-IQ), the Hamilton Anxiety Rating Scale (HAMA), the Hamilton Depression Rating Scale (HAMD) and the Mini-International Neuropsychiatric Interview (MINI) were used for assessment. The specific relationships between ACEs and “risky drinking” were explored.P < 0.001). The “risky drinking” group was more likely to have experienced a major depressive episode (P < 0.05), nonalcohol psychoactive substance use disorder (P < 0.01) and bulimia nervosa (P < 0.05), and they also experienced more physical abuse (P < 0.05), community violence (P < 0.001) and collective violence (P < 0.01). In a single factor logistic regression, physical abuse, community violence and collective violence were associated with a two to 11- fold increase in “risky drinking” in the adult COA, and in multiple factor logistic regression, community violence showed a graded relationship with “risky drinking”.Respondents ranged in age from 29.70 ± 6.72 years; 74.5% were females; 94.7% were of Han nationality; 56.7% had a level of education above high school; 12% had no formal or stable job. There was difference in attitude to self-drinking between two groups (The childhood adverse experiences contribute to “risky drinking” in COA. This finding in the Chinese context have significant implications for prevention not only in China but in other cultures. There must be greater awareness of the role of ACEs in the perpetuation of alcoholism. Alcohol dependence was marked by compulsive drinking, withdrawal symptoms and increased tolerance to alcohol . The preAlthough the number of offspring with alcoholic parents had not been counted worldwide, limited research had shown that alcohol dependence had an intergenerational impact with psychological and social implications. The offspring of those with alcoholism were at risk of experiencing the negative effects of parental alcohol dependence . CompareACEs included emotional abuse, physical abuse, sexual abuse, emotional neglect, physical neglect, and family dysfunction  . In a meExisting studies examining COA came mostly from Western countries, which had quite a different cultural background than Asian countries . It was In this case–control study, participants were enrolled via an advertisement and screened from August 2020 to February 2021 at Peking University Sixth Hospital.biological father could be diagnosed as alcohol dependence based on ICD-10  criteria;participant was aged 18 to 45;participant had an AUDIT score ≥ 7 .The inclusion criteria for those in the risky drinking group included the following:severe physical or neurological disease;history of loss of consciousness or learning disability;mother drank during pregnancy or could be diagnosed with maternal alcohol use disorder based on ICD-10 criteria;either parent was diagnosed with schizophrenia, bipolar disorder or dementia.The exclusion criteria for those in the risky drinking group included the following:The inclusion criteria for those in the non-risky drinking group differed from the risky group was that AUDIT score < 7. The exclusion criteria for those in the non-risky drinking group were the same as for the risky drinking group.A total of 213 participants were enrolled after the first screening, A total of 161 persons provided informed consent in writing, and the remaining persons did via WeChat. Eighty-four participants were not in Beijing and were interviewed via video phone, and 77 persons came to Peking University Sixth Hospital and were interviewed face to face. The protocol took one hour to complete. Finally, 150 participants met all inclusion criteria and did not meet the exclusion criteria.The two-dimensional code of the advertisement was sent to discharged inpatients, follow-up groups, or posted in the outpatient department. There was a three-phase screening process. One attending psychiatrist, trained to perform the evaluation, interviewed the participants and completed all questionnaires. Simple feedback was given to the participants.Demographic form recorded data including sex, age, race, education, occupation, income, marriage, attitudes toward their father's drinking, attitudes toward self-drinking and knowledge of self-help groups (see Table The Alcohol Use Disorder Identification Test (AUDIT) was used to divide the participants into the “risky drinking group” and “non-risky drinking group” using a cutoff of 7, which had been tested in China and found to be the best score to identify “risky drinking” in the Chinese population. Cronbach’s alpha was 0.782, and the item-level content validity index was 0.83 .A Chinese version of the ACE adapted from World Health Organization (WHO) ACE-IQ , was useP < 0.01). The reliability index was 0.36 [The Hamilton Anxiety Rating Scale (HAMA) was used to rate anxiety with a cutoff score of 14 to divide the different classes. The validity index for the total score was 0.93, and the validity correlation index of the subscales was 0.83–1 (was 0.36 .P < 0.01). The reliability index was 0.92 [The 17-item Hamilton Depression Rating Scale (HAMD-17) was used to rate the degree of depression with a cutoff score of 17 used to divide the participants into depressed and nondepressed groups. The validity index of the total score was 0.99, and the validity correlation index of the subscales was 0.78–0.98 (was 0.92 .P < 0.01) and 0.97 (P < 0.01)respectively, and the criterion validity was between 0.764 and 0.880 [The Mini-International Neuropsychiatric Interview (MINI) was used to screen for mental disorders . The connd 0.880 .Six questionnaires were used in the interview.The study was approved by the Institutional Review Board of Peking University Sixth Hospital (No. 202046). The Clinical Report Form contained an introduction to the study and informed consent, which stated that the participants joined the study voluntarily. Confidentially was assured. The participants who were not in Beijing verbally agreed to the informed consent by WeChat. No payment was given to the participants for joining this study. All the authors gave their consent for publication.P-values below 0.05 were considered statistically significant.SPSS 26.0 was used to perform the statistical analysis. We compared the variables of demographic data, ACE-IQ, HAMA, HAMD-17, MINI, AUDIT between two groups. Nonparametric tests were used to compare the only continuous variable of age. Chi-square analyses and Z analysis were used to compare the remaining variables. The simple factor binary logistic regression compared risky drinking (AUDIT ≥ 7) as a dependent variable and adverse childhood experience and other variables that showed significant differences between the two groups as independent variables. In a multifactor binary logistic regression, we took all independent variables as covariates. In both regression analyses, we calculated the adjusted odds ratios (OR) and 95% confidence intervals (95% CI) for the associations between risky drinking and adverse childhood experiences. In all analyses, n = 112), and 94.7% were of Han nationality (n = 142). Seven percent graduated from middle school (n = 11) or technical school, and 56.7% graduated or were currently in college or university (n = 85). There were 12% with no formal education and stable job (n = 18), and 43.3% earned less than 5000 RMB per month (n = 65). A total of 66.7% were single or divorced (n = 100) . A total of 74.5% were females . There was statistical difference in attitude to self-drinking between two groups . The “risky drinking” group was more likely to enjoy drinking, while the “non-risky drinking” group was more likely to hate drinking. More “risky-drinking” people had suffered a current major depressive episode , nonalcohol psychoactive substance use disorder , and bulimia nervosa . More people in the “risky drinking” group experienced physical abuse , community violence . All other ACEs and mental disorders screened by MINI showed no significant differences. Comparing the number of ACEs showed that both groups suffered more than one kind of ACE, with most of them having suffered 4 or more ACEs; the risky drinking group suffered more ACEs, but the difference was not statistically significant , and more COA in the same group suffered community violence with frequencies of “a few times” and “many times” . The item “Did you experience the deliberate destruction of your home due to any of these events?” showed a difference between the two groups , because a few people in the risky drinking group suffered the experience one or more times, but no participant in the non-risky drinking group had this kind of experience. There was no significant difference between the two groups in emotional neglect, physical neglect, domestic violence, emotional abuse, sexual abuse or bullying , current major depressive episode , nonalcohol psychoactive substance use disorder , bulimia nervosa , physical abuse , community violence  and collective violence . Adverse childhood experiences showed a graded relationship with “risky drinking” in adult COA. Depression, nonalcohol psychoactive substance use and bulimia nervosa increased the risk of “risky drinking” two to five fold, while physical abuse, community violence and collective violence increased the risk of “risky drinking” two to 11- fold in adults with COA  , it mighThe findings of this study are meant to help clinicians focus more on the family of COA and, for the first time in our country, provide data and a theoretical basis for the needed healthcare, psychological support and societal understanding. Schools and medical professionals need to perform more evaluations of the negative childhood experiences of COA and provide interventions for COA. The implications of this study are particularly strong for Asian cultures where awareness and prevention efforts lag.COA engaging in “risky drinking” experienced more childhood adversities, such as physical abuse and environmental violence, and had more comorbidities, such as depression and bulimia nervosa. On the basis of the high prevalence of ACEs among the whole sample of COA, even after controlling for the covariates, one pattern of ACEs still had an apparent association with risky drinking. In China, this study was one of very few studies that have focused on COA who were at high risk of developing drinking and other mental health problems. In this case–control study, we aimed to understand the impact of factors influencing the lives of COA and explored possible targets for intervention and prevention. Raising awareness of the association between alcoholism and ACEs has significant implications not only in China but in other Asian cultures. It is essential for the prevention of the intergenerational “transmission” of a propensity to pursue “risky drinking” and alcoholism.