A large library of derivatives based on the scaffold of 2-(benzylsulfinyl)benzoic acid were synthesised and tested as atypical inhibitors against four different isoforms of human carbonic anhydrase . The exploration of the chemical space around the main functional groups led to the discovery of selective hCA IX inhibitors in the micromolar/nanomolar range, thus establishing robust structure-activity relationships within this versatile scaffold. HPLC separation of some selected chiral compounds and biological evaluation of the corresponding enantiomers was performed along with molecular modelling studies on the most active derivatives. The researches of the last years showed all the consequences of their altered activity, either in the case of excessive or deficient onesmilieu of hypoxic tumours, promoting drug resistance as well as proliferation, migration, invasion and metastasis of tumour cellsOne of the most studied alterations has been observed in tumours, where some hCA isoforms appear overexpressed; the so-called tumour-related isoforms, hCA IX and XII which are transmembrane enzymes, participate along with cytosolic hCA II in the complex pH machinery which controls the ,out” conformation. This amino acid, which exists in two conformations called “in” and “out”, acts as a proton-shuttle system transferring a proton from the zinc-coordinated water molecule to the environment, to reconstitute the catalytic active form. Interfering with this process  is equal to block or strongly reduce the enzymatic activity, obtaining the enzyme inhibition. Our lead compound was inactive against hCA I and XII (Ki hCA I/XII >10 µM), while showed inhibitory activity in the low micromolar range towards both hCA II and IX, with a slight preference for the former . With this in mind, we developed a novel series of derivatives based on the lead compound scaffold, pursuing some changes on its structure in order to improve activity and selectivity , prompted the discovery of four different mechanisms of inhibition for hCAsectivity .N-methyl amide, hydroxamic acid and ketone. The selected functionalities possess different hydrogen-bond acceptor/donator profiles if compared with the carboxylic acid one; furthermore, except for amide and hydroxamic acid moieties that still conserve a “deprotonatable” group, the others lack this feature whose importance has been in this way deepened.The carboxylic acid moiety red in  was repl3–C5 alkyl chains. The aim of these changes was to understand the importance of the hydrophobic interactions with Phe231 and Asn232 that the phenyl ring established in the hCA II-inhibitor adduct. The addition of substituents on this phenyl ring changed the π-system charge distribution along with its hydro/lipophilicity. On the contrary, moving the phenyl group away from the sulphur atom with the addition of a carbonyl group (in the benzoylmethyl or phenylacetic derivatives) shed light on the importance of this system in that position and its conformational freedom. Attempts with unsaturated alkyl chains with increasing lengths have been done in order to evaluate if the presence of groups, able to give hydrophobic interactions, could work similarly to the phenyl ring. The role of sulphur atom was also challenged ethan-1-one and 2-nitrobenzamide, respectively  at reflux, in the presence of K2CO3. The so obtained compounds (1 and 16) were treated in the next oxidative reaction with meta-chloroperbenzoic acid (mCPBA). Although many routes able to afford sulphur oxidation existIn order to obtain the novel compounds, we followed the synthetic strategies reported in Schemes 1–4. For the synthesis of derivatives ectively ; both of7–12 and 19–21 were obtained with a nucleophilic substitution reaction between methyl 2-mercaptobenzoate and (un)substituted benzyl bromides, followed by the oxidative step. The carboxylic acid derivatives were obtained through the hydrolysis of compound 7 with aqueous 2 N sodium hydroxide (NaOH) in a mixture of equal amounts of water and 1,4-dioxane  to give 4, that was subsequently oxidised to 5 and 6 benzoate, was commercially available). Only two of the E intermediates bearing butyl and pentyl chain respectively (E4 and E5), underwent the oxidative step obtaining the sulphinyl derivatives 22–23. All the E1–E5 intermediates were hydrolysed with aqueous 2 N sodium hydroxide in a mixture of equal amounts of water and 1,4-dioxane . After synthesis completion, the reactions were quenched with hydrochloric acid (HCl), giving the intermediates A1–A5 (2) in the presence of triethylamine (Et3N) excess. Monitoring the reaction with thin layer chromatography (TLC) we detected the disappearance of the acidic intermediate, until the completion of anhydride formation. Then, ammonium chloride (NH4Cl) was added. The presence of the triethylamine excess involved the “de-blocking” of NH3 from NH4Cl, giving the final products .In fact, controlling the amount of oxidant added during the reaction we obtained both the species that were easily separated owing to the different chromatographic profiles. Compounds  5 and 6 . Compounsly seen . The synes A1–A5 . The actAlthough chiral properties are often linked with the presence of a quaternary carbon atom binding different substituents, even the sulphur atom can have chiral behaviour under appropriate conditions.5, 8, 11, 17, 20) obtained in of the synthetic pathway shown in The sulfoxides have a pyramidal structure where one vertex contains the sulphur electron pair, which can be considered as the fourth substituent bound to the sulphur atom. Therefore, if the two groups bound on the sulphinyl moiety are diverse, there is a chiral centre and the presence of two enantiomers. Differing from tertiary amines, which had low pyramidal inversion energy barrier, the sulfoxides possess energy barrier that allows the existence of two stable enantiomers, which can be resolved. Some sulfoxides , nitrogen atom , or alkyl chain . Compounds 39–41 and 43 (anthranilic acid) were commercially available and used as purchased.In compounds 42 was obtained starting from anthranilic acid, which was treated with sodium hydride (NaH) in dry THF. After 10 min, the phenylacetyl bromide was added at room temperature under nitrogen, giving the desired amide  in boiling dry methanol to give the corresponding methyl esters , to afford the desired amides . Analytical thin-layer chromatography was carried out on Sigma-Aldrich® silica gel on TLC aluminium foils with fluorescent indicator 254 nm. Visualisation was carried out under UV irradiation (254 and 365 nm). 1H NMR spectra were recorded on a Bruker AV400 . Chemical shifts are quoted in ppm, based on appearance rather than interpretation, and are referenced to the residual non deuterated solvent peak. Missing signals must be attributed to overlapping peaks. Infrared spectra of the most representative compounds were recorded on a Bruker Tensor 27 FTIR spectrometer equipped with an attenuated total reflectance attachment with internal calibration.Solvents were used as supplied without further purification. Starting materials and other chemicals were purchased by Sigma-Aldrich  and used in the syntheses and in the biological assays without further purification. All synthesised compounds have been fully characterised by analytical and spectral data. Column chromatography was carried out using Sigma-Aldrichmax) are reported in wavenumbers (4000–400 cm−1). Elemental analyses for C, H, and N were recorded on a Perkin-Elmer 240 B microanalyzer obtaining analytical results within ± 0.4% of the theoretical values for all compounds. All melting points were measured on a Stuart® melting point apparatus SMP1 and are uncorrected (temperatures are reported in °C). Where given, systematic compound names are those generated by ChemBioDraw Ultra® 12.0 following IUPAC conventions. Mass spectra on the most representative compounds were performed on a LCQ (Thermo Finnigan) ion trap mass spectrometer  equipped with an electrospray ionisation (ESI) source. The capillary temperature was set at 300 °C and the spray voltage at 4.25 kV. The fluid was nebulised using nitrogen (N2) as both the sheath gas and the auxiliary gas. All the characterisation data for each compound were reported as Supplemental material.Absorption maxima (ν3.2.2 hydration activity4 (for maintaining constant the ionic strength), following the initial rates of the CA-catalysed CO2 hydration reaction for a period of 10–100 s. The CO2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. In particular, CO2 was bubbled in distilled deionised water for 30 min till saturation. A CO2 kit  was used to measure the concentration in serially diluted solutions from the saturated one at the same temperature. For each inhibitor at least six traces of the initial 5–10% of the reaction have been used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (1 µM) were prepared in distilled-deionised water and dilutions up to 0.1 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to assay to allow for the formation of the E-I complex or for the eventual active site mediated hydrolysis of the inhibitor. The inhibition constants were obtained by non-linear least-squares methods using PRISM 3 and the Cheng-Prusoff equation,An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalysed CO3.3.2NH-) and the ligands were energy minimised (MMFF94x force field). All protein structures were obtained from the RCSB protein databank: hCA I , hCA II , hCA IX  and hCA XII . The protein atoms and the active site zinc ions were retained and all other atoms were omitted. The remaining structure was protonated using the protonate 3 D functionality of MOE and subsequently, the obtained structure was energy-minimised (AMBER14:EHT)2+-ions, zinc-binding histidines and the overall backbone atoms superposed well . Docking calculations were performed using the FlexX docking tool  within MOE. The binding pocket was defined as all residues within 6.5 Å of the reference ligand acetazolamide. The sulphonamide tail of the ligands was forced to adopt a similar orientation and interactions to the Zn2+ ion as observed for acetazolamide using a pharmacophore model. All ligands were docked fifty times and the best scoring three poses were subjected to refinement calculationsThe three-dimensional structures of all ligands were prepared in their lowest energy conformation using the MOE software package . The sulphonamide nitrogen atoms of the ligands were assigned a negative charge (R-SO4.Ki >100 µM) and some of them were more active against the tumour-related isoform hCA IX if compared with the parent drug 2-(benzylsulfinyl)benzoic acid  and was ineffective against the two off-targets (Ki hCA I/II >100 µM). The oxidation to sulfoxide (2) impaired the selectivity “restoring” the inhibitory activity owned by the lead compound for hCA II, although in the high micromolar range, while the affinity towards hCA IX  was slightly inferior than 1. The sulphone 3 displayed affinity exclusively for hCA IX, although weaker than 1 and 2 .All the tested compounds had no affinity for the common off-target hCA I isoform  but still conserving the selectivity (Ki hCAI/II >100 µM). The oxidation to sulfoxide ((R/S)-5) and sulphone (6) improved inhibitory activity in the low micromolar range against the tumour-related isoform. However, while 6 bearing the sulphonyl moiety was inactive towards the two off-target actings selectively against hCA IX , the two sulphinyl enantiomers of 5 exhibited their preference for hCA II ((R)-5, Ki hCA II = 0.21 µM; (S)-5, Ki hCA II = 0.093 µM) rather than hCA IX ((R)-5, Ki hCA IX = 1.4 µM; (S)-5, Ki hCA IX = 1.9 µM). Comparing the inhibition data against hCA II of 5 enantiomers, it was quite clear the preference of the enzyme for (S)-5, which was the eutomer. Indeed, after incubation with the racemic mixture of 5, only the adduct between hCA II and (S)-5 was observed7–9 were endowed with a methyl ester functional group, keeping constant the benzyl group bound to the sulphur atom (in the different oxidation states). These derivatives showed reduced inhibitory activity against hCA IX, regardless the sulphur oxidation number. Furthermore, compounds 7 and (R/S)-8 were also weakly active against hCA II (65.7 < Ki hCA II (µM)<84.9). The unfavourable effect of the additional methyl group was mitigated by the insertion of a chloro atom at the para-position of the phenyl ring (10–12); this substitution counteracted the detrimental effects of the methyl group, probably enforcing other interactions inside the enzyme. Compound 10 was a good inhibitor of hCA IX exhibiting Ki against hCA IX of 1.0 µM, with a high micromolar residual activity against hCA II (Ki hCA IX = 45.7 µM). The oxidation to sulphinyl group ameliorated selectivity, because the two enantiomers (R/S)-11 were ineffective against both of hCA I and II (Ki hCA I/II >100 µM). These two optical isomers showed different affinity towards hCA IX, with the (R)-enantiomer that inhibited this isoform better than the (S)-one ((R)-11, Ki hCA IX = 1.4 µM; (S)-11, Ki hCA IX = 18.1 µM). The further oxidation to sulphone (12) gave a detrimental effect, reducing both activity against hCA IX (Ki=21.1 µM) and selectivity (Ki hCA II = 40.1 µM). The formal insertion of a carbonyl moiety between the methylene group and the phenyl ring in order to move the latter away from the sulphur atom, led to the 13–15 derivatives. The presence of this benzoyl moiety was not tolerated by this scaffold, giving compounds which did not exhibit effects towards all the tested isoforms (Ki>100 µM).The replacement of 16–18). Compound 16, containing a non-oxidised sulphur atom, was the best inhibitor of the series against hCA IX displaying Ki of 0.046 µM. This result was slightly impaired by the oxidation to sulphinyl derivatives ((R)-17, Ki hCA IX = 2.2 µM; (S)-17, Ki hCA IX = 1.9 µM), while the corresponding sulphone (18) completely lost the affinity for hCA IX. All the amidic derivatives showed affinity for hCA II, albeit the sulphide (16) and sulphone (18) inhibited this isoform better than the sulphinyl enantiomers, which exhibited Ki values around 50 µM . The presence of a methyl group on the amidic moiety was detrimental for the inhibitory activity towards hCA IX. Indeed, the derivatives 19–21, endowed with monomethyl amide, were less effective against the cancer-related isoform (19.8 < Ki hCA IX (µM) <25.8) than the analogues containing primary amide. Furthermore, the activity against hCA II was also lowered in the high micromolar range (36.9 < Ki hCA II (µM)<86.3). This behaviour was very similar to that observed for molecules containing a methyl ester moiety, which were less effective compared with their carboxylic acid analogues.The consequence of the amidic moiety insertion was also evaluated (22–38) and testing the influence against the two off-target hCA I and II, and the two tumour-related isoforms hCA IX and XII. The first attempts were done with the two compounds 22 and 23. These derivatives, bearing methyl ester with the sulfoxide group linked to butyl and pentyl chain respectively, were inactive against all the tested isoforms, underlying the detrimental effects of this structural combination. However, better results were obtained when the amide moiety was placed instead of methyl ester one (24–38). The derivatives 24–38 displayed their effect exclusively against hCA IX, without any inhibitory activity against hCA I, II and XII. Only for derivative 30, bearing propyl chain bound to the sulphur atom, we observed activity against hCA II, but with a very low affinity (Ki hCA II = 90.9 µM). Compound 32, the sulphone analogue of sulphide 30, was the best inhibitor against hCA IX among the compounds substituted with alkyl chains, inhibiting the cancer-related isoform with a Ki value of 2.3 µM. Other good results were found with 33 and 36, containing a sulphur atom and bearing butyl or pentyl chain, respectively . Except for derivatives with propyl chain (30–32), the compounds containing the non-oxidised sulphur atom showed better inhibitory activity than the related sulfoxide and sulphone analogues (39–53), keeping constant the functional groups constituting the scaffold seen above . Some of the obtained compounds showed structures completely comparable to the lead compound . As one can see from The effect and the importance of the phenyl moiety were also challenged by substituting it with linear alkyl chains of increasing length , has an own steric hindrance along with specific bond angles and a stable dipole; all these features could affect negatively the interaction with the isoform IX. Finally, the substitution of the sulphur atom with oxygen, nitrogen one, or methylene underlined the importance of the sulphur atom for the activity of these compounds, because all the obtained derivatives were inactive against all the tested isoforms. Furthermore, some selected compounds were also tested against hCA XII to preliminary provide information on this target. All the derivatives, except compounds 5.5.1.KI value for hCA IX was measured for compound 16 (Ki = 46 nM), which is lower compared to the reference compound acetazolamide (Ki = 250 nM). Two different binding interactions with the hCA IX active site were suggested. In the first docked pose, the ligand directly formed an interaction with the Zn2+-ion via its carbonyl group  can also adopt poses in which a direct interaction with the Zn2+ is possible. In the second docked pose, the ligand carbonyl group formed a hydrogen bond to the zinc-bound water molecule  and the second-lowest Ki value was measured for compound 16 (Ki = 2.66 µM). Two docked poses have been obtained for these compounds: both formed an interaction with the Zn2+-ion via the ligand carbonyl groups benzoic acid, an innovative and atypical hCA inhibitor. We have designed, synthesised, and tested 53 derivatives against the most important hCA isoforms establishing robust SARs within this scaffold. Most of them were selective hCA IX inhibitors in the low micromolar/high nanomolar range. The stereochemistry of the sulfoxide group had no impact on the biological activity, whereas the isosteric replacement of the sulphur atom with oxygen, nitrogen or methylene led to a total loss of activity. Molecular modelling studies demonstrated different binding poses of the inhibitors within the active site, differently from that observed for the parent compound.


Despite renewed focus on biomedical prevention strategies since the publication of several clinical trials highlighting the efficacy of pre-exposure prophylaxis (PrEP), knowledge of postexposure prophylaxis (PEP) and PrEP continues to remain scarce among women, especially among African American women who are disproportionally affected by HIV. In an effort to address this barrier and encourage uptake of PEP and PrEP, an electronic health  video was created using an entertainment-education format.The study aimed to explore the feasibility, acceptability, and preference of an avatar-led, eHealth video, PEP and PrEP for Women, to increase awareness and knowledge of PEP and PrEP for HIV in a sample of African American women.A cross-sectional, Web-based study was conducted with 116 African American women aged 18 to 61 years to measure participants’ perceived acceptability of the video on a 5-point scale: poor, fair, good, very good, and excellent. Backward stepwise regression was used to the find the outcome variable of a higher rating of the PEP and PrEP for Women video. Thematic analysis was conducted to explore the reasons for recommending the video to others after watching the eHealth video.P=.004), not having unprotected sex in the last 3 months , higher income , lower level of education , and lower exposure to sexual assault since the age of 18 years . After watching the eHealth video, reasons for recommending the video included the video being educational, entertaining, and suitable for women.Overall, 89% of the participants rated the video as good or higher. A higher rating of the educational video was significantly predicted by: no current use of drugs/alcohol (beta=−.814; Utilization of an avatar-led eHealth video fostered education about PEP and PrEP among African American women who have experienced insufficient outreach for biomedical HIV strategies. This approach can be leveraged to increase awareness and usage among African American women. African American women are disproportionately affected by HIV in the United States, with 91% of new infections among this population being attributed to heterosexual transmission . IncreasFor sexually active individuals, the use of the male condom has remained the most cost-effective and readily accessible prevention tool since the start of the AIDS epidemic . The useLittle is known about the interest of biomedical prevention strategies among African American women ,11, suggRecent advances in eHealth have enabled the development of innovative HIV prevention strategies that have created a large number of opportunities for connecting to others to improve health and awareness about HIV prevention -19. eHeaGrimley and Hook tested a computer-delivered intervention with primarily African American females who were sexually transmitted infection (STI) clinic patients . At the Sisters Informing Healing Living and Empowering (SiHLE), a 4-session CDC-DEBI group-level HIV prevention intervention for teenage African American females aged 14 to 18 years, was translated into a 2-hour computer-delivered individual intervention called multimedia SiHLE . The resPEP and PrEP for Women video, which was designed to increase the knowledge of PEP and PrEP using entertainment-education format eHealth targeting African American women.Despite the fact that computer-based interventions have been efficacious among African American women , currentThis study was guided by the social cognitive theory through the use of the entertainment-education communication strategy -36. The Entertainment-education narratives exert influence by reducing the audience’s resistance to health messages . The cenPEP and PrEP for Women eHealth video study is intended to engage learners through digital storytelling and critical thinking on issues about (1) HIV risk, (2) sexual violence, (3) relationship dynamics, (4) patient-provider communications, (5) initiation of PEP and PrEP, and (6) female-controlled HIV prevention methods. A collaborative process among the research team members resulted in an initial 9-min and 3-second script that was based on a review of the HIV risk literature on African American women and their male sexual partners [In our application of the social cognitive theory framework and entertainment-education narratives, the cations,  initiatipartners ,41.This study utilized data collected over a 6-week period (February and March 2015) for a cross-sectional health survey conducted on the Web. Participants were recruited through a social marketing campaign, which included Web recruitment via social media, flyers posted in community settings, and snowball sampling. Web-based venues used in Web recruitment included Facebook, Twitter, LinkedIn, and Craigslist. Flyers were posted in community venues that African American women frequent . As this is a historically difficult-to-reach population, snowball sampling was used to recruit African American women . Study eThose who were deemed ineligible were thanked for their time and exited the site. Eligible participants were directed to the Web-based consent form, which outlined the study’s purpose, risks, and benefits followed by a prevideo survey, a 9-min eHealth video (PEP and PrEP for Women), and a brief postvideo survey. Those who completed the study were entered into a random drawing to receive an Amazon gift card  and asked to forward the link to potential eligible participants in their network. The institutional review board at Teachers College Columbia University reviewed and approved all study procedures.PEP and PrEP for Women eHealth video.We assessed a comprehensive range of sociodemographic variables, including age, employment status (unemployed vs employed), annual income level , education level (high school/GED or less vs some college vs college or more), current student (yes/no), relationship status (single vs partnered), and gender of current sexual partners . We also assessed previous knowledge of both PEP and PrEP. Additional information related to sexual risk behaviors including drug/alcohol use, drug/alcohol use during sex, HIV testing history, relationship dynamics , condom PEP and PrEP for Women eHealth video was created using Goanimate Technology [The 2-part chnology . Goanimachnology . Researcchnology  to facilPart 1 of the video addresses a storyline that warrants the initiation and use of PEP. In scene 1, the main character, Tania, informs her friend, Michelle, that she has been sexually assaulted . In scenPart 2 addressed a storyline that warrants the use of PrEP to prevent HIV transmission. In scene 4, we introduce 2 new characters for this narrative, Kia and Jackie. The main character Kia has a discussion with her friend, Jackie, about her boyfriend Mike . Mike haAfter the participants watched the video, we conducted a brief survey to measure the participant’s perceived acceptability of the video on a 5-point scale. First, we accessed their dose of exposure to the video to identify if the participants watch all, most, some, or none of the video. Second, we asked participants to rate the video with the following response anchors: poor, fair, good, very good, or excellent. Next, participants were asked if they would recommend the video to other women. A dialogue box was provided to ascertain why or why not they would share this video and information with other women. Finally, they were asked about their intentions to use PrEP and PEP in the future, if needed, and if they would recommend PrEP and PEP to other women.PEP and PrEP for Women eHealth video. To refine our model to exclude insignificant or highly correlated variables, backward stepwise regression was used. The rationale for using this approach was based on the work by Mantel who explained that backward selection serves to reduce the degrees of freedom, has joint predictor capability, and removes noise caused by including unrelated variables or variables that may be highly correlated with each other [P value) being removed when the model was refitted. Then, a new model is built in the absence of that 1 independent variable and the evaluation process is repeated again, removing the least significant variable. This removal process and the equation-reconstruction process were continued until only significant independent variables (P<.05) remained—as the final model reported for the backward stepwise regression.Quantitative data were downloaded from SurveyMonkey, transferred to SPSS, and analyzed using SPSS 21 . Descripch other . This wach other , (21) bech other , and  derived from relevant literature pertaining to entertainment-education narratives [opencode). Next, the open-coded data were organized under and integrated into the closed code list. Third, the data under each thematic code  were reread, and, if needed, recoded into subcategories to refine the analytic categories used. As the themes of the responses emerged, special attention was paid to data that did not confirm emerging themes, noting these for exploration in future research. In addition to using these methods of analysis, we also ensured analytic rigor by engaging several peer reviews of early analytic claims [Analyses of the open-ended questions were thematic, focusing on dominant themes that emerged and were organized by the question asked; attention was paid to the level of endorsement of a theme across the sample. For the qualitative analysis, a grounded theoretical approach was used, permitting the use of categorizing strategies to code and analyze the data ,49. Firsrratives . Second,c claims . For thiIn our sample (N=116), the mean age was 34 years. Most women were born in the United States , identified as black or African American , were in a relationship , and were intimate with male partners only . Approximately one-third  of them had a high school education, and a similar proportion  of them were current students. The majority  of them reported either current full-time or part-time employment. Almost half  of them reported a household income of US $50,000 or more. Before watching the video, only 18% (21) of the participants reported that they heard of PrEP and 22% (26) reported that they heard of PEP .Overall, 78% (91/116) of the eligible participants watched all of the video . In addiOverall, 89% (81/91) of the participants rated the video as good or higher; 29% (26) of them rated the video as excellent, 32% (29) rated the video as very good, and 29% (26) rated the video as good. Only 10% (9) of participants rated the video as fair and 1% (1) rated the video as poor. The mean rating for the video was very good . Among the sample, 91% (83) of participants reported that they would recommend the video to others, whereas 9% (8) of them reported that they would not recommend the video.After watching the video, 97% (88/91) of participants reported that they would seek out PEP if they felt that they might have been exposed to HIV, including taking the recommended oral medication for 28 days. In addition, 93% (85) of them reported that they would recommend to another woman to seek out PEP if they knew the woman might have been exposed to HIV. Regarding the use of PrEP, after watching the video, 76% (69) of them reported that they would seek out PrEP. In addition, 91% (83) of participants reported that they would recommend to another woman to seek out PrEP .P=.004), using condoms all the time in the past 3 months , a higher household income , lower education level , and less experience of sexual abuse as an adult . For this model, the adjusted R square was .224 , meaning that 22.4% of the variance was explained by this regression model . The backward stepwise regression analysis showed that the 5 variables that best predicted a high rating of the video were not engaging in any current drug use  of the participants expressed that they would recommend the video to other women. Our analysis yielded 3 themes about the eHealth video, which were (1) educational, (2) entertaining, and (3) empowering .at-risk scenarios were relatable to women’s current relationship situations, and it may encourage them to change their behaviors. Other participants commented that the video was nice to watch and entertaining. For the third theme, empowering, subthemes covered 3 areas: (a) female-controlled HIV prevention methods, (b) culturally relevant, and (c) heightened perception of risk. The participants reported that they would recommend the video because it provided information on an HIV prevention method that was female controlled. In addition, the video was considered to be culturally relevant based on the scenarios depicted in the video, and it was not judgmental. Finally, the participants reported that the video provides scenarios that would heighten women’s awareness of their own risk because of their partner’s behavior in sexual relationships.Under the first theme, educational, subthemes covered how the video provided awareness or knowledge of PEP and PrEP and how it was adaptable. The participants reported that they were not aware of PEP and PrEP before watching the video and that the video was informative and easy to follow. For the second theme, entertaining, subthemes covered how the video had characters that were relatable, and the information was provided via a video with a storyline and characters. The participants reported that they would recommend the video because the In addition, 8 participants reported that they would not recommend the video to other women (information was excluded from the table). There were 3 emergent themes, including subthemes. First, participants expressed criticism of the script—with the subtheme of their identification with all of the characters. One participant wrote regarding the characters in part 2:I would prefer more back and forth between women who are considering it, not a doctor promoting it.Participants stated that they would have preferred for the health information to not be delivered by the doctor character in the video. Second, participants reported problems maintaining attention because of the length of the video:Although this video is very informative, it is too long. I doubt people will watch the whole thing.Third, participants expressed a preference for other modes of communication—with subthemes of age appropriateness and video format. The participants reported that the animated format was too cartoonish and would be more appropriate for younger women. As expressed by 1 of the participants:While it was educational, I feel the avatar format is better suited for middle-older adolescent/college age women. The video was done very well though.edutainment) and engaging, while having diverse characters suited for connecting to African American women [PEP and PrEP for Women, the viewer learns through a conversation between 2 friends (Kia and Jackie) that the main character, Kia, does not have to rely on her male partner to use condoms to prevent HIV. In this scene, Kia learns about PrEP from her friend, and she is referred to a clinic that provides PrEP. The video does not attempt to answer the question of personal responsibility or criticize complex relationship dynamics that influence HIV risk, but rather it increased the viewer’s knowledge and awareness of an alternative HIV prevention option via involvement with the story characters [This intervention aimed to increase the knowledge and initiation of PEP and PrEP among African American women by modeling situations and discussions that would be relevant to the target population. The goal was to also make the video entertaining , the vast majority found the avatar video product to be informative, accessible, and engaging. Those individuals who have a positive response to the avatar video were willing to adopt the innovation and engage in the diffusion of it within their social networks, which includes disseminating on social media networking sites. Studies have shown that HIV prevention programs grounded in the diffusion of innovation theory can be useful for increasing HIV/AIDS risk reduction knowledge among early adopters and diffusing information throughout social networks; therefore, preventing HIV among vulnerable populations . This stFinally, this study offers recommendations and suggestions for Web-based research as well as Web-based recruitment using modern-day social networking. Although the sample may not be representative of all African American women in the United States with respect to socioeconomic status, this strategy allowed for rapid recruitment of the sample population with minimum resources, such as staff and funding. Furthermore, this strategy potentially provides opportunities not only for recruitment purposes but for eHealth intervention purposes. Researchers and practitioners should consider the benefits of not only Web-based survey research but also of Web-based health education, health promotion, and disease prevention to reach more diverse samples of subpopulations affected by the HIV epidemic. eHealth intervention has proven to be extremely useful in educating African American women on a number of topics related to HIV/AIDS or STI prevention. Web-based health education can be beneficial for African American women if they use the internet to access and share information with their peers. In addition, as previous research studies have shown, using eHealth interventions may help reach a subgroup of African American women who are not usually addressed in the literature pertaining to HIV risk and prevention .This study had some limitations. First, this was a small, cross-sectional, Web-based sample of African American women, thus making it difficult to reach generalizations from this data source. Second, internet access familiarity with Web-based technology may have influenced the decision to participate in the study. Women who use a different type of electronic device  to access the internet may be able to navigate websites easily and may encounter challenges with technical issues. Third, the video and script were not previously tested with different groups of African American women. Having focus groups or concept-mapping sessions before the launch of the study would have enhanced the script of the video for tailoring of the message. Future research should involve conducting focus groups with different age groups, socioeconomic statuses with regard to the scripting, video display, and avatar characters to reach a broader audience of African American women. Fourth, given that this is a feasibility and acceptability study, it is not possible to make any conclusion regarding the efficacy of the video. In addition, participants’ responses to PEP and PrEP adoption intentions were based on a hypothetical situation rather than their actual experience. Increasing knowledge does not necessarily correlate with motivating behavioral changes. Finally, the results could have been influenced by social desirability bias . To miniPEP and PrEP for Women video using eHealth and entertainment-education health message narratives for a targeted, culturally centered, HIV/AIDS preventative education intervention for African American women can be readily adopted and diffused as an innovation, while still using low-cost technology in the public domain. Findings from this eHealth video study demonstrated that (1) a culturally tailored eHealth video could be used to increase awareness of HIV prevention methods and (2) it is feasible to recruit and engage African American women into an eHealth intervention that is delivered via the internet and social media. The PEP and PrEP for Women video was viewed as culturally centered Web-based content by the participants and capitalized on the widespread use of the internet to reach a population that is not only underserved in eHealth interventions but also in traditional HIV research targeting African American women, therefore, adding to the limited body of research on computer-mediated HIV interventions targeting African American women [This study has demonstrated that the ted that  a culturan women . The eHean women .Given the disproportionate impact of the HIV epidemic on African American women, the lack of awareness of both PEP and PrEP , and poo


Differentiating carotid near-occlusion  from conventional stenosis is the first step when grading carotid stenoses with NASCET method. The internal carotid artery (ICA) can be asymmetrically associated with Circle of Willis variations. When such ICA asymmetry coincides with stenosis, it may mimic near-occlusion. We studied ICA anatomical variant prevalence in 4042 consecutive CTA exams from all indications, 53 excluded due to carotid occlusion, 814 with any ≥ 50% steno-occlusive disease intra- or extracranially, 3228 without. Of the 3989 included cases, 568 (14%) had ICA asymmetry, of which 335 (59%) were from associated with Circle of Willis variations. Of 3228 patients without ≥ 50% stenosis or other steno-occlusive disease intra- and extracranially; 257 (8.0%) demonstrated ICA asymmetry associated with Circle of Willis variations, equally common among sexes and age unrelated and most frequently attributed to an ipsilateral A1 hypoplasia/aplasia, less often attributed to large contralateral posterior communicating artery. As ICA asymmetry associated with Circle of Willis variations are common, caution should be exercised diagnosing near-occlusion on asymmetry alone. When grading carotid stenosis with NASCET-method, the first step is to exclude near-occlusion with or without full collapse by determining that there is distal internal carotid artery (ICA) luminal reduction caused by proximal ICA stenosis –3.In prognostic and management studies, separation of near-occlusion and conventional ≥ 50% carotid stenoses is made by assessing several features of near-occlusion –5. KoskiOne observer (EJ) assessed a 5-year consecutive sample of 4402 CTA exams from 4042 consecutive patients aged ≥ 18 years sent to or performed at the University Hospital of Northern Sweden, Umeå, Sweden. CTA protocols varied across 12 sites, reflecting clinical practice. Cases with extracranial carotid occlusion were excluded. A blinded second observer (AF) assessed all cases with stenosis requiring separation between near-occlusion and ICA anatomical variants. Disagreements were settled by consensus. The most representative exam was used for cases with several exams. Demographic data obtained was age and sex. Artery diameter measurements  and CoW evaluations were performed for all cases with any ICA asymmetry or ≥ 50% carotid stenosis and randomly selected controls with ICA symmetry and < 50% carotid stenosis. The study was approved by the ethical review board in Umeå, Sweden, with the need for informed consent waived due to the observational nature of the study.ICA asymmetry was defined as a visible side-to-side difference in extracranial ICA diameter well beyond the bulb (approximately at the C2 vertebra level) caused by a pathology or anatomical variance proximal and/or distal to this artery segment. Causes of ICA asymmetry were categorized as the following: near-occlusion, distal disease , ICA hypoplasia , CoW anatomical variance and uncertain. CoW anatomical variance co-existing with moderate carotid stenosis was separated from near-occlusion by systematically interpreting several features of near-occlusion: ICA size, stenosis severity, and ICA/external carotid artery ratio , 8. Apprt test, and χ2 test using SPSS 24.0.The side with the smaller ICA was considered the index side. We used 95% confidence intervals (95%CI), mean, standard deviation (±), n = 335), near-occlusion , distal disease , ICA hypoplasia , and uncertain .A total of 3989 cases without extracranial occlusion were included, 53 occlusions excluded. There were 568 cases with asymmetric ICAs, associated with CoW anatomical variants  had conventional stenosis and symmetric ICAs, 78 (12%) conventional stenosis and ICA asymmetry associated with CoW anatomical variants , 155 (24%) near-occlusion, and 34 (5%) ICA asymmetry of uncertain cause.p = 0.17) or mean age differences . When grouped by decade, ICA anatomical variants were similarly common in all groups (range 6.3–10.8%). In these 257 asymmetric cases, mean smallest ICA diameter was 3.6 ± 0.5 mm, compared to 4.1 ± 0.5 mm in controls (p < 0.001). An ICA ratio ≤ 0.88 was 98% sensitive and 99% specific for separating the 257 asymmetric and 257 control cases.Of 3228 cases without any ≥ 50% steno-occlusive disease, 257  had asymmetric ICA associated with CoW anatomical variants. There were no sex  had a ≥ 1.0-mm side-to-side difference in ICA diameter, which was also seen in 93% of near-occlusions. CoW anatomical variants were also noted in 32% of controls with symmetric ICAs and in 50% of near-occlusions  is insuCoW anatomical variance was also quite common among controls without ICA asymmetry. However, a small ipsilateral A1 seemed to be more strongly associated with ICA asymmetry compared than a large contralateral Pcom Fig. .In near-occlusion, asymmetric CoW is often noted, presumably caused by pathology , whereas in CoW anatomical variations, the cause is presumably congenital (hypoplasia/aplasia). Since the mechanism of CoW appearance  is very difficult to determine with CTA, CoW appearance is likely of limited value when distinguishing between these causes if ICA asymmetry; rather, a systematic approach of several extracranial features seems reasonable , 8. GiveICA anatomical variance seemed to be more common when coinciding with a stenosis than when not, especially ipsilateral stenosis. This was possibly caused by our conservative estimate of near-occlusions, categorizing some actual near-occlusions as caused by anatomical variance. This is sometimes a difficult determination, with some cases eluding certain diagnosis.Limitations of this study are non-uniform exam protocols although protocols are reflective of standard clinical practice and therefore generalizable to a broader population. Additional limitation is lack of postoperative imaging that otherwise might improve the certainty in near-occlusion  and near-occlusion mimic . Visual assessment was used to determine if a case was asymmetric as there is no well-accepted measured threshold for ICA asymmetry; however, the outcome (a ≤ 0.88 ICA ratio) was very similar to a previous study assessing near-occlusion (≤ 0.87 ICA ratio) [In summary, ICA asymmetry associated with CoW anatomical variation commonly mimic near-occlusion when coinciding with stenosis. Thus, when determining if carotid stenosis is a near-occlusion , it is important to not rely solely on ICA asymmetry but interpret several features of near-occlusion. It seems disappointing that a measurement or measurements cannot be recommended to solely diagnose near-occlusion .


Autonomous sensory meridian response (ASMR) is a sensory phenomenon commonly characterized by pleasant tingling sensations arising from the back of the head and accompanied by feelings of relaxation and calmness. Although research has found ASMR to have a distinct physiological pattern with increased skin conductance levels and reduced heart rate, the specific tingles felt in ASMR have not received much investigation. The aim of the present study was to investigate the physiology and characteristics of ASMR further by examining whether experiencing ASMR is visible from the pupil of the eye. A total of 91 participants were recruited and assigned to three different groups based on their experience of ASMR (ASMR vs. non-ASMR vs. unsure). Participants were instructed to watch a control video and an ASMR video and to report any tingling sensations by pressing down a button on the keyboard. Pupil diameter was measured over the duration of both videos using a tower-mounted eye tracker. Data was analyzed on a general level, averaging pupil diameter over each video, as well as on a more specific level, comparing pupil diameter during reported episodes of tingling sensations to pupil diameter outside of those episodes. On the general level, results revealed no significant differences between the groups. On the specific level, however, the tingling sensations experienced in ASMR were found to cause statistically significant increases in pupil diameter, demonstrating that they have a physiological basis. The results of the study further reinforce the credibility of ASMR and suggest that the tingles felt in ASMR are at the very core of the experience itself. Autonomous sensory meridian response (ASMR) refers to a sensory phenomenon that has received a large amount of popular attention over the past few years. The first scientific study on the topic defined experiencing ASMR as static-like tingling sensations felt on the skin accompanied by a mix of positive feelings and relaxation . The tinASMR has been studied in relation to various other sensory phenomena, including synesthesia, misophonia, and aesthetic chills. One study found that ASMR participants showed a slightly greater prevalence of synesthesia than the general population . Other sEven though ASMR has gained in popularity and research into ASMR has been done to some extent, debate whether it is a real phenomenon still exists. In a study of how expectancy effects influence ASMR, participants previously unfamiliar with ASMR were more likely to experience ASMR after their expectations were manipulated . ASMR paDue to the controversial nature of ASMR, researchers have become increasingly interested in whether the subjective experience of ASMR can be linked to more objective physiological measures. A study by Poerio et al. found that ASMR videos promoted feelings of both calmness and excitement, while reducing heart rate and causing elevations in skin conductance levels . ContrarThe underlying mechanisms in ASMR are still unknown. The increased activation of brain areas related to social engagement observed in ASMR as well as the commonly social nature of ASMR videos has led to suggestions that ASMR may be similar to social grooming , 11, andEye-tracking research has become increasingly popular over the years due to its potential in exploring various different topics as well as its noninvasive nature. As proposed by Daniel Kahneman and later reinforced by a multitude of studies, the pupil of the eye expands in response to increased mental effort –15. OtheThe aim of the present study was to examine the physiological profile of ASMR further. Using eye tracking, we sought to investigate ASMR by measuring pupil diameter while simultaneously inducing it in participants. We wanted to know whether experiencing ASMR in general and during tingling sensations was visible in pupillary changes. In order to accomplish this, we obtained real-time pupil diameter recordings during both an ASMR and a control video from participants who experience ASMR, participants who do not experience ASMR, and participants who are unsure about their ASMR experience. By allowing participants to report tingling sensations via a button press, we were able to further extract recordings of pupil diameter during the actual tingling sensations felt in ASMR. Data was analyzed on a general level, examining pupil diameter over the whole duration of the videos, as well as on a more specific level, examining pupil diameter during the tingles themselves. We expected participants who experience ASMR to show a greater average pupil diameter increase from the control video to the ASMR video when compared to participants who report to not experience ASMR. Moreover, we expected pupil diameter during reported episodes of tingling sensations to be greater when compared to pupil diameter outside of those episodes.A total of 91 participants were recruited for the experiment through posters and flyers spread around the campus, social media recruitment channels, and mailing lists. Sixty-three participants were female while 28 were male. Mean age was 25 years with a range of 18 to 60. All participants provided informed consent and were comfortable watching a computer screen at a distance of 57 cm without the need for eyeglasses. Participants were assigned to either the ASMR group, the non-ASMR group, or the unsure group based on their response to the question “Having watched these videos or just from your everyday life, would you classify yourself as someone who experiences ASMR?”. Out of the 91 participants, 37 reported to experience ASMR, 35 reported to not experience ASMR, and 19 reported that they were unsure whether they experience ASMR. The study was approved by the local ethics committee of the Faculty of Social and Behavioural Sciences of Utrecht University (FETC18-089).The experiment involved two short videos and a questionnaire for each participant. All participants first viewed the same two videos in a counterbalanced order and then responded to the same questionnaire and were assigned to their respective group after completing the experiment. Participants were measured on three different variables over the course of each video: pupil diameter, the number of tingling sensations, and the total duration of tingling sensations.https://www.youtube.com/watch?v=B8jUVci17vE). In order to further assess pupillary reaction in response to differing levels of lighting, each participant was shown an additional five-second presentation of a static grey, black, and white image.Two videos were used for the experiment: an ASMR video and a control video. The video used as the ASMR video was a popular ASMR video with over 15 million views on YouTube. It was chosen due to its popularity, static background, static colors, and constant lighting. Permission was obtained by the video creator, and the video was downloaded and edited down to an appropriate length of two minutes. The edited video portrayed a woman providing personal attention toward the viewer in the form of whispering, speaking in a soft voice, hair brushing, and some hand movements. Since different lighting conditions are known to impact pupil diameter , 24, we Pupil diameter was measured using the SensoMotoric Instruments (SMI) Hi-Speed tower-mounted eye tracker. Pupil diameter and gaze position was recorded from the right eye at a sampling rate of 240 Hz for the duration of each video and presentation of the static images. All videos and static images were presented at a distance of 57 cm on a 19-inch HP monitor using a resolution of 1280 x 1024 pixels. Gaze position was recorded in horizontal and vertical coordinates. Pupil diameter was recorded in pixels and converted into millimeters using the recording of a printed-out black circle with a diameter of five millimeters to determine the conversion ratio (1 mm = 17.2 pixels).Participants were instructed to press down a button whenever they experienced tingling sensations anywhere in their body during each video. The start point (i.e. when the button was pressed down) and end point (i.e. when the button was released) of each button press was recorded to identify and extract episodes of tingling sensations as well as to count the number and duration of the tingling sensations felt by each participant.At the end of the experiment, each participant was given a questionnaire with a variety of questions related to their demographic, experience of the videos, and experience of ASMR in general. An overview of responses to the questionnaire is presented in the supporting information .Testing of participants took place at the laboratories of the Department of Psychology at Utrecht University. Each session began with a brief oral explanation of the experiment process provided by the experimenter. After providing informed consent, participants were asked to place their head in an appropriate way on the tower-mounted SMI eye tracker and put on a pair of Bose QuietComfort 25 noise cancelling headphones. Prior to the presentation of the videos, each participant completed a nine-point gaze calibration procedure. After successful calibration, an instruction screen was presented. Participants were instructed to keep their head as still as possible, to refrain from speaking, to fixate their gaze at the center of the screen, and to report any tingling sensations felt anywhere in the body by pressing down a specified button on the keyboard. The instruction screen was followed by a static grey image for a duration of five seconds, after which one of the videos was played. To avoid potential effects that might result from the presentation order of the videos (e.g. participants who saw the control video first might experience the ASMR video in a different way than participants who saw the ASMR video first), the order of the videos was counterbalanced so that half of the participants saw the ASMR video first while the other half saw the control video first. The first video was always followed by a subsequent five-second presentation of a static black image immediately followed by a static white image. The second video was played directly after the static white image. After finishing the second video, participants were asked to fill in a questionnaire with items related to their demographic, experience of the videos, and experience of ASMR in general. Upon completion of the experiment, participants were offered an adequate reward as compensation.Pupil data from two participants failed to record due to technical problems with the eye tracker. These participants were excluded from all of the following analyses. For the eye-tracking data of each participant, periods of eye blinking were identified as instances of missing gaze data. Upon examination of the data around these periods, we noticed that the velocity of pupil diameter change was greatly increased in their immediate proximity, likely representing measurement error during the closing and opening of the recorded eye. In attempt to remove these instances of high velocity pupil change, the periods of missing data were expanded on both sides, meaning that four samples of data  both before and after each missing data point were removed. Total data loss percentage for each participant was determined by calculating the amount of data loss due to missing gaze data and adding the additional data loss percentage of the remaining data due to the expansion of missing data points to that value. Participants with over 50 percent data loss were excluded from the analyses. The cleanup process was automated using MATLAB, and all subsequent analyses were conducted using IBM SPSS Statistics 25.0. The final sample used for the analyses consisted of 30 ASMR participants, 33 non-ASMR participants, and 19 unsure participants.F = 0.64, p = .531 for the horizontal signal and F = 1.29, p = .281 for the vertical signal). To further confirm that participants showed similar pupillary responses to different levels of lighting, we ran a 3 x 3 repeated measures ANOVA with group type as the between-subjects variable, static image type (grey vs. black vs. white) as the within-subjects variable, and average pupil diameter during the static images as the dependent variable. A total of four participants did not have data for at least one of the image types and were therefore excluded from the analysis. The interaction between static image type and group type was not found significant, F = 0.93, p = .448.To determine whether participants in the three groups showed different patterns of gaze fixation, the standard deviation of the horizontal and vertical gaze signal for each participant was calculated. A 3 x 2 repeated measures analysis of variance (ANOVA) with group type (ASMR vs. non-ASMR vs. unsure) as the between-subjects variable, video type (ASMR vs. control) as the within-subjects variable, and the standard deviation of the horizontal and vertical signals as the dependent variables did not reveal a significant interaction between video type and group type  = 48.07, p < .001. Participants in the ASMR group , the non-ASMR group , and the unsure group (Mdiff = -.21 p < .001) all showed significantly greater pupil diameter during the ASMR video. The interaction between group type and video type was not significant, F = 0.13, p = .878. A visualization of these results along with means and standard deviations are presented in In order to examine pupil diameter between the three different groups and the two videos, we conducted a 3 x 2 repeated measures ANOVA with group type as the between-subjects variable, video type as the within-subjects variable, and pupil diameter as the dependent variable. A significant main effect on video type was observed, t(25) = 3.61, p = .001  = 16.49, p < .001. Participants in the ASMR group  and the unsure group  reported significantly longer tingle duration for the ASMR video compared to the control video, which was not the case for non-ASMR participants   = 15.84, p < .001; both ASMR  and unsure  participants experienced significantly more episodes tingling sensations for the ASMR video when compared to the control video, while non-ASMR participants did not   See . Similar.05) See . The meaPrevious studies have revealed ASMR to be a complex emotional experience with feelings of both relaxation and calmness as well as excitement. Physiological responses to ASMR-inducing content have shown decreases in heart rate and increases in skin conductance levels, but solely on a general level (i.e. over the whole period of watching an ASMR video) . On a moTo answer these questions, it was important to first confirm that the ASMR video used in the study resulted in more tingling sensations when compared to the control video for ASMR participants but not for participants who do not experience ASMR. Results of the statistical analyses showed that both the ASMR and unsure group differed significantly on both number and total duration of tingles on the variable video type, indicating that the ASMR video resulted in more tingling sensations for both the ASMR and unsure group, but not for the non-ASMR group. As seen from Figs We hypothesized that the increase in pupil diameter from the control video to the ASMR video would be greater for participants who reported to experience ASMR than for participants who reported to not experience ASMR. Notably, all three groups showed a significant increase in pupil diameter between the control and the ASMR video. This increase can likely be explained by the absence of an audio track in the control video; simply presenting an auditory stimulus has been shown to cause increases in pupil diameter . ContrarWe not only hypothesized that the increase in pupil diameter between the videos would be greater for ASMR participants, but also that pupil diameter would likely increase during self-reported tingles. To examine tingles on an individual level, we compared mean pupil diameter during reported episodes of tingling sensations to mean pupil diameter outside of reported tingling episodes for each participant. Pupil diameter during reported episodes of tingling sensations was significantly greater when compared to pupil diameter outside of those episodes even when correcting for pupil diameter increase due to the manual response of pressing a button, suggesting that the tingling sensations in ASMR are accompanied by dilation of the pupil. The uncorrected increase in pupil diameter during tingles we observed in our results is similar to that of musical chills  and sligIn the present study, we examined the tingling sensations in ASMR further and found them to be accompanied by increases in pupil diameter. We observed a significant difference in pupil diameter only during reported episodes of tingling sensations, but not throughout the entire ASMR video. We see this pattern as reflective of the importance of tingling sensations in ASMR; the whole experience of ASMR might involve multiple components resulting in different reactions, and the tingling sensations are likely at the very core of the experience itself. The relaxing and calming component of ASMR could potentially be a result of a more general effect accompanied by decreases in heart rate, while the more exciting and arousing aspects of ASMR might be a more direct effect of the tingling sensations that is observed in increased skin conductance levels and pupil dilation. Whether or not this is the case, the tingles felt in ASMR seem to be reflected in the pupil. An increased pupil diameter during both ASMR and aesthetic chills add to the already many similarities these two phenomena have between them, suggesting that the release of norepinephrine in the LC-NE system might be involved in ASMR as well. Further research into the concentrations of neurotransmitters during the tingles in ASMR is likely to yield interesting results.The present study is the first to examine pupil diameter in relation to ASMR and one of the few studies to dissect the ASMR experience into its more specific levels (i.e. the tingling sensations it is often characterized by). We tested a relatively large sample and employed a control group. When interpreting the results, however, some notable limitations should be kept in mind. Although we included a control group and an ASMR group, a third group of participants unsure about their ASMR experience emerged. The unsure group is likely to be a heterogenous group consisting of both ASMR and non-ASMR participants, and not reflective of either population. Consistent with this idea, our results showed that the unsure group placed in between the ASMR and non-ASMR group in terms of their experience of the number and total duration of tingles. The large number of unsure participants is most likely due to ASMR being a still relatively unknown phenomenon: people unfamiliar with it will have a hard time distinguishing whether they actually experience it. Another reason for this could be the inherent indecisiveness of participants who are prone to respond unsure whenever such an option is offered. To complicate things further, ASMR is quite an ambiguous experience without clearly defined criteria; it is not an easy task to determine what counts as a tingle and what does not. To avoid such ambivalent attitudes towards the ASMR experience, future research needs to find ways to define the criteria for ASMR more clearly.A limitation of the more general analyses of the present study is the auditory difference between the control video and the ASMR video. Although ASMR videos without sound might be viable options as control videos, the effect of sound on the dependent variable needs to be considered carefully. The increase in pupil diameter between the videos that was observed in the present study likely due to sound only could potentially have masked an interaction effect. In situations where the control video needs to be as similar as possible in levels of lighting as the ASMR video, a better option might be to switch the audio track of the ASMR video to an audio track from a different video known to not induce ASMR, or to include background music, which has been suggested to have an inhibitory effect on ASMR . A furthBy examining ASMR on both a general and specific level, we have provided evidence that the tingling sensations in ASMR are accompanied by reliable changes in physiology. These results were persistent even when corrected for the effect of the manual response. ASMR is most commonly characterized by the presence of static tingling sensations that can spread throughout the body. By providing evidence that the tingling sensations themselves are coupled with changes in physiology, we have found more support for the idea that they are an essential component of ASMR, potentially reflecting the more exciting and arousing aspect of it. This finding gives further credibility to ASMR and particularly the tingles associated with it, suggesting that it is a real phenomenon that warrants further research. By dissecting and examining the components of ASMR further, better understanding of its underlying mechanisms can be obtained. We suggest future researchers to examine the physiological and emotional responses both during and outside of tingles to better understand the mixed physiological and emotional profile of ASMR and to determine whether certain responses are more likely to be associated with certain components of ASMR. With more understanding of the underlying physiological mechanisms of ASMR, its potential use as a therapeutic tool in various different settings will become clearer.S1 AppendixResults of the follow-up experiment and an overview of the questionnaire responses.(DOCX)Click here for additional data file.S1 DatasetAll data for the main experiment.(CSV)Click here for additional data file.S2 DatasetAll data for the follow-up experiment.(CSV)Click here for additional data file.S1 Data SupplementInformation on each dataset variable.(DOCX)Click here for additional data file.


The debilitating effects of recurrent stroke among aging patients have urged researchers to explore medication adherence among these patients. Video narratives built upon Health Belief Model (HBM) constructs have displayed potential impact on medication adherence, adding an advantage to patient education efforts. However, its effect on medication understanding and use self-efficacy have not been tested.The researchers believed that culturally sensitive video narratives, which catered to a specific niche, would reveal a personalized impact on medication adherence. Therefore, this study aimed to develop and validate video narratives for this purpose.This study adapted the Delphi method to develop a consensus on the video scripts’ contents based on learning outcomes and HBM constructs. The panel of experts comprised 8 members representing professional stroke disease experts and experienced poststroke patients in Malaysia. The Delphi method involved 3 rounds of discussions. Once the consensus among members was achieved, the researchers drafted the initial scripts in English, which were then back translated to the Malay language. A total of 10 bilingual patients, within the study’s inclusion criteria, screened the scripts for comprehension. Subsequently, a neurologist and poststroke patient narrated the scripts in both languages as they were filmed, to add to the realism of the narratives. Then, the video narratives underwent a few cycles of editing after some feedback on video engagement by the bilingual patients. Few statistical analyses were applied to confirm the validity and reliability of the video narratives.P<.001) for both learning outcomes and questions which indicated good agreement between members. Each statement’s Cronbach alpha was above .8 with SD values within a range below 1.5 that confirmed satisfactory content and construct validity. Approximately 75% (6/8) of members agreed that all chosen statements were relevant and suitable for video script content development. Similarly, more than 80% (8/10) of patients scored video engagement above average, intraclass correlation coefficient was above 0.7, whereas its Kendall W was about 0.7 with significance (P<.001), which indicated average agreement that the video narratives perceived realism.Initially, the researchers proposed 8 learning outcomes and 9 questions based on HBM constructs for the video scripts’ content. However, following Delphi rounds 1 to 3, a few statements were omitted and rephrased. The Kendall coefficient of concordance, W, was about 0.7 (The Delphi method was proven to be helpful in conducting discussions systematically and providing precise content for the development of video narratives, whereas the Video Engagement Scale was an appropriate measurement of video realism and emotions, which the researchers believed could positively impact medication understanding and use self-efficacy among patients with stroke. A feasibility and acceptability study in an actual stroke care center is needed.Australian New Zealand Clinical Trials Registry ACTRN12618000174280; https://www.anzctr.org.au /Trial/Registration/TrialReview.aspx?id=373554&isReview=true Medication nonadherence is prevalent at large especially in major chronic diseases, despite patient education and advanced knowledge and methods . RegardlIn educational strategy, 75% of information is engaged visually and about 13% of it is engaged using our hearing senses . Hence, The researchers proposed a patient education intervention at an outpatient stroke clinic as it may be a perfect venue for focused recurrent stroke education because it provides access to a common variation of people who are at high risk for recurrent stroke. In a quest for cost-effectiveness, the researchers utilized the prolonged waiting time in the clinic as an opportunity to deliver the intervention adjunct to the current medication therapy adherence clinic’s (MTAC) effort that may benefit the patients with stroke. Time spent in the waiting area is a potential period for patients to gain knowledge and confidence in managing their medication . This edpersuasive power would be instilled whereby the individual’s perception influenced by previous learning experience would have a change. The Social Learning Theory explains that an individual’s behavior depends on the conditioning of the mind, influenced by his or her environment, which then controls the action of the doer [The researchers believed that video narratives shown simultaneously with patient education modules are expected to have a positive impact on self-efficacy. Consequently, if the video is incorporated with theoretical behavioral constructs, it could induce self-reflection and simulation by a role model. In addition, if the video is repetitively seen, a the doer . The plathe doer . It makeThis study hypothesized that providing video narratives incorporated with theoretical behavioral constructs adjunct to the existing MTAC’s patient education effort, informational brochures, counseling, and medication review would result in better stroke awareness, medication understanding, and use self-efficacy toward improved adherence. This study was the intervention development and validation phase of a randomized controlled trial  . This stThe Delphi method originated from RAND Corporation studies from the 1950s and aimed to develop a reliable technique to obtain consensus from experts. Since then, many researchers have applied this organized method for expert problem-solving issues. They have also developed systematic guidelines of the process and analysis of the Delphi Method ,12.The researchers in this study applied a Delphi method to obtain anonymous consensus on learning outcomes, Health Belief Model (HBM) constructs, and content of video scripts which took place from October 2017 to December 2017 among experts experienced in stroke patient education. The consensus procedure incorporated 3 rounds of questionnaires via email to finalize expert panelists’ viewpoints.The process started with literature findings on the local need for stroke survivors. Most patients’ crucial need encompassed feelings of being independent to have a good quality of life, reducing the severity and preventing recurrent stroke -16. To bto take action on their medication-taking habit.Fundamentally, the design of the content was based on the most widely used framework, HBM . HBM hasThe core of the Delphi method was the selection of a knowledgeable and experienced expert panel of members within the specific need of content development . TherefoThe expert panel team of 8 comprised 2 neurologists, 2 pharmacists, 2 medical educationists, and 2 patients who had experienced a stroke. The neurologists were selected based on their 10 to 12 years of professional experience of diagnosing and prescribing medications to patients with stroke. The pharmacists were also selected based on their 10 to 12 years of professional experience of reviewing and dispensing prescribed medications to patients with stroke at the hospital and community level. Whereas, the medical educationists, who were also knowledgeable in developing curricular pedagogy, contributed to the suitability of learning outcomes for stroke according to local context and sensitivity. Finally, the patients with stroke for about 5 years had experiences and an awareness of the need for emotional support to enhance self-efficacy.There is no specific sample size recommendation for the Delphi method in this area of study as different disciplines and purpose of discussion often result in dissimilar response rates and time ,20. HoweA fruitful discussion with the panel of experts led to the video narrative script development. The researchers developed the scripts in English and translated them into the Malay language with the help of a professional bilingual translator. Then, back translation was performed by another bilingual researcher who was not exposed to the initial scripts to verify the similarity of meanings. Both scripts (a neurologist’s and a patient’s version) addressed a brief summary of (1) the debilitating impact of stroke; (2) related risk factors of recurrent stroke, its prevention strategy, and benefit; (3) belief in self-confidence; and (4) real-life cues of successful recovery regardless of the severity of stroke. The Flesch-Kincaid reading level for the narrative scripts scored an average grade level of 6 . Though The researchers believed that it was ideal and realistic to have actual actors  to narrate the scripts. Meanwhile, the video was taken at the Arts and Social Sciences School, Monash University, Malaysia, with the help of a technical officer. They narrated each video script, both in English and Malay language within 2 min, and the manner of speech was according to communication principles . The narThe researchers drafted the initial narrative script content guide from literature findings, which comprised 8 learning outcomes and 9 HBM-related questions linked to individual perceptions, cues to action, the likelihood of action, and self-efficacy. The panel of experts was given options  and an open-ended question to add any other relevant information to the list or justify any redundancy. Hence, this round helped to establish the initial content and construct development of the list, clarification of meaning, and rephrasing or merging of a redundant statement. They were given 3 weeks’ time to respond to the Delphi method coordinator.It was accepted that, approximately, an 80% agreement from the panel  for response frequencies for each learning outcome and HBM question was to be accepted or omitted. This percentage cut off was an appropriate reference point to attain content and construct validity . Hence, P values less than .05 are considered statistically significant.The researchers repeated the same procedures and timeline as the previous discussion except that the panel of experts was asked to rank the level of relevance using a 7-point Likert scale . They were asked to justify their choice of rank if it was 4 points and lower. Kendall W coefficient of concordance was used to measure the nonparametric rankings  for a beThe coordinator received comments and feedback to rephrase a few statements to illustrate appropriate meanings. The coordinator asked the expert panelists if they were willing to continue the rounds until the W value rises and all agreed. Hence, a final edition of learning outcomes and HBM questions were resent via an online survey questionnaire for the Delphi method round 3 discussion.weaker strength statements. The coordinator decided to run the final round of discussion, round 3, and the purpose was to hint the panel experts to confirm and justify revision of specific individual scores, which showed some discrepancies. Inter-rater reliability was determined with the ICC, whereby a 2-way mixed model (fixed raters) with absolute agreement was applied. An ICC value with 0.7 and greater indicated moderate to good reliability. W and SD values were then calculated, and subsequently, a full-detailed report of the discussion was sent to all expert members.Round 2 discussion and analysis produced a summary of responses and clarification from the panel of experts, which gave an overall picture of final scoring and the current level of consensus of the A purposeful sample of 10 bilingual patients with stroke  were requested to provide written feedback on the comprehension of the English and Malay video narratives scripts. The informed and consented patients were asked to reply either via email or via a prepaid postal service. Their responses contributed to face and content validity. They also viewed the video narratives in both languages and responded to the Video Engagement Scale (VES) that was presented to them face-to-face during their follow-up clinic visit. Test-retest was not appropriate as these patients were exposed to patient education materials, which could affect their follow-up responses. We expected occurrences of revision in every round of iteration. Therefore test-retest was not applicable to the Delphi method.To the researchers’ knowledge, there were no fixed guidelines to validate a video narrative for patient education; however, there has been a link between the construct of engagement and persuasive communication . TherefoApprovals for this development and validation study have been obtained from the Malaysian Medical Research and Ethics Committee (NMRR ID-15-851-24737) and the Monash University Human Research Ethics Committee (ID 9640) whereas the MyStrokeStory trial was registered with the Australian New Zealand Clinical Trials Registry .The researchers made no addition to the initial draft of the learning outcomes and HBM questions before the Delphi method round 1. We omitted statements that were redundant, had less than 80% agreement , or were rephrased . Whereas, few other statements or questions had only a minor correction. Therefore, 8 learning outcomes and 9 HBM questions were edited to 6 statements with 6 questions each for the Delphi method round 2.In round 2, the W value was below 0.7. The mean ranking for learning outcomes and HBM questions also varied .However, in round 3, the list of learning outcomes and HBM questions was finalized . KendallThe researchers received positive feedback on the scripts , but there were not many comments on the structure of sentences or usage of words. Therefore, the researchers concluded that the scripts were suitable to the local context; hence, the narrative scripts were finally confirmed.The VES scores were above average, which exhibited a good link with perceived realism . Out of This study explicitly developed and validated video narratives to be used as intervention materials in a randomized controlled trial  whereby There were some apparent limitations in this video narrative development. Face-to-face discussion was unable to be carried out in the Delphi method rounds owing to the lack of interval time and slow responses from the expert panel despite constant reminders. Hence, the Delphi method discussion ended in round 3 whereby force agreement would have occurred. The researchers were also aware that face validity and video engagement responses lacked the required number of participation from poststroke patients because of specific inclusion and exclusion criteria via purposive sampling method. Therefore, the video narratives’ validation and study aim were skewed toward particular samples only, and hence, results could not be generalized to the whole population of patients with stroke. In addition, responses from nonbilingual patients were also not assessed owing to the delay during the purposive sampling period and having the VES available in the English version only.Nevertheless, the Delphi method proved to be a versatile and helpful technique in conducting discussions systematically and reaching a consensus unanimously, eliciting precise ideas, and providing rich, in-depth data in defining an intervention strategy. In addition, the video narrative development processes were found to be useful as a guideline for other behavioral studies, which use video as their intervention, samples with chronic illness, and study sites other than health care centers.no stone had been left unturned in this development and validation process. The VES had helped to reveal the preliminary understanding of the patients’ video engagement styles and emotions that were being affected ; however, we believed that bigger samples would produce far more significant data. The researchers recommend that the VES be summarized and translated in various languages in the future to test its effectiveness in distinguishing the video engagement style of multicultures. A future test of the video narratives’ feasibility and acceptability in an actual stroke care center would undoubtedly add significance to its validation and effectiveness.The researchers believed that


Withdrawal from binge-drinking increases negative affect, coinciding with increased expression of the metabotropic glutamate receptor 5 (mGlu5) within the shell of the nucleus accumbens (AcbSh). Supporting a causal-effect relationship, systemic treatment with the mGlu5 receptor antagonist MTEP [3-((2-Methyl-4-thiazolyl)ethynyl)pyridine] is anxiolytic in binge-drinking adult and adolescent mice. Here, we employed neuropharmacological approaches to examine the functional relevance of AcbSh mGlu5 for behavioral indices of alcohol withdrawal-induced hyper-anxiety. Adult (PND 56) and adolescent (PND 28) male C57BL/6J mice consumed alcohol under modified Drinking-in-the-Dark procedures  for 14 days. At an alcohol withdrawal time-point when mice manifest robust behavioral signs of hyper-anxiety , mice were infused intra-AcbSh with 0, 1 or 10 μg MTEP and then affect was assayed in the light-dark shuttle box, marble-burying and forced swim tests. Brain tissue was collected to evaluate changes in Egr1 (early growth response protein 1) induction to index AcbSh neuronal activity. As expected, alcohol-experienced mice exhibited behavioral signs of hyper-emotionality. The anxiolytic effects of intra-AchSh MTEP were modest, but dose-dependent, and varied with age of drinking-onset. In adult-onset mice, only the 1 μg MTEP dose reduced withdrawal-induced hyper-anxiety, whereas only the higher dose was effective in adolescent-onset animals. MTEP reduced Egr1 expression within the AcbSh, irrespective of alcohol drinking history or age of drinking-onset. However, only the high MTEP dose reduced Egr1 expression in adolescent-onset binging mice. These results implicate AcbSh mGlu5 in modulating alcohol withdrawal-induced negative affect and suggest age differences in the neurobiological effects of alcohol withdrawal and behavioral responsiveness to mGlu5 blockade within the AcbSh. Binge-drinking is defined as a pattern of consumption that elevates blood alcohol concentrations (BAC) to ≥80 mg/dl, which equates to approximately 4–5 drinks in a 2-h period . In bothIndeed, age-related differences in the manifestation of alcohol withdrawal-induced negative affect are apparent in murine models of binge-drinking , 2017c. In mouse models, withdrawal from a chronic history of alcohol consumption up-regulates the expression of the metabotropic glutamate receptor 5 (mGlu5) within the nucleus accumbens shell (AcbSh), present at as early as 24-h withdrawal . This inThe binge-drinking and behavioral testing procedures used in this study were identical to those employed previously in our laboratory , 2017b aad libitum, except during the 2-h alcohol-drinking period. The study design consisted of 2 age of drinking-onset groups (adults versus adolescents), 2 drinking groups , and 3 intra-AcbSh treatment groups . The sample size was 12 per group.The subjects in this study were male C57BL/6J mice, purchased from The Jackson Laboratory . Mice were allowed 7 days to acclimate to our vivarium and were either PND 28 (adolescents) or PND 56 (adults) at the onset of our binge-drinking procedures. Animals were identified using small animal ear tags  and housed in age-matched groups of 4 in a climate-controlled vivarium under a reverse light/dark cycle (lights off at 10:00 h). Food and water were available Half of the animals from each age of drinking-onset group were subjected to 14 consecutive days of binge-drinking under 3-bottle DID procedures. Control animals received water only. As in our more recent studies  alcohol-Submandibular blood samples were collected from all alcohol-drinking animals on day 11 of alcohol presentation, immediately upon conclusion of the 2-h drinking period. The scheduling of the blood sampling was selected to ensure that the animals’ intakes had stabilized, while also allowing ample time for recovery prior to behavioral testing. BAC was determined using an Analox alcohol analyzer .Based on our previous work showing age-related differences in the emergence of a withdrawal phenotype , 2017c, TM . General locomotor activity was also assessed by measuring the total distance traveled during the trial. We also employed the marble-burying test as an alternate index of anxiety  were placed in the animals’ home cage, 5 at each end. The total number of marbles with at least 75% of their surface area covered with sawdust bedding were counted at the end of the 20-min trial. Each animal was video recorded and an experimenter blind to the treatment of the mice scored the videos using a stopwatch to determine the latency to begin burying marbles and the total time spent burying.TM tracking software. Behavioral testing was conducted during the animals’ circadian dark phase.In this assay, each mouse was placed into an 11-cm diameter cylindrical container and the latency to first exhibit immobility , total time spent immobile, and the numbers of immobile episodes were monitored throughout the entire 6-min trial period using Any-mazeBoth adult and adolescents underwent surgery at the same age, approximately 3 weeks prior to behavioral testing (PND 42–48). Thus, surgery occurred prior to the 14-day drinking period in adults and afterward in adolescents. In addition to maintaining consistency in the surgery timing across age groups, this also allowed us to avoid the practical limitations of performing craniotomies on juvenile mice. Surgical procedures were similar to those conducted previously in our laboratory e.g., . Under 1MTEP  was dissolved in sterile water and infused at a volume of 0.25 μl/side, such that animals were infused with either 1 or 10 μg/side. These doses were selected based on prior neuropharmacological studies by our group demonstrating effectiveness at reducing binge-drinking in mice , 2014a. ®  and transcardially perfused with phosphate-buffered saline (PBS), followed by 4% paraformaldehyde. Brains were removed and post-fixed for 24 h in 2% paraformaldehyde in PBS, then cold-stored in cryoprotectant  until slicing. Tissue was sectioned (40 μm) along the coronal plane on a vibratome at the level of the striatum . Egr1 expression was analyzed in the AcbSh, as well as the adjacent core subregion (AcbC) for comparison. DAB staining intensity was quantified using ImageJ , using the ‘mean gray’ function. The mean gray intensity value of the entire image was divided by an internal background control value for each animal obtained from a region lacking any Egr1+ cells, yielding a normalized measure (arbitrary units) of staining intensity  was used to analyze intake data for all alcohol-drinking animals to assess age differences in alcohol consumption across the 14-day drinking period. A Pearson’s correlational analysis was conducted to determine the relationship between alcohol intake and resulting BACs sampled on day 11 of drinking. Two-way ANOVAs were used to analyze the behavioral data and immunohistochemistry, with Drinking  and Treatment  as factors. Tukey–Kramer multiple comparison tests were used to further deconstruct group differences for ANOVAs yielding significant findings. Tukey–Kramer tests were also applied to data for which the results of the ANOVAs failed to indicate significant interactions between our independent variables to address al power , and then = 9–11 per group. Data identified as statistical outliers were also omitted from analysis, resulting in the exclusion of 0–2 animals per test.Animals were omitted entirely from data analysis based on incorrect microinjector placement, difficulties encountered during staining, and attrition due to surgical complications, resulting in final samples sizes of F = 14.93, p < 0.001]. On day 11 of drinking, adults consumed an average of 3.69 ± 0.31 g/kg resulting in an average BAC of 69.93 ± 3.17 mg/dl and adolescents consumed an average of 4.94 ± 0.34 g/kg resulting in an average BAC of 78.29 ± 3.01 mg/dl. Although the correlation between alcohol intake and resulting BAC on day 11 did not quite reach significance, there was a strong statistical trend for a positive relationship .Adults consumed an average of 3.88 ± 0.20 g/kg and adolescents an average of 5.00 ± 0.21 g/kg across the 14-day drinking period Figure ,b,c, sugF = 3.31, p = 0.044; Figure post hoc comparisons showed that in vehicle-infused animals, adult-onset alcohol-drinking mice made fewer light-side entries, compared to their water controls (p = 0.001). Infusion of 1 μg/side MTEP significantly increased the number of light-side entries in the alcohol-drinking mice, compared to vehicle infusion (p = 0.039). In contrast, this low MTEP dose did not alter the number of light-side entries in water drinkers and the 10 μg/side dose did not alter this measure in either alcohol- or water-drinking mice (p’s > 0.10). There was a similar treatment × drinking interaction in the time spent on the light side . Tukey–Kramer comparison analyses showed that alcohol drinking did not alter the latency to begin burying among vehicle-infused animals, relative to water-drinking controls (p > 0.10). However, in both water- and alcohol-drinking animals, 1 μg/side MTEP significantly increased latency to begin burying , while the 10 μg/side dose was ineffective in either group (p’s > 0.10).A significant main effect showed that adult-onset alcohol-drinking animals buried more marbles overall compared to water control animals . Although no significant interaction was detected between these variables, inspection of the data in Figure p = 0.001). Compared to vehicle infusion, 1 μg/side and 10 μg/side MTEP significantly reduced Egr1 expression in both alcohol- and water-drinking animals .In adult-onset animals, a significant main effect showed that alcohol increased Egr1 expression within the AcbSh relative to water control animals , but no interaction between these factors. As conducted for the data for adult-onset animals, Tukey–Kramer comparisons were conducted to examine the dose-selective of MTEP’s effects in adolescent-onset alcohol-drinking mice. This re-analysis showed that among vehicle-infused mice specifically, alcohol-drinking mice exhibited higher Egr1 expression, compared to their water-drinking controls (p = 0.009). Among water-drinking animals, both 1 μg/side and 10 μg/side MTEP reduced Egr1 expression, relative to vehicle infusion . However, in adolescent-onset alcohol-drinking mice, the reduction in Egr1 expression was significant at the 10 μg/side MTEP dose (p = 0.003), but not at the 1 μg/side dose (p = 0.20). The ANOVAs revealed no significant effects of adult or adolescent alcohol drinking or intracranial drug treatment on Egr1 expression within the AcbC  is effective at reducing binge-drinking  and alcogreater sensitivity to MTEP’s effects upon binge-drinking (In contrast to adult-onset drinkers, intra-AcbSh infusion of the 1 μg/side MTEP dose was ineffective at reducing any behavioral sign of the negative affective state observed during protracted withdrawal in adult mice with an adolescent-onset drinking history Figures –8. Also drinking . In our drinking . As suchAs in our prior study employing systemic MTEP administration , the ageWhile alcohol withdrawal-induced hyper-anxiety in mice with a history of binge-drinking during adulthood correlates with AcbSh mGlu5 expression , adolescIt is interesting to note that despite exerting effects on several anxiety-like measures in the light-dark shuttle box and the marble-burying tests, intra-AcbSh MTEP did not influence the behavior of male mice in the forced swim test, irrespective of the age of alcohol-drinking onset. Admittedly, the water-alcohol differences in behavior manifested on the forced swim test were smaller in this study than that reported previously by our group previously , 2017a,cc-fos within the AcbSh during early withdrawal in animals with alcohol experience ranging from a single alcohol exposure (c-fos immunoreactivity within specific brain regions, including the AcbSh, that also respond to anxiety-provoking stimuli such as an air-puff challenge (c-fos activation within the AcbSh (When determined during adulthood, alcohol withdrawal increased Egr1 induction within the AcbSh of both adult- and adolescent-onset drinking mice, coinciding with the presence of hyper-anxiety. These results contrast with those from our earlier report in which we failed to detect withdrawal-induced changes in Egr1 expression within the AcbSh of adult mice with a 30-day history of binge-drinking . The disexposure  to as loexposure . In facthallenge  and averhallenge . Additiohe AcbSh . Therefohe AcbSh .c-fos induction within the AcbSh (Although the hyper-anxiety expressed by adult-onset alcohol-drinking mice during early withdrawal is insensitive to higher MTEP doses . Moreovehe AcbSh . Howeverhe AcbSh –8. It ishe AcbSh , as wellhe AcbSh  when infAs a final point of clarification, the focus of the present study was the AcbSh specifically, given its role in extended amygdala circuitry, and thus its potential involvement in the negative affective consequences of alcohol abuse. This being said, we also examined Egr1 expression within the adjacent AcbC and found that it was not affected by a history of alcohol drinking during either adulthood or adolescence, which is consistent with and extends our previous findings for alcohol regulation of Egr1 expression in adult animals . There wMany people who abuse alcohol report that anxiety reduction is a key motivator for drinking  and in tThe results of the present study highlight an important, albeit modulatory, role for mGlu5 within the AcbSh in the age-related manifestation of hyper-anxiety during alcohol withdrawal. Adolescent-onset alcohol-drinking appeared to render mice less sensitive to MTEP’s anxiolytic effects during adulthood than adult-onset drinking, extending earlier indications that the age of drinking-onset is a critical subject factor influencing the behavioral sequelae of excessive drinking, including the severity of affective symptoms and therapeutic responsiveness. Further, we provide confirmatory evidence that heavy drinking during adolescence produces enduring behavioral and neurobiological changes within extended amygdala structures that potentially contribute to an increased vulnerability to addiction and/or affective disorders later in life.KL and KS designed the experiments, conducted the data analyses, and composed the manuscript. KL, MiC, MaC, KS and MB performed the experiments. All co-authors edited the manuscript.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.