Background: Diarrheagenic Escherichia coli (DEC) strains are a main cause of diarrhea worldwide in children under 5 years old. DEC virulence is strongly regulated by environmental conditions and metabolites produced by the gut microbiota in the intestinal tract. In this study, we evaluated changes in gut microbiota-metabolome in children with or without diarrhea produced by DEC pathotypes.Goal: To determine gut microbiota composition and metabolome in stool samples obtained from healthy children and children with diarrhea positive for DEC pathotypes.Methods: We analyzed a total of 16 age-paired stool samples: 8 diarrheal samples positive for one DEC pathotype and 8 stool samples from healthy children. To identify the microbiota composition, we sequenced the V3-V4 region of the 16S rRNA and determined operational phylogenetic units (OPU). OPU were then used to predict metabolic pathways using the PICRUSt2 software. The presence of metabolites in stool samples was determined by LC-MS. A correlation analysis was performed with the main genera from each group and main metabolites. Bacteria associated with variance of main metabolites were identified using the MIMOSA2 software.Results: DEC and healthy groups showed a statistically different microbiota composition. A decrease in Firmicutes together with an increase in Bacteroidetes and Proteobacteria was found in the DEC group compared to the healthy group. Metabolic pathway predictions based on microbiota diversity showed that pathways involved in histidine and L-ornithine metabolism were significantly different between groups. A total of 88 metabolites detected by LC-MS were included in the metabolome analysis. We found higher levels of histamine and lower levels of ornithine in DEC samples than in the healthy group. Histamine and L-ornithine were associated with a specific microbiota species and the corresponding metabolic pathways.Conclusion: Stool samples from healthy children and children positive for DEC displayed a differential metabolome and microbiota composition. A strong correlation between a gut microbiota species and certain metabolites, such as histamine and L-ornithine, was found in the DEC group. This information might be useful to identify mechanisms and signaling molecules involved in the crosstalk between microbiota and DEC pathotypes. Escherichia coli (DEC) is the most common bacterial etiological agent of diarrhea in diverse subpopulations, both in developing and industrialized regions, and it primarily affects children under 5 years of age  and the HLCM daycare center, respectively, located in Santiago, Chile. All stool samples were stored at −80°C. Frozen stool samples were screened for 22 enteric pathogens using FilmArray® GI testing and selected samples were grouped as healthy  and DEC . We excluded from the study children who received antibiotics, probiotics, steroidal and non-steroidal anti-inflammatory drugs 2 months prior to enrollment. Eight age-paired samples for each group were chosen for this study .This study was conducted in accordance with Declaration of Helsinki guidelines. The study protocol was approved by the Ethics Committee of the Universidad de Chile. Written informed consent was obtained from all parents on behalf of their children.Total DNA was extracted from each stool using the QIAamp Fast DNA Stool Mini kit (Qiagen), quantified using a Synergy HT® spectrophotometer (Biotek™) and stored at −20°C. DNA samples were shipped to Molecular Research LP  for DNA amplification and sequencing of the V3-V4 regions of the 16S rRNA, using the Illumina miSeq 2x300 PE.Vegan  at MS-Omics (Denmark). Briefly, PCR grade water was added to 500 mg of stool sample, vortexed and centrifuged. Supernatants were filtered with a 0.2 μm filter and filtered supernatants were shipped to MS-Omics. A total of 156 metabolites were detected. We used the KEGG database  package . At phylum level, the DEC group presented a decreasing number of Firmicutes , as well as an increasing number of Proteobacteria  compared to the healthy group. We also found differences in the proportion of Bacteroidetes . All the taxa comparisons are shown in We identified a total of 755 OPU within 16 stool samples. The redundancy analysis showed that microbiota composition was statistically different among DEC and healthy groups, with a distinctive community structure clustering . Using a univariate analysis of samples by volcano plot, we found 13 metabolites with a fold change >2 and p-value of t-test <0.05 (p = 0.05). Dendrograms and heatmaps of the significantly associated metabolites described above displayed a hierarchical organization of samples similar to the a priori grouping of samples based on health status , Escherichia (p = 0.003), Prevotella (p = 0.027), Erwinia (p = 0.010), and Buttiauxella (p = 0.016) were the genera significantly associated with the DEC groups. Instead, Faecalitalea (p = 0.001), Lactococcus (p = 0.032), and Clostridium (p = 0.010) were the genera significantly associated with the healthy groups. Correlation between the abundance of these main genera and the 15 metabolites with a fold change >2 showed a clear pattern of link between the group's associated genera and metabolites in each group  variations depend on the presence of specific taxa. Among the taxa associated with the positive contribution of histamine and ornithine levels, abolites . Within in vitro infection experiments to evaluate the role of metabolites significantly associated with the DEC group as signaling molecules involved in DEC virulence regulation.Our study has limitations. We analyzed a small number of samples and therefore our results should be considered as an exploratory approach. However, our data provided valuable information about the importance of gut microbiota and metabolome analysis in understanding the regulatory mechanism of DEC virulence. It is important to note that our metabolome analysis did not include metabolites that have been previously proven to have an impact on DEC virulence, such as SCFAs, because the primary goal of this study was to identify new pathways or metabolites that might be important to DEC pathogenicity. From this study we obtained an effect size value of 0.4377, which will be used to obtain the required sample size for further studies to confirm our observations, as well as In conclusion, our study showed that stool samples from healthy children and children positive for DEC displayed a differential metabolome and microbiota composition. We found a correlation between gut microbiota species and certain metabolites, information that might be useful in identifying mechanisms and signaling molecules implicated in the crosstalk between microbiota and DEC pathotypes that might participate in the virulence of these pathogens.https://www.ncbi.nlm.nih.gov/, PRJNA623942.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The studies involving human participants were reviewed and approved by Ethical Committee of Universidad de Chile. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.PG participated in sample processing, performed sequences and metabolites analyses, interpreted the data, and participated in manuscript writing. MI participated in data analysis and manuscript writing. RV participated in study design and data analysis. FS participated in data analysis and manuscript writing. JO participated in data analysis. MF participated in study design, data acquisition and interpretation, manuscript writing, and final approval of the manuscript. All authors contributed to the article and approved the submitted version.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.


To develop and evaluate a fast respiratory navigator (fastNAV) for cardiac MR perfusion imaging with subject‐specific prospective slice tracking.A fastNAV was developed for dynamic contrast‐enhanced cardiac MR perfusion imaging by combining spatially nonselective saturation with slice‐selective tip‐up and slice‐selective excitation pulses. The excitation slice was angulated from the tip‐up slice in the transverse plane to overlap only in the right hemidiaphragm for suppression of signal outside the right hemidiaphragm. A calibration scan was developed to enable the estimation of subject‐specific tracking factors. Perfusion imaging using subject‐specific fastNAV‐based slice tracking was then compared to a conventional sequence  in 10 patients under free‐breathing conditions. Respiratory motion in perfusion images was quantitatively assessed by measuring the average overlap of the left ventricle across images  and the average displacement of the center of mass of the left ventricle (avCoM). Image quality was subjectively assessed using a 4‐point scoring system .P < .001) and reduced avCoM . There were no statistically significant differences between the 2 sequences in terms of image quality .The fastNAV calibration was successfully performed in all subjects . Prospective motion correction using fastNAV led to higher avDice (0.94 ± 0.02 vs. 0.90 ± 0.03, fastNAV enables fast and robust right hemidiaphragm motion tracking in a perfusion sequence. In combination with subject‐specific slice tracking, fastNAV reduces the effect of respiratory motion during free‐breathing cardiac MR perfusion imaging. Slice‐selective pulses can have a small temporal footprint, whereas angulation of these slices with respect to each other was used to localize the navigator signal to the RHD in the transverse plane. By applying the tip‐up pulse immediately following the saturation pulse and exciting the fastNAV with a low flip angle, signal variations were minimized. To enable the estimation of a subject‐specific tracking factor for fastNAV, a calibration scan was also developed. Perfusion imaging using subject‐specific fastNAV‐based slice‐tracking prospective motion correction was then compared to a conventional perfusion sequence in patients under free‐breathing conditions.22.1The diagram of the proposed fastNAV prototype pulse sequence is shown in Figure 2.2The proposed perfusion sequence acquires 3 short axis slices per heartbeat, with prospective fastNAV‐based slice tracking applied to each slice Figure . The RHD2.3In order to calibrate subject‐specific tracking factors, a sequence was designed to allow the near‐simultaneous measurement of fastNAV signal and LV displacement in a coronal image Figure . The calThe fastNAV‐based RHD displacement values and coronal DICOM images were exported offline to estimate a subject‐specific tracking factor using a custom‐based software MedIACare27 in MA2.4Ten patients  were included in this study. These patients were referred for an outpatient elective CMR study on clinical grounds, and their indications for the study included suspected cardiomyopathy (7) and suspected myocarditis (3). This study was approved by the National Research Ethics Service (15/NS/0030), and written informed consent was obtained from all patients for the scan and for inclusion in this study. Imaging was performed at 1.5 T  using a 32‐channel spine array and an 18‐channel body array coil.2, slice number: 1, slice thickness: 10 mm, matric size: 192 × 192, voxel size: 2.1 × 2.1 mm2, temporal GRAPPA acceleration factor: 3, bandwidth: 1085 Hz/pixel, and dynamics: 30.Each subject underwent a fastNAV calibration scan Figure  prior to2, slice number: 3, slice thickness: 8 mm, matrix size: 192 × 144, voxel size: 1.9 × 1.9 mm2, partial Fourier factor: 3/4, GRAPPA acceleration factor: 2 (24 integrated reference lines), bandwidth: 1085 Hz/pixel, total acquisition duration per heartbeat: 603.9 ms, effective saturation recovery time: 133 ms, and dynamics: 60. The FOV was adjusted according to patient size. Both sequences were planned on the systolic phase of 2‐, 3‐, and 4‐chamber cine images to ensure coverage of the base, mid‐, and apical slices. The patients were instructed to breathe normally for all scans  in this study.Two rest dynamic contrast‐enhanced perfusion protocols were then performed within the same CMR examination using 1) the proposed prospective motion correction approach, and 2) a conventional approach without motion correction. A dose of 0.075 mmol/kg gadobutrol  was injected at a rate of 4mL/s, followed by a 25 mL flush of normal saline by a power injector  for each perfusion protocol. To allow for contrast washout, the second perfusion protocol was performed 10 min after the first injection. The order of the 2 perfusion protocols was randomized across subjects. A BIR‐4 adiabatic saturation pulse was used in all cases, with no additional delay time between the fastNAV and the image acquisition to maximize the time available for data sampling of the perfusion image. Both perfusion acquisitions used an electrocardiogram‐triggered balanced steady‐state free precession saturation recovery sequence prescribed in the short axis orientation with the following parameters: TR/TE: 2.38/1.04 ms, FA: 50°, FOV: 360 × 270 mm2.5Although RHD NAV is expected to jointly reduce through‐plane and in‐plane motion, only in‐plane motion can confidently be assessed in 2D short axis perfusion images. To quantify any potential benefit of the proposed prospective motion correction, the temporal alignment of the LV in dynamic images was assessed by measuring the average Dice similarity coefficient2.6s.n. and a.c.) with more than 5 and 10 years of CMR experience, respectively. The readers were blinded from both patient information and acquisition scheme. Images were exported in the DICOM format and visualized using OsiriX . Image quality was assessed for each dataset using a 4‐point scoring system .To investigate whether fastNAV may have introduced additional image artefacts, subjective assessment of image quality was performed in consensus by 2 expert readers  or Wilcoxon signed‐ranks test . Qualitative image quality scores were compared between the 2 sequences using the Wilcoxon signed‐ranks test. All statistical tests were 2‐tailed, and P values < .05 were considered significant.All results of continuous variables are expressed as mean ± SD, whereas results concerning nonparametric variables are presented as median value and interquartile range. Normality of distribution of continuous variables (avCoM and avDice) was assessed using the Shapiro‐Wilk test. Continuous variables were compared between the 2 sequences using paired 33.1Examples of the fastNAV signal and images acquired during a calibration scan in 1 patient are given in Figure The fastNAV calibration was successfully performed in all subjects and is summarized in Table 3.2Figure For all 10 subjects, fastNAV signals from the first 19 dynamics from the start of the perfusion imaging are shown in Figure P < .001) and reduced avCoM .Both fastNAV and conventional perfusion sequences were successfully acquired in all patients. Figure P = 1).All presented slices were scored either as “excellent” or “minor artefact but not limiting diagnosis.” Overall, there was no statistically significant difference between the 2 methods in terms of image quality , which was shorter than previously reported navigators used for CMR perfusion imaging.Slice tracking using fastNAV was shown to successfully reduce in‐plane motion. Because the slice location adjustment was in the superoinferior direction, the in‐plane and through‐plane motions for images acquired in the short axis orientation were geometrically linked, and it can be inferred that through‐plane motion was also reduced. Moreover, from studies with RHD NAV, it is already known that slice tracking reduces through‐plane motion.A 10 min delay was employed between the 2 perfusion scans, which was not sufficient to allow for the complete washout of the contrast agent from the myocardium. To minimize the effect this had on our measurements, the acquisition order of the 2 sequences was randomized across patients. We did not compare fastNAV to other prospective motion correction strategies because it is difficult to perform more than 2 perfusion protocols per subject in the same session. A conventional perfusion protocol without prospective motion correction was used as reference, which is the most common approach used in the clinic. Perfusion quantification was not performed in this study due to the high contrast agent dose and lack of suitable approach for sampling of the arterial input function. However, the accuracy of perfusion quantification has already been demonstrated to improve with the presence of prospective slice tracking  and accurate in‐plane motion correction.In this study, the calibration process relied on a linear model of the RHD motion and the superoinferior heart motion, as commonly used clinically.The fastNAV technique has been used with an adiabatic saturation pulse and is compatible with conventional nonselective saturation pulses. However, composite pulses have also been proposed to further improve the saturation efficiency.The feasibility of fastNAV was demonstrated during rest perfusion in a small number of patients with no perfusion defects, thereby opening the way for improved perfusion image acquisition and analysis in the clinic. Future larger studies will be needed to characterize the clinical benefit of fastNAV under stress conditions in patients with coronary artery diseases. The validity of the tracking factor estimated at rest and used during stress conditions remains to be demonstrated. It may also be possible to acquire the fastNAV calibration scan immediately before the stress perfusion scan to avoid such potential limitation, which would require an online and automatic calculation of the tracking factor. Other potential applications for fastNAV‐enabled prospective motion correction include: drift correction during prolonged breath‐hold perfusion protocols, correction of large respiratory motion occurring during exercise stress perfusion protocols, improvement of retrospective motion correction, improvement of reconstruction techniques exploiting temporal information, and possibly even reconstruction algorithms simultaneously solving image reconstruction and motion correction problems.5The fastNAV enabled fast and robust RHD motion tracking in a CMR perfusion sequence. The use of fastNAV combined with real‐time slice tracking and a subject‐specific tracking factor reduced the effect of respiratory motion during free‐breathing dynamic contrast‐enhanced CMR perfusion imaging.Dr. Radhouene Neji is an employee of Siemens Healthcare.VIDEO S1 Video of the entire dynamic perfusion imaging series acquired with the conventional non‐motion corrected sequence for subject #3. Base, mid, and apical images are displayed at 10 frames per second. The video shows the position of the myocardium moving due to respiratory motionClick here for additional data file.VIDEO S2 Video of the entire dynamic perfusion imaging series acquired with the fastNAV‐enabled prospective motion corrected sequence for subject #3. Base, mid, and apical images are displayed at 10 frames per second. The video demonstrates that the position of the myocardium remains relatively fixed, with little residual motionClick here for additional data file.VIDEO S3 Video of the entire dynamic perfusion imaging series acquired with the conventional non‐motion corrected sequence for subject #5. Base, mid, and apical images are displayed at 10 frames per second. The video shows the position of the myocardium moving due to respiratory motionClick here for additional data file.VIDEO S4 Video of the entire dynamic perfusion imaging series acquired with the fastNAV‐enabled motion corrected sequence for subject #5. Base, mid, and apical images are displayed at 10 frames per second. The video demonstrates that the position of the myocardium remains relatively fixed, with little residual motionClick here for additional data file.VIDEO S5 Video of the entire dynamic perfusion imaging series acquired with the conventional non‐motion corrected sequence for subject #10. Base, mid, and apical images are displayed at 10 frames per second. The video shows the position of the myocardium moving due to respiratory motion and non‐rigid deformation of the myocardium in the apical sliceClick here for additional data file.VIDEO S6 Video of the entire dynamic perfusion imaging series acquired with the fastNAV‐enabled motion corrected sequence for subject #10. Base, mid, and apical images are displayed at 10 frames per second. The video demonstrates that the position of the myocardium remains relatively fixed, with little residual motion but also non‐rigid deformation of the myocardium in the apical sliceClick here for additional data file.


The current study examined the predictors of the onset of alcohol use as well as predictors of remission and relapse, both from heavy drinking and from alcohol dependence. Similarities and differences in both clinical and psychosocial predictors across the transitions were examined.A sample of men from the Vietnam Era Twin Registry (N = 1769) completed an assessment of lifetime drinking history, which allowed age markers for starting and stopping different drinking patterns. The men also completed various assessments regarding personality, alcohol motives, and psychiatric diagnoses. Survival analyses were used to examine the predictors of the three transitions of onset, remission, and relapse for the phenotypes of heavy drinking and of alcohol dependence, censoring the individuals who had not yet experienced an event.As expected, predictors of onset for drinking, heavy drinking, and alcohol dependence were largely consistent and included externalizing symptomology, nicotine dependence, and cotwin history of drinking as risk factors. Predictors of remission from heavy drinking, somewhat similarly to remission from alcohol dependence, included the risk factor of externalizing disorders but also, as predicted, included more risk and protective factors in the psychosocial realm that were not predictors of onset. Contrary to our prediction, relapse to heavy drinking and alcohol dependence were predicted largely by unique psychosocial risk and protective factors including social and coping motives.Current findings extend the findings of past research to remission and relapse in the later decades of life and have implications for treatment of alcohol use problems. Researchers and clinicians agree that the course of alcohol dependence is better characterized as a widely varying process with varying consequences than as a uniform pattern of steadily declining health and functioning. Differences in the course of alcohol problem use and dependence have been identified and characterized via work on subtypes, etiology, treatment, and trajectories of alcohol use disorders –6. For eAlthough the prevalence of alcohol use disorders (AUD) is highest during adolescent and young adult years, treatment seeking for AUD is twice as likely to occur after age thirty , and morMoving beyond association studies of specific milestones in alcohol use are studies of the rate of progression from one stage of use to another. In such models, risk factors would be those influences that accelerate a person’s progression to the next stage and protective factors would be those that delay progression. Evidence shows that different psychiatric and psychosocial factors increase (or decrease) the rate of progression through a specific stage –29. As wA key limitation of association studies is that the predictors of any given milestone will necessarily include the predictors of all preceding milestones. That is, the predictors of AD will also include the predictors of initiation of drinking since initiation always precedes AD. Studies that model the rate of progression though a series of distinct sequential periods defined as beginning at one milestone and ending at another can isolate predictors of each transition. Survival analysis, which can examine the time it takes to experience a given event and what predicts a quicker or slower progression, is one such model. For example, in a study of young adults, Sartor et al.  used conThe same risk factors associated with initiation of drinking will also be associated with progression to heavy drinking and progression to AD (the three “onset” transitions).Factors associated with remission from heavy drinking and remission from AD will be different from those predicting initiation and progression, with psychosocial factors being prominent influences.Factors associated with relapse will be similar to those associated with onset and progression of heavy drinking and AD.Specifically, the current study examined predictors associated with the duration of different stages in the progression of alcohol use in a sample of male veteran twins with AD history. After examining risk factors for age of first drink, the three successive transitions of onset, remission, and relapse were examined for both heavy drinking and AD. Thus, seven survival analysis models examined significant predictors of the time function, which permitted identification of risk and protective factors that accelerated or delayed the rates of transition through each distinct developmental period. The current study examined psychiatric variables, family history of AD, personality traits, alcohol expectancies, and drinking motives as possible predictors of transitions. Prediction from heavy drinking to remission was of particular interest for clinical treatment, as physicians and counselors could benefit from knowing which factors are most influential in supporting treatment success, especially if such predictors can be modified via therapy. We had the following general hypotheses, which are exploratory in nature but are based on patterns seen in past research on predictors of different aspects of alcohol use:The participants in this study were twin veterans who were members of the Vietnam Era Twin (VET) Registry, although the twins were treated as individuals in the study. The VET Registry has been described in detail elsewhere –34, so oProcedures for obtaining verbal informed consent were approved by institutional review boards. Data collection was conducted by the Institute for Survey Research at Temple University. Interviewers, who were blind to the twin’s substance use history, used a computer-assisted telephone interview system with standardized interview questions and probes. After personal identifiers were removed by the VET Registry, data were released to the investigators. Interviews for the twin fathers in COA/TAP included a lifetime drinking history (LDH) assessment , 40 thatEach veteran’s other lifetime psychiatric diagnostic information was obtained from their 1992 interview using DSM-III-R criteria. Nicotine dependence was used as a risk factor. Additionally, both externalizing and internalizing factors, which are dimensional measures used as alternatives to categorial diagnoses , 46 wereIndividual variable data, collected via a self-report mailed questionnaire in 2000, included the big five personality traits  assessed with the revised NEO Personality Inventory , 48, sevSurvival analyses were completed in SAS  using Prp < .05 in Table p < .01 level so as to not over-interpret effects.The predictor variables included a range of demographic, psychiatric, and individual variables derived from various VET Registry assessments. The basic demographic variables were entered first in each model . The demographic variables that were significant predictors of the time lapse variable were then also included in each subsequent survival analysis as covariates. Otherwise, first five different analyses for a transition stage were completed, each including one set of predictors: (a) the three psychiatric risk variables, (b) the three family history risk variables, (c) the five personality traits, (d) the four drinking motives, or (e) the seven alcohol expectancies. Once predictors were identified within each of the five domains, two across-domain survival analyses were completed for each transition stage to see which predictors remained significant across domains: one model included the significant psychiatric and family risk factors together  and one included the significant individual predictor variables together . It is these across-domain model results that are presented here; the results from the within-domain models can be found in the Additional file SD = 2.9, range 43–63), had 13.7 (SD = 1.8) years of education on average, and had a mean household income of about $54,000 per year. This high-risk sample had a substantial family history of heavy alcohol use in that 49% reported that their father, 11% that their mother, and 48% that their twin brother had a history of excessive drinking. In terms of lifetime psychiatric history, 12% met criteria for PTSD, 11% met criteria for MDD, 5% met criteria for ASPD, 3% met criteria for GAD, and 2% met criteria for Panic Disorder. Concerning lifetime substance history, 56% met criteria for nicotine dependence, 47% for AD, and 22% for DD.This sample of 1769 COA/TAP male-male twin veterans had a mean age of 51.8 years  and the “Individual Predictors” model included all personality, drinking motives, and expectancy variables. As stated above, hazard ratios greater than one reflect “risk” for onset and relapse outcomes but “protection” for remission outcomes.Table Hypothesis 1 examined the consistency of factors predicting the three transitions of onset of alcohol use problems: years to first drink and subsequent years to either heavy drinking or AD. Four risk factors were identified as being associated with accelerating all three onset variables (hazard ratios greater than 1.0): nicotine dependence (ORs = 1.19–1.40), externalizing disorders (ORs = 1.14–1.41), cotwin drinking history (ORs = 1.28–2.22), and the alcohol expectancy of risk and aggression (ORs = 1.08–1.14). Another risk factor, the drinking motive of enhancement, predicted both earlier onset of heavy drinking and of alcohol dependence . Significant consistency was evident across these three transitions in risk, but four single-stage specific risk and protective factors were also identified. For drinking onset, both agreeableness (OR = 0.97) and neuroticism (OR = 0.98) were protective factors that delayed years to first drink. The unique association for years from first drink to onset of heavy drinking was the risk factor of a tension reduction expectancy (OR = 1.10). For years from first drink to first AD, the specific risks of internalizing disorders (OR = 1.38), father drinking history (OR = 1.30), and coping drinking motives (OR = 1.05) were related to quicker transition.p < 0.05 level, although with the same effect size as for heavy drinking. In comparing predictors across remission and onset transitions, there were seven significant predictors of either remission variable that were unique and not shared with the onset risk and protective factors, while three factors were consistent across the two remission transitions and also significant for at least one onset transition .Hypothesis 2 evaluated factors associated with remission in contrast to the various transitions of progression towards higher levels of use. Those factors that were related to accelerated remission were considered protective factors; those connected to delaying remission were risk factors. For years to remission of heavy drinking, significant risk for delayed remission came from externalizing disorders (OR = 0.89), the personality trait of extraversion (OR = 0.97), and the three drinking motives of enhancement (OR = 0.97), social (OR = 0.97), and coping (OR = 0.97) while significant protective factors accelerating remission were internalizing disorders (OR = 1.16), the traits of agreeableness (OR = 1.03) and conscientiousness (OR = 1.02), the drinking motive of conformity (OR = 1.05), and the expectancy of cognitive and behavioral impairment (OR = 1.14). Many fewer significant predictors of years to remission of AD were found but they were the same as the predictors of remission from heavy drinking: coping motives (OR = 0.95) again delayed remission and agreeableness again accelerated remission (OR = 1.03); externalizing was a risk factor only at the p < .05 level.Hypothesis 3 compared the predictors of onset transitions  with the factors associated with relapse after remission. Few factors predicted remission with a significant effect: only the risk factor of social drinking motives (OR = 1.10) and the protective factor of expectancy of liquid courage (OR = 0.74) for heaving drinking and the risk factor of coping motive (OR = 1.15) and protective factor of conformity motives (OR = 0.86) for AD. Of these, only two worked in similar ways during other transitions: social motives had also delayed remission of heavy drinking and coping motives had also accelerated initiation and slowed remission of AD. Surprisingly, the major onset risk factors of externalizing and cotwin drinking history predicted relapse of AD and predicted relapse of heaving drinking only at a Using conditional survival analysis, the current study examined the amount of time between transitions in the progression to initiation of drinking, heavy drinking, and diagnosis of alcohol dependence, and then further examined two later transitions, remission and relapse, thus characterizing the natural course of an alcohol use trajectory. This unique, time-based research method examined the predictors identified in current alcohol use disorder literature and their consistency in predicting progression from one transition to the next. Unlike other event-based research methods, this method examined each transition independent of earlier effects, thus providing a more precise characterization of factors relevant to that timeframe alone. Results will be discussed in terms of the hypotheses.Hypothesis 1 was supported in that there was consistency across predictors of the three onset transitions  with four risk factors associated with acceleration to all three onset transitions and one consistent across the two more problematic drinking transitions. Of note, however, is that some overlap in significant predictors may not be surprising because some of the same cases would be included, albeit with potentially different years-to-event, in the heavy drinking and AD models . The first two predictors were psychiatric variables that are comorbid with alcohol use: nicotine dependence and externalizing disorders. Research supports the idea that not only is there overlap between the occurrence of problematic use of nicotine and alcohol, but that common genetic underpinnings underlie risk for nicotine, alcohol, and other drug use , 55. In The third predictor was a family history variable: co-twin’s history of drinking. A substantial literature documents the role of family history in alcohol use , 5. ThesFourth, and turning to psychosocial influences, current findings implicated the alcohol expectancy of risk and aggression as a factor accelerating onset progression. In a sample of men, Patrick and Schulenburg  found thFifth, current findings implicated the drinking motive of enhancement as associated with accelerated progression toward heavy drinking and AD onset transitions. Enhancement motives have been shown to predict increased drinking quantity and drinking misuse from adolescence to middle-adulthood . Given tTaken together, these findings demonstrate the consistency of five well-recognized onset predictors of accelerated progression toward three key onset transitions: years to first drink, years from initiation to heavy drinking, and years from initiation to first AD. These results confirm and clarify current models of problem alcohol use etiology using a more precise methodology, and results at least partially confirm hypothesis 1 that onset risk factors are similar across the three onset variables. However, there were some stage-specific risk and protective factors as well. For example, the alcohol expectancy of tension reduction was related to accelerated progression to heavy drinking but not initiation or AD. Patrick and Schulenburg  used groHypothesis 2, that the predictors of delayed remission would be different than the predictors of accelerated onset and would likely be more psychosocial in nature, was supported. Of the five onset risk factors identified above, only externalizing disorders and the drinking motive of enhancement affected remission from heavy drinking while none affected remission from AD. There were a few other risk factors that delayed remission that were not consistent onset risk factors: namely the personality trait of extraversion and the social and coping drinking motives.Additionally, five other variables were identified as protective factors that were not significantly related to onset transitions. Thus, it was other aspects of our measured domains that had important connections to a quicker decrease in alcohol use. First, in terms of clinical variables, internalizing disorders speeded progress toward remission of heavy drinking. Internalizing disorders are well-substantiated risk factors that are central to the internalizing, negative affect pathway to alcohol use , 65. ThuThe other four factors related to accelerated remission were psychosocial in nature. Beginning with personality, agreeableness was found to accelerate remission from heavy drinking and AD and conscientiousness was found to accelerate remission from heavy drinking. The general personality profile associated with alcohol dependence is low agreeableness, low conscientiousness (related to impulsivity and disinhibition), and high neuroticism , 21, 64,Remission from heavy drinking was also accelerated by the drinking motive of conformity. Remission generally occurs later in one’s drinking career, whereas enhancement (enjoyment) motives may be most apparent at earlier ages and stages (as it was with onset of heavy drinking and AD in our sample), drinking motives change over time . In the p < .05 level, although sample size (as discussed below) was problematic for relapse transitions and could have affected significance levels. The other consistent onset risk factors of cotwin nicotine dependence, risk and aggression expectancies, and enhancement motives were also not significant for relapse. Instead, there were four predictors for relapse transitions that were not consistent onset predictors.Hypothesis 3 turned to the predictors of relapse, with the hypothesis that the factors associated with relapse would be similar to those associated with onset and progression of heavy drinking and AD finding little support. In the current data, after remission had been achieved, findings implicated that few, if any, of the consistent risk factors for onset were also risks for the relapse transitions. Externalizing disorders and co-twin history were surprisingly not strongly predictive, being significant only at the One of these was the risk factor of social drinking movies for accelerated relapse of heavy drinking. It appears that social drinking motives are not what is needed when one is in remission from an alcohol use disorder. The social aspect of drinking is potentially central to the ongoing debate between those who argue that moderation of drinking is the best path to recovery and Alcoholics Anonymous (AA) that insists on complete abstention from alcohol use . Social The factor that was found to be significantly related to accelerated relapse to AD was the drinking motive of coping. The results related to the risk factor of coping were interesting, in that coping was a consistent risk factor for all AD transitions  though not a significant predictor for most heaving drinking transitions (except remission). This finding underscores the greater risk of “coping by drinking” for those who have met criteria for AD and who are probably later in their alcohol use career. Other research has also found that drinking to avoid problems was related to heavier drinking in later early adulthood (age 22–30) . The curFor protective factors against relapse, the expectancy of liquid courage for heavy drinking and the motive of conformity for AD were fairly unique to these transitions. Of these, recall that a conformity motive was related to accelerated remission from heavy drinking. It may be that greater age and maturity increases interpersonal responsiveness (“conformity”) that, in turn, encourages both remission (from heavy drinking) and the delay of relapse (back to AD).In addition to the support found for hypotheses 1 and 2 and lack of support for hypothesis 3, another comparison in the current data could be interesting. Although we thought that more psychosocial variables would affect remission more than onset and relapse (part of hypothesis 2), which has some support, the data also suggested that there were more individual psychosocial predictors for first drink and heavy drinking transitions than for AD transitions. It is possible that more individual or cultural motives and expectancies could drive drinking frequency and quantity while more physiological components of addiction  may be predictive for diagnosable drinking issues. The conclusion here is tentative, given the smaller sample size for the AD transitions, but may be of potential interest for further investigation.This study was not without its limitations. The current sample included only men so it is unknown if the same variables would be associated with transitions in women: Research has suggested that late-life transitions of alcohol use may differ in timing for women compared to men . The samUsing time-based survival analyses to identify faster or slower progression to key developmental transitions in alcohol use, the current study examined domains of risk identified in current alcohol problem use etiology literature. Results confirmed that known risk factors were associated with faster progression toward three key onset transitions and then extended examination to subsequent remission and then later relapse. Further research should continue the effort to identify the predictors that hasten or slow the progression of drinking throughout an alcohol use career. The study of remission and relapse may be particularly informative to clinicians and others involved in the treatment of substance disorders. Importantly, the role of psychosocial variables as predictors of remission and the delay of relapse may provide empirically-identified avenues of treatment intervention that may inform developing treatment programs.Additional file 1: Table S1. Hazard ratios from survival analyses predicting rate of progression through stages of alcohol use, where predictors were entered in domain groups . Variables that were significant in these models were included in the models discussed in the manuscript and shown in Table


Of the 150,000 patients annually undergoing coronary artery bypass grafting, 35% develop complications that increase mortality 5 fold and expenditure by 50%. Differences in patient risk and operative approach explain only 2% of hospital variations in some complications. The intraoperative phase remains understudied as a source of variation, despite its complexity and amenability to improvement.The objectives of this study are to (1) investigate the relationship between peer assessments of intraoperative technical skills and nontechnical practices with risk-adjusted complication rates and (2) evaluate the feasibility of using computer-based metrics to automate the assessment of important intraoperative technical skills and nontechnical practices.This multicenter study will use video recording, established peer assessment tools, electronic health record data, registry data, and a high-dimensional computer vision approach to (1) investigate the relationship between peer assessments of surgeon technical skills and variability in risk-adjusted patient adverse events; (2) investigate the relationship between peer assessments of intraoperative team-based nontechnical practices and variability in risk-adjusted patient adverse events; and (3) use quantitative and qualitative methods to explore the feasibility of using objective, data-driven, computer-based assessments to automate the measurement of important intraoperative determinants of risk-adjusted patient adverse events.The project has been funded by the National Heart, Lung and Blood Institute in 2019 (R01HL146619). Preliminary Institutional Review Board review has been completed at the University of Michigan by the Institutional Review Boards of the University of Michigan Medical School.We anticipate that this project will substantially increase our ability to assess determinants of variation in complication rates by specifically studying a surgeon’s technical skills and operating room team member nontechnical practices. These findings may provide effective targets for future trials or quality improvement initiatives to enhance the quality and safety of cardiac surgical patient care.PRR1-10.2196/22536 Nearly 150,000 coronary artery bypass grafting (CABG) procedures are performed annually in the United States, and it is a procedure associated with a high rate of major adverse events (35% of patients) that vary by hospital . While uPrior research has evaluated the association between technical skills  and opeNontechnical practices  are botA surgeon’s nontechnical practices, manifesting as diagnostic failure  or a breThis study will evaluate how operative skills and nontechnical practices impact CABG outcomes. Patients undergoing CABG are at risk of harm due in part to the (1) reconstruction of anatomical structures under high magnification, (2) multiple high-risk phase transitions of care between team members , and (3) need for communication and teamwork  across many team members.Our proposed study is novel and innovative for three important reasons. To our knowledge, this study will be the first (1) multicenter intraoperative evaluation of both technical skills and operative team nontechnical practices at scale, (2) study to relate evaluation of intraoperative nontechnical practices with important clinical outcomes, and (3) study to apply a video-understanding platform to high fidelity recorded surgical videos to assess feasibility of automated objective assessments of technical skills and nontechnical practices.Preliminary Institutional Review Board (IRB) review has been completed at the University of Michigan by the Institutional Review Boards of the University of Michigan Medical School. This study will include a single IRB to govern research activities conducted across the collaborating hospitals.Our population will include adult patients undergoing electively scheduled CABG operations using cardiopulmonary bypass performed by attending surgeons at six hospitals participating in the Multicenter Perioperative Outcomes Group (MPOG) Collaborative, a national physician-led collaborative of academic and community hospitals, and specialty-specific peer assessors. Surgeons who have operated at their hospital for less than 2 years will be excluded.We will record 506 CABG operations at six hospitals. The study coordinator will use a randomization protocol from the Data Coordinating Center (DCC) to select, by week, different cardiac surgical operating rooms for video recording. The coordinator will synchronize the cameras with other operating room data sources  . Three CKey transitions in phases of patient care are routinely documented within the intraoperative electronic health record of participating MPOG hospitals. These data are validated and mapped to universal MPOG concepts . DigitalStudy data will be transmitted to the DCC, which will conduct audio and video quality checks across hospitals and recordings. Initially, investigators at the DCC will review the entire recording to fine tune the MPOG event timestamping to the exact second. Given the input operative data, a Hidden Markov Model ,26 or deThe DCC will provide monthly reports to hospitals, including number of digitally recorded operations, quality of transmitted digital recordings, and adherence to study operational protocols.We will use a two-stage process for recruiting candidate assessors as follows: (1) we will poll the Michigan Society of Thoracic and Cardiovascular Surgeons Quality Collaborative (MSTCVS-QC), MPOG, and the Michigan Perfusion Society membership for potential assessors working outside of Michigan or at hospitals not participating in MPOG, and (2) if unable to secure sufficient assessors, we will recruit from the MSTCVS-QC, MPOG, and Michigan Perfusion Society membership.Assessors, blinded to the hospital and operative team, will provide technical (aim 1) and nontechnical (aim 2) video-based assessments of cardiac surgical operations using a web-based Health Insurance Portability and Accountability Act (HIPAA)-compliant assessment platform.Each operation will receive at least 12 assessments (three for each provider group), with 20% of assessments rereviewed. Surgeons will assess a surgeon’s (1) technical skills (modified OSATS plus cardiac surgery–specific skills) via a validated 5-point behaviorally anchored scale and (2) nontechnical practices using NOTSS . AnestheWe will resubmit 20% of edited segments to the same (test-retest reliability) or other assessors (interrater reliability) using an intraclass correlation coefficient α ≥.670 for good concordance .We will use a web-based training and readiness module that will include (1) foundational knowledge of the relevant tool and (2) video examples of correct identification, categorization, and assessment. Assessors will have to reach 70% agreement with gold standard (investigative team) assessments to contribute to the study. A median 70% agreement with reference assessments will serve as a basis for assessor eligibility to conduct real case assessments .We will calculate each surgeon’s adverse event rate for the previous 2 years using each hospital’s Society of Thoracic Surgeons (STS) data.We will conduct peer-reviewer assessments of recorded CABG operations at six MPOG hospitals (representing 36 surgeons) to associate technical skills with major adverse events.Each surgical operation will be divided (using our video segmentation protocol) into prespecified phases containing the most critical operative portions. The DCC will distribute video segments for surgeon assessment via our HIPAA-compliant assessment platform that will provide assessors with a view of the operative field  rate. We will use clinical data from each center to adjust for covariates incorporated within the STS risk prediction models ,31.We will use linear mixed effect models to model assessments of surgical procedures where assessors and surgeons are included as random effects. We will quantify variation in peer-assessor assessments of a surgeon’s technical skills and use the intraclass correlation coefficient to measure interassessor reliability. We will use predictions of each surgeon’s technical skills from the linear mixed effect models as summary measures of a surgeon’s technical skills in subsequent analyses. Generalized linear mixed effect models with a logit link will then be used to associate a surgeon’s technical skills with our composite outcome. We will model surgeons and hospitals as random effects, accounting for the nesting structure of the data . We will adjust for patient and surgeon factors by including them as fixed effects in the models. The factors of interest are summary measures of a surgeon’s technical skills, which are included as surgeon-level explanatory variables. We will consider the overall assessments of a surgeon’s technical skills, averaged across three assessors and each domain individually.We use simulations to evaluate statistical power for a two-sided test (α=.05). Our analysis will be based on outcomes for approximately 7200 operations over 2 years from 36 surgeons (approximately 100 operations per surgeon) at six hospitals. We estimate having approximately 98% power in detecting an odds ratio of 0.85 per one unit (standardized) increase in a surgeon’s technical skills for the rate of adverse events.We will leverage each hospital’s intraoperative electronic health record system for video segmentation, using in part precomputed, validated, publicly available MPOG phenotypes ,32. SegmOur primary exposure will be the average summary peer assessment of each provider’s nontechnical practices. Similar to aim 1, the primary outcome will be the rate of major morbidity or mortality, adjusting for clinical covariates ,31.We will use generalized linear mixed effect models with a logit link to associate peer-assessor assessments of nontechnical practices of the surgeon with the surgeon’s STS composite score for major morbidity and mortality. Models will be similar to those described in aim 1, although we will include average summary measures of surgeon’s nontechnical skills as surgeon-level explanatory variables and hospital-level average summary measures of anesthesiologists, perfusionists, and scrub nurses. Both overall summary measures and individual scale domains will be considered. We will focus primarily on assessing the effects of nontechnical practices on morbidity and mortality rates, while adjusting for patient-level risk factors and a surgeon’s technical skills. We will explore the influence of nontechnical practices on the relationship between a surgeon’s technical skills and our composite endpoint by including nontechnical practices as an interaction term in models with technical skills.The power analysis is based on approximately 7200 cases across 36 surgeons from six hospitals. As surgeon’s nontechnical practices are considered a surgeon-level variable, there will be sufficient power in detecting the same effect sizes as reported in aim 1.High-dimensional computer-based assessments of digital recordings will be used to recognize and track human activity (computer vision). Computer vision focuses on training computers to derive meaning from visual imagery. Video understanding, a specialty within computer vision, focuses on identifying and tracking objects over time from video and developing mathematical models to train computers to extract the meaning within these moving images. This field may offer unparalleled capabilities for conducting objective peer assessments by automatically identifying and tracking human activity comparable to that of expert human assessors.Video understanding may address some of the limitations in traditional mentored or simulation-based approaches for assessing a surgeon’s technical skills, including human assessor bias and limited scalability. Prior investigations have documented the reliability of video-based surgical motion analyses for assessing laparoscopic performance as compared to the traditional time-intensive human assessor approach ,34. AzarNontechnical practice assessments have predominantly occurred within simulated environments and relied on trained human observers ,38. InveWe will explore the feasibility of using a video-understanding platform to identify important features associated with assessor ratings in recorded operations. To support developing the video-understanding platform, we will conduct interviews and site visits at a subset of low- and high-performing hospitals to enhance the understanding of a hospital’s contextual characteristics  and important “usual practices.”elements within and across video angles. We will use proven methods for video understanding , which will be applied to identify and measure surgeons’ technical skills (aim 1) and team-based nontechnical practices (aim 2). We will apply ambiguity reduction across the three time-synchronized video recordings to harmonize (rather than duplicate) boosting  and deepboosting ). We wilboosting ) and modboosting . We willboosting  and quanConcurrent with developing and testing the video-understanding platform, we will randomly select up to four of the six hospitals  participating in aim 2 for more detailed investigation. We will conduct semistructured interviews with interdisciplinary cardiac surgery operating room team members. To enhance our understanding of technical and nontechnical operating room practices, we will collect data (through interviews with intraoperative team members) concurrent with conducting analyses. We will develop a semistructured interview guide to encourage new and/or unexpected ideas or concepts to surface. For each interview, the interviewer will play back video segments from an operation involving the interviewee and ask the interviewee to describe his/her role within that operative phase. The interviewer will ask questions seeking to better understand team member roles and influences on technical skills and nontechnical practices. We expect the guide will consist of seven to nine open-ended questions with probes. Interviewers will participate in a 3-day training program at the University of Michigan Health Communications Laboratory. Interviews will continue until reaching informational redundancy “saturation” at each hospital. We will (1) conduct 40 to 60-minute interviews in private rooms, (2) digitally record and transcribe transcripts verbatim, (3) compare 10% of transcripts (and correct as needed) against the recordings, and (4) provide interviewees with a gift certificate. We expect that (1) in reviewing the videos, providers will complement peer assessments regarding how and why contextual factors influence performance  and (2) interviewees will validate the video content to maximize our video understanding algorithm’s fidelity. Thus, our interview findings will improve our interpretation of the video content to iteratively inform and enhance our video-understanding platform’s training.Our primary outcome will be the features derived from the video-understanding platform, which will be compared to a gold standard human identifying the same features. Features, as economy of motion, are derived from the raw output of the video-understanding platform, which naturally performs visual detection and visual tracking. The gold standard uses the analogous “raw output” from humans and the same method for the computation of the derived feature.We will assess our video-understanding platform’s ability to correctly identify and track features within our testing data set. Using the raw video in the testing set, we will provide the necessary bounding-box annotations for each feature, which will be compared to the automatically generated features from the video-understanding system using standard metrics . Results of aims 1 and 2 will likely yield assessments that identify a wide range of variations in both surgeons’ technical skills and nontechnical practices as has already been documented in the literature. Where our study will make an important contribution is in associating these assessments with adverse event rates. The novel contribution of aim 3 will be to associate computer-based assessments with adverse event rates, as a more objective and reliable replacement for human peer assessors, moving us closer to our overall goal of improving outcomes for cardiac surgery patients. We will use the study results to develop data-driven technical skills and nontechnical practice coaching interventions across a subset of hospitals. We plan to undertake our study over a 5-year period .There is increasing demand from the public and payers to improve health care value . Despite wide variability in cardiac surgical quality and robust clinical data from the STS for risk adjustment and outcomes ascertainment, only 2% of hospital variability in some outcomes are explainable by currently recorded data elements . AnalysiOur proposed study, leveraging the infrastructure and track record of two established physician-led quality collaboratives integrated with a cutting-edge scalable video-understanding platform, will advance our understanding of how surgical skills and nontechnical practices impact outcomes. Our approach aimed at identifying key modifiable intraoperative determinants of major adverse events may likely be applied to approximately 200,000 additional cardiac surgical procedures involving valve repair or replacement, aortic procedures, and percutaneous cardiac procedures  or other high-risk noncardiac surgical specialties .Although unlikely, there are a few potential challenges with this study.There is a remote possibility that we will not find that the investigated technical skills are associated with adverse events. If needed, we will expand our review of surgical operations to include (1) hospitals with lower operative volume, (2) longer segments for peer rating, (3) an expanded list of operative phases that might distinguish between high- and low-performing surgeons, and (4) high-risk or technically challenging operations. We will consider expanding to other hospitals if (1) hospital variability in adjusted adverse events is less than anticipated or (2) we are unable to amass sufficient digital recordings from our initial six hospitals.If needed, we will (1) expand our sampling pool of assessors to include providers who have expressed desire to partner on this project but were not selected initially, (2) provide monthly feedback and engagement support to participating assessors, and (3) provide expanded assessor training and calibration.Our video-understanding platform may not be completely automated. Alternatively, we will consider a semiautomated platform that relies, for instance, on a human periodically manually annotating the relevant features in the video at a certain segment and then allowing the video-understanding platform to interpolate those annotations.


Scophthalmus maximus), a commercially valuable flatfish species, combining transcriptomic and histopathological analyses. The myxozoan parasite Enteromyxum scophthalmi, which represents a major challenge to turbot production, was used as a model of infection. The thymus tissues of healthy fish showed overrepresented functions related to its immunological role in T-cell development and maturation. Large differences were observed between the transcriptomes of control and severely infected fish. Evidence of inflammatory response, apoptosis modulation, and declined thymic function associated with loss of cellularity was revealed by both genomic and morphopathological analyses. This study presents the first description of the turbot thymus transcriptome and provides novel insights into the role of this organ in teleosts’ immune responses.The thymus is a primary lymphoid organ that plays a pivotal role in the adaptive immune system. The immunobiology of the thymus in fish is considered to be similar to that of mammals, but it is actually poorly characterized in several cultured teleost species. In particular, while investigations in human and veterinary medicine have highlighted that the thymus can be affected by different pathological conditions, little is known about its response during disease in fish. To better understand the role of the thymus under physiological and pathological conditions, we conducted a study in turbot ( The thymus is a primary lymphoid organ that plays a critical and unique role in the development of T cells. This organ provides the necessary microenvironment for the precursors of lymphocytes to proliferate, rearrange their receptors, and finally differentiate into mature T lymphocytes ,2,3. OncThe immunobiology of this organ, particularly its pivotal role in the adaptive immune response as the main site of T-cell development, is considered to be similar in fish and mammals ,7. DespiIn terrestrial species, it has been demonstrated that different pathological conditions, such as malnutrition and infectious diseases, can affect the thymus, disrupting its architecture and function and having a detrimental effect on the ongoing immune response ,10,11. TScophthalmus maximus, a commercially important flatfish species, the gross anatomy and histology of the organ in juvenile fish was described by Vigliano et al. [The thymus in fish usually appears as a paired organ located in the dorsal region of each gill chamber ,15. In to et al. . In thiso et al. ,17. NeveEnteromyxum scophthalmi, is a fast-spreading parasitic disease with a great impact on turbot aquaculture. E. scophthalmi is transmitted directly between animals, triggering a cachectic syndrome associated with poor growth performance and high mortality rates [Enteromyxosis, caused by the myxozoan ty rates . The maity rates . Recentlty rates ,21. The ty rates . NeverthHere, we employed a multidisciplinary approach to improve the knowledge of this primary lymphoid organ in teleosts under physiological and pathological conditions. We analyzed the transcriptomic profiles and performed histopathological studies of the thymuses of healthy and experimentally infected turbot, using enteromyxosis as the disease model.The experimental design was described in a previous paper (see Reference ). BrieflE. scophthalmi parasites, whereas control fish were inoculated with the same amount of HBSS alone. Donor turbot came from an experimentally infected stock maintained at IATS . Tissue samples from control and infected fish were collected at 7, 24, and 42 days postinoculation (dpi) and were preserved in Bouin’s fluid and RNAlater for histopathological and transcriptomic analyses, respectively. The experimental infection was carried out by oral route; receptor fish received 1 mL of intestinal scraping homogenates in Hank’s balanced salt solution (HBSS) from 20 donor fish containing live The health status of control and infected fish was assessed by light microscopy on H&E- and toluidine-blue-stained sections. Challenged fish were classified into three groups  according to the histopathological grading described by Bermúdez et al., which considers the lesional degree and parasite burden . For theThe experiment was carried out following international , national , and institutional regulations .Thin sections (3 µm thick) were placed on slides treated with silane to improve section adherence and dried overnight at 37 °C. Immunohistochemical markers of cell proliferation , apoptosis (active caspase-3), and T cells (CD3ε) were used, together with antibodies against the proinflammatory molecules tumor necrosis factor alpha (TNF-α) and inducible nitric oxide synthase (iNOS) . The antEndogenous peroxidase activity was quenched by incubation with a peroxidase-blocking solution . Background prevention and antigen retrieval were performed as required. After incubation with primary antisera, slides were incubated with peroxidase (HRP)-labeled polymer conjugated to rabbit secondary antibody and peroxidase reaction was developed using a diaminobenzidine-positive chromogen . All incubations were performed in a humid chamber at room temperature. Finally, sections were counterstained with hematoxylin, dehydrated, and coverslipped with DePeX mounting medium. The sections were washed three times for 5 min in 0.1 M phosphate-buffered saline containing 0.05% Tween-20 between all subsequent steps, and positive and negative controls were included in each assay to assess its specificity. ® ND-1000 spectrophotometer , respectively. Samples were barcoded and prepared for sequencing by the Wellcome Trust Centre for Human Genetics  on an Illumina HiSeq 4000  as 150 bp paired-end reads. RNA extraction was performed using the RNeasy mini kit  with DNase treatment following the manufacturer’s instructions. RNA quality and quantity were evaluated in a Bioanalyzer  and in a NanoDrophttp://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Quality filtering and removal of residual adaptor sequences were conducted on read pairs using Trimmomatic v.0.38 [Raw sequencing data were deposited in NCBI’s Short Read Archive (SRA) under BioProject ID PRJNA623212. The quality of the sequencing output was assessed using FastQC v.0.11.5 (c v.0.38 . Specifihttp://www.R-project.org/). Gene count data were used to estimate differential gene expression using the Bioconductor package DESeq2 v.3.4 [p-value < 0.05 were considered to be enriched.Filtered reads were mapped to the most recent turbot genome assembly  using Sq2 v.3.4 . Transcrq2 v.3.4  and Kyotq2 v.3.4 . GO and The histological evaluation of the thymuses did not reveal any significant lesion in controls or in experimentally infected fish graded as slightly or moderately infected. Conversely, most fish with severe infections showed some degree of lymphocyte depletion in the outer zone of the organ, sometimes accompanied by an alteration of tissue architecture, namely a loss of differentiation between the inner and outer zones A,B.The immunohistochemistry for CD3ε and PCNA was strongly positive in healthy (control) thymus, especially in the outer zone, where most labeled cells were consistent with thymocytes C,E. No dThe immunochemical staining pattern of TNF-α in turbot thymus under physiological conditions has been previously described . AccordiSimilarly, few iNOS-immunoreactive cells were found in the thymus tissues of control and slightly infected turbot. The immunoreactivity was mainly observed in the epithelial layer and in the cytoplasm of some mucous and macrophage-like cells C. SevereRegarding the apoptotic marker active caspase-3, the immunolabeling was mainly detected in scattered cells consistent with thymocytes in the outer zone of the organ. The result was similar when comparing control and slightly infected turbot, while a mild increase of caspase-3 was detected in severely infected fish, which showed a higher density of positive thymocyte-like cells E,F. To characterize the turbot thymus transcriptome, we studied different subsets of highly expressed genes, selected based on their expression levels in control animals , and performed GO term and KEGG pathway enrichment analyses . The enrOnly four genes showed differential expression between slightly infected and control fish, whereas a total of 4640 differentially expressed genes (DEGs) were detected between the thymus transcriptomes of control and severely infected turbot: 2150 upregulated and 2490 downregulated . p-value: 0.087, p-value: 0.0055; GO term and KEGG pathway enrichment analyses were performed to study these DEGs between control and severely infected turbot . Both seE.-scophthalmi-infected turbot according to a histopathological grading system [The structural and functional characterization of the thymus has scarcely been addressed in many teleost species to date. In particular, the response of the thymus during diseases has barely been studied in cultured species, although several diseases of relevance in aquaculture are known to compromise the immune response and/or cause malnutrition. Infectious diseases and malnourishment are conditions markedly connected to thymus alterations in mammals ,10,11. Ig system . This apMost of the functions enriched in the thymus tissues of control turbot were related to energy production, protein synthesis, and cell proliferation. This reflects the high anabolic environment of this organ in physiological conditions, also described for terrestrial vertebrates and associated with lymphoblastic proliferation . AccordiThis study was focused on turbot juveniles, which we expected to have fully functional thymuses. In future studies, it would be interesting to assess the potential histological and transcriptomic changes related to aging in turbot. Age-associated thymic involution is a process that apparently occurs in all terrestrial vertebrates ,37, but The lesions associated with disease-related effects on thymus tissue and the underlying pathogenic mechanisms are beginning to be understood from studies in human and murine models. Malnutrition and infection by a variety of pathogens, including parasites, have been shown to induce similar atrophy-related alterations in the thymus, consisting of cell depletion, reduction of the cortex:medulla ratio and loss of corticomedullary limit distinction ,11,39. TE. scophthalmi showed intense transcriptomic changes at advanced stages of infection, whereas hardly any changes were noticed in fish with incipient infection, whether with morphological or with transcriptomic techniques. This might be related to a delayed response of the organ to the infection, which is characterized by a long pre-patent period and circumscribed to the gastrointestinal tract [The thymus tissues of turbot infected by al tract . Furtheral tract . Turbot enteromyxosis is a serious disease against which the host is incapable of triggering an effective immune response. Previous studies (reviewed in Reference ) have shMycobacterium avium [Trypanosoma cruzi [Plasmodium berghei in mice, which have been related to thymus dysfunction via the alteration of extracellular matrix (ECM) proteins [In mammals, pro-inflammatory mediators have been suggested to be responsible for the deleterious effects of infection on the architecture and function of the thymus, inducing apoptosis or alterations of the thymic microenvironment ,12. Nitrum avium , and TNFma cruzi . It has proteins . In thatOn the other hand, enteromyxosis induces a cachectic syndrome, which is particularly evident in advanced stages of infection, during which fish show severe weight loss and anorexia . LymphoiE. scophthalmi (The adipocyte-secreted hormone leptin has been indicated as a possible mediator of malnutrition-induced thymic atrophy in mammals ,12. Decrphthalmi . T. cruzi and P. berghei [Both infectious diseases and malnutrition have been shown to ultimately provoke alterations of chemokines and ECM proteins of the thymic microenvironment ,12,13,14 berghei ,11,13. E. scophthalmi. However, the mechanisms by which this process is activated and the extent of its role in the development of thymus atrophy should be carefully investigated. A recent study on the thymus tissues of malnourished mice infected by Leishmania infantum showed that the alteration of the thymic microenvironment was the main cause underlying thymic atrophy, rather than an increased thymocyte apoptotic rate [Regarding apoptosis, the transcriptomic data revealed a modulation of this pathway at advanced stages of enteromyxosis. Moreover, the immunohistochemical technique for the detection of active caspase-3, an effector protease in the apoptotic process, indicated an increased presence of this protein. Thymocytes physiologically undergo apoptosis during the processes of positive and negative selection , and ourtic rate . Furthertic rate ,14. Indetic rate ,11,12. IAlthough further investigations on the underlying mechanisms are necessary, strong evidence of declined thymic function during enteromyxosis was found in our study. Many genes found to be highly expressed in the thymus tissues of control fish, and putatively associated with thymic function, were found to be downregulated in infected fish. These genes were related to the immunological role of the organ, such as antigen presentation or lymphocyte differentiation, as well as to the processes of protein synthesis and cell proliferation. The transcriptomic profile was concordant with immunohistochemical and histopathological findings. The immunohistochemistry to PCNA confirmed a minor cell proliferation activity, which is a common feature observed in the thymus during infection and malnutrition ,14,39. FE. scophthalmi in turbot has been previously associated with the cell depletion suffered by the lymphohematopoietic organs [E. scophthalmi. It is plausible that common pathogenic mechanisms of cell depletion occur in all lymphoid organs during enteromyxosis, with a probable synergistic effect of inflammatory mediators and malnutrition. A comprehensive understanding of these mechanisms might help to implement therapeutic measures [Interestingly, cell depletion is also typically observed in spleen and kidney tissues at advanced stages of enteromyxosis . This obc organs ,49,50, ameasures ,39.The present investigation provides the first description of the turbot thymus transcriptome and gene expression profiling integrated with morphological observations. The results indicate the functional relatedness of this organ with that of mammals in physiological conditions. The significant changes found in advanced enteromyxosis suggest that thymic function is strongly affected, pointing towards the development of thymus atrophy as a consequence of the disease. This situation might contribute to the failure of the turbot immune response in fighting the disease. More studies are needed to increase our knowledge of the physiological and pathological thymus microenvironment and to investigate thymus atrophy during diseases in fish. This future research will benefit from combining genomic and proteomic approaches as well as from the identification of further immunohistochemical markers for the characterization of the thymus microenvironment and T-cell subsets. Overall, the present findings highlight that the role of the thymus in pathological conditions should not be overlooked, as it can contribute to a deeper understanding of host–pathogen interactions in infectious diseases.


A qualitative study among gay and bisexual men examining shigellosis-related knowledge, attitudes, and practices revealed low knowledge levels, limited concern about shigellosis, and mixed intention to engage in prevention behaviors. Shigella. Little is known about shigellosis-related knowledge, attitudes, and practices among gay, bisexual, and other MSM.Shigellosis is a highly contagious enteric bacterial disease transmitted through the fecal-oral route. It is primarily transmitted through person-to-person contact and via contaminated food and water. Outbreaks of shigellosis among men who have sex with men (MSM) attributed to sexual person-to-person contact have been reported. These outbreaks are of concern because they are often caused by multidrug-resistant strains of Six focus groups were conducted among self-identified gay or bisexual men in Atlanta, GA, in Fall 2017. Participants were asked about shigellosis-related knowledge, attitudes, and practices. Focus groups were audio recorded, and the transcribed audio was analyzed using inductive and deductive thematic coding.Shigella was low, with most never having heard of the disease or bacteria. Participants did not perceive shigellosis to be a serious health concern, especially when compared with HIV; however, they did perceive gay and bisexual men to be at risk of Shigella infection. Participants reported mixed intentions to change sexual behaviors to prevent shigellosis or talk with sexual partners about diarrhea.Among the 24 focus group participants, most perceived that diarrheal illness was caused by contaminated food. Knowledge of shigellosis and Health communication and education efforts could be used to increase knowledge about shigellosis and shift perceptions about the severity of shigellosis among gay, bisexual, and other MSM. Additional work is needed to identify effective ways to promote shigellosis-related prevention behaviors among gay, bisexual, and other MSM. Shigella bacteria and is the third most common bacterial enteric disease in the United States, causing an estimated 500,000 cases annually.1Shigella bacteria are transmitted through the fecal-oral route, and people can become sick if they come into contact with contaminated food, water, objects, hands, or through sexual contact.3 Most outbreaks of shigellosis in the United States are attributed to person-to-person contact,4 as are most sporadic cases of Shigella infection in the United States.5 Although most shigellosis cases in the United States occur among children younger than 10 years,6 other populations at risk include international travelers7 and men who have sex with men (MSM).10Shigellosis is an acute diarrheal illness caused by 11 Since that time, cases, clusters, and outbreaks of shigellosis among MSM have been reported in the United States13 and elsewhere,16 with many of these outbreaks believed to be associated with spread of Shigella bacteria through sexual contact.3 The burden of sporadic shigellosis among MSM in the United States is not well characterized, and the number of clusters or outbreaks of shigellosis among MSM in the United States is also not known. Shigellosis among MSM is of concern because multiple outbreaks and clusters have been caused by drug-resistant strains of Shigella.15 Drug-resistant Shigella is an emerging public health threat,17 and resistant infections can be more difficult to treat and can result in more severe health outcomes.Outbreaks of shigellosis have been reported among MSM since the early 1970s.16 In addition, coinfection with a sexually transmitted infection or with HIV has also been reported among MSM cases of shigellosis.13 Less is known about unique risk factors for shigellosis among MSM, but some that have been identified include being HIV positive and having direct anal contact before illness onset.9 Knowledge and awareness of shigellosis among MSM seem to be low. In one study conducted in the United Kingdom among 3646 MSM, only 26% had heard of Shigella, 16% were aware that Shigella can cause stomach upset and can be spread by food and through sex, and 16.3% were aware that Shigella can spread easily.18 In addition, in 2 outbreak investigations in the United Kingdom, few MSM had heard of shigellosis before their illness.10 For this study, we sought to better understand shigellosis-related knowledge, attitudes, and practices (KAPs) among gay and bisexual men, and identify opportunities for health communication and education efforts.Outbreak and other epidemiological investigations among MSM have identified that a range of behaviors cases have engaged in before becoming sick with shigellosis. This includes oral and anal sex, direct oral-anal contact , multiple sex partners, use of drugs or alcohol before or during sex, and finding sex partners through online dating applications or in sex venues like bath houses.Six focus group discussions were conducted between November and December 2017 in Atlanta, GA. Participants were eligible for the study if they (1) were 18 years or older, (2) were cisgender male, (3) self-identified as gay or bisexual, (4) reported having sex with another man in the past 3 months, and (5) were able to speak and understand English. The term MSM will not be used to refer to participants in this study as all participants in this study self-identified as gay or bisexual.Focus group participants were recruited using 2 strategies. First, Georgia State University (GSU) team members partnered with local organizations and health care providers serving gay and bisexual men in the greater Atlanta area to distribute an informational flyer describing the focus groups. Second, study staff asked individuals interested in participating in the focus groups to share information about the project within their social networks. Individuals interested in participating in the focus groups were directed by recruitment materials to call project staff, who screened potential participants for eligibility. Eligible individuals were invited to attend 1 of 6 focus group sessions that best fit with their schedules.Before the start of the focus groups, participants completed a brief demographic questionnaire that contained questions about age, sex, race, ethnicity, education level, and use of mobile dating applications. A semistructured moderator guide was used to facilitate the focus group discussions. The interview guide included questions on health-related help-seeking behaviors, KAPs related to shigellosis, and health education messaging preferences. Focus groups were audio recorded, and field notes were taken to capture nonverbal responses and emergent focus group themes. Audio recordings of the focus groups were transcribed verbatim, and the transcripts were used as the basis of all analyses.Thematic analysis was used to analyze focus group transcripts, with segments of texts assigned both deductive and inductive codes. A preliminary codebook was developed after one transcript was collectively coded by 4 researchers. All transcripts were then coded by a minimum of 2 coders. Each transcript was coded deductively using the codebook, and new inductive codes were also applied to transcripts during the coding process. All discordant deductive codes were discussed until 100% agreement between coders was achieved. Newly identified inductive codes were also discussed until 100% agreement on code name, definition, and instructions for use was obtained among coders. These newly identified inductive codes were then added to the codebook and used prospectively.19Once all transcripts were coded and all new inductive codes were added to the codebook, all 6 transcripts were recoded a second time. During this second round of coding, a minimum of 2 coders discussed all codes applied in each transcript, and codes were updated to reflect any changes to the codebook made during the first round of coding. All data management, coding, and analyses were completed using qualitative data management software MAXQDA 12.3.5.Human subjects research approval was obtained from the GSU institutional review board). Informed consent was obtained from all participants, and copies of informed consent documents were provided to all participants. Participants were provided with a $40 Visa gift card as compensation for their time and were compensated even if they chose to withdraw from the focus group during the session. All recruitment and data collection activities were conducted by GSU.A total of 24 participants were recruited for the 6 focus groups. Each focus group discussion lasted approximately 1 hour, and the number of participants in each group ranged from 2 to 6. Participants ranged in age from 21 to 59 years, with a mean age of 36 years. More than half of participants  were non-Hispanic African American, 38% (n = 9) were non-Hispanic white, and 8% (n = 2) were Hispanic, of any race. Less than half  of participants had completed a 4-year college degree, another 25% (n = 6) reported having postgraduate education, and 25% (n = 6) had completed high school or some college  during sexual activity, washing genitals and the anus before sex, and avoiding sex while sick with shigellosis. For these sex-related prevention strategies for shigellosis, more research is needed to determine which are most effective for preventing shigellosis and which are most effective at preventing the spread of shigellosis among people who are sick or recently sick. In addition, both qualitative and quantitative research is needed to understand the determinants of both general and sex-related shigellosis prevention behaviors among MSM. Finally, theory-driven research should be conducted to identify variability in determinants of behaviors among different subpopulations of MSM who may be at particular risk of becoming sick with shigellosis , of spreading shigellosis to others , or of having more severe health outcomes .KAPs identified in this study, including lack of knowledge and openness to learning more about shigellosis, may be different from shigellosis-related KAPs among gay and bisexual men in other parts of the United States and may be different from MSM who do not identify as gay or bisexual. This was a small qualitative study conducted among gay and bisexual men in Atlanta, GA, and results are not generalizable to other populations. Additional qualitative and quantitative work is needed to examine shigellosis-related KAPs among a wider range of MSM in the United States and among subpopulations of MSM who may be at particular risk.Shigellosis prevention efforts among gay, bisexual, and other MSM could include health education and communication aimed at increasing knowledge about shigellosis and shifting perceptions about the severity of shigellosis. Future research that describes effective shigellosis prevention methods among gay, bisexual, and other MSM, including strategies for promoting prevention behaviors, is needed.


Coffee drinking has been inversely associated with liver cancer consistently in prospective studies. Yet, the specific compounds underlying this association, and whether associations vary by preparation method, are unknown. Associations with other sites within the gastrointestinal tract are also unclear. A recent study by Tran et al. leverages the resources of the UK Biobank to begin answering these questions, and suggests important avenues for future work. Such conclusions are consistent with those of a recent meta-analysis3 that indicated evidence for inverse associations with cancers of the breast, colorectum, endometrium, liver, prostate and skin.Coffee is one of the most widely consumed beverages worldwide, despite often being considered a guilty pleasure due to long-standing concerns that coffee drinking may increase risks of cancer, cardiovascular disease and other chronic diseases. Yet, the accumulated research literature paints a different picture. In 2015, the U.S. Dietary Guidelines Advisory Committee concluded that drinking up to five cups of coffee per day was compatible with a healthy diet.4 Whether the differences in chemical composition influence coffee-cancer associations is largely unknown, but exploring such exposure heterogeneity may provide important clues about disease aetiology.Nevertheless, there remain numerous questions regarding the impact of coffee drinking on health. The chief among them is a lack of understanding as to the mechanisms by which coffee drinking may be related to disease. A cup of coffee is a mixture of hundreds, possibly thousands, of compounds, and the levels of these compounds are altered by a variety of production and preparation processes, including decaffeination, freeze-drying, roasting and brewing. A recent metabolomics study investigating the differences in the chemical composition of commonly consumed coffee types found that instant coffee, with particularly high levels of diketopiperazines and lower levels chlorogenic acids among other differences, markedly differed from other coffee types.5 examine the association of self-reported coffee drinking with the risk of digestive cancer in the UK Biobank cohort. Despite the large size of the cohort, follow-up remains relatively short, so the numbers of individual cancers and associated statistical power was relatively low for most sites. However, unlike most cohort studies, UK Biobank participants reported whether they typically consumed decaffeinated, instant, ground or other types of coffee. In this way, Tran et al.5 provide an important methodological advance over previous studies.Within this context, Tran et al.5 found no evidence that the inverse association between coffee drinking and HCC varied by coffee type. Despite small case numbers, these results are consistent with a few prior studies that evaluated brewing type, such as in the ATBC study of Finnish smokers, where inverse associations for coffee drinking with incident liver cancer were observed for both boiled and filtered coffee.6 The results for other end points may differ, however. For example, a study on coffee drinking and mortality, which also used data from the UK Biobank cohort, observed stronger inverse associations for ground than for instant coffee intake, particularly for cardiovascular disease mortality.7In line with earlier studies, they found that coffee drinking was associated with a lower risk of hepatocellular carcinoma (HCC), the most common type of primary liver cancer, although associations with other types were largely null. Importantly, Tran et al.https://epi.grants.cancer.gov/dhq3/) and the Automated Self-Administered 24-Hour  ask for additional details on the type of coffee consumed.As follow-up in the UK Biobank continues and the cohort matures, it will be important to update these analyses. In addition, we encourage future cohorts to comprehensively assess coffee type and preparation. To aid in this effort, current versions of dietary assessment tools like the National Cancer Institute’s Dietary History Questionnaire III  Cancer Screening Trial cohort, biomarkers of caffeine identified using metabolomics were associated with incident colorectal cancer,8 whereas associations with self-reported coffee drinking were null. A study on coffee-related metabolites, liver cancer and liver disease mortality in Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study cohort was recently published in the Journal of National Cancer Institute.9 This study found that metabolites that were positively correlated with coffee drinking, including trigonelline and serotonin, were associated with lower risk of liver cancer and liver disease death, whereas metabolites that were negatively correlated with coffee drinking, including two bile acids, were associated with higher risk of liver cancer and liver disease death. A separate investigation, published in the same journal, identified an association between pre-diagnostic bile acid concentrations and colon cancer risk in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort.10 These observations are supported by experimental models demonstrating that bile acids are critical to the gut–liver–axis, and may contribute to the pathogenesis of liver, colorectal and perhaps other digestive diseases.11 These exciting results provide plausible mechanisms linking coffee drinking to gastrointestinal cancer, and merit replication in future studies. The impact of coffee roasting and brewing method on biomarkers of exposure, and response to coffee drinking, remains to be determined; however, we advocate for future studies coupling improved assessment of coffee drinking with biochemical measurements to provide insight into the potential role of coffee drinking on cancer.Studies using biomarkers present another promising line of inquiry in coffee-cancer research. Biomarkers can serve both as objective markers of coffee intake and as indicators of the underlying mechanisms. Coffee drinking has consistently been inversely associated with biomarkers of diabetes and insulin resistance, and inflammation.5 begin the important process of examining the impact of coffee preparation method. Improved self-report via automated 24-h recalls, and cloud computing coupled with advances in studying the human microbiome, genetic variation and metabolism, as well as high-throughput and reproducible biochemical assays suggest an emerging renaissance in nutritional studies.12 Future studies incorporating these new approaches have great potential to clarify the impact of coffee drinking on health, and identify the underlying mechanisms. It is time that we brew a fresh pot and get to work.Despite long-standing concerns that coffee drinking may cause cancer, the body of evidence suggests that coffee drinking may instead have some beneficial qualities. Yet, the mechanisms by which coffee drinking may affect cancer have not been established. Tran et al.