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Table S4

High-grade serous ovarian cancer (HGSC), the principal cause of death from gynecologic malignancies in the world, has not significantly benefited from advances in cancer immunotherapy. Although HGSC infiltration by lymphocytes correlates with superior survival, the nature of antigens that can elicit anti-HGSC immune responses is unknown. The goal of this study was to establish the global landscape of HGSC tumor-specific antigens (TSA) using a mass spectrometry pipeline that interrogated all reading frames of all genomic regions. In 23 HGSC tumors, we identified 103 TSAs. Classic TSA discovery approaches focusing only on mutated exonic sequences would have uncovered only three of these TSAs. Other mutated TSAs resulted from out-of-frame exonic translation (n = 2) or from noncoding sequences (n = 7). One group of TSAs (n = 91) derived from aberrantly expressed unmutated genomic sequences, which were not expressed in normal tissues. These aberrantly expressed TSAs (aeTSA) originated primarily from nonexonic sequences, in particular intronic (29%) and intergenic (22%) sequences. Their expression was regulated at the transcriptional level by variations in gene copy number and DNA methylation. Although mutated TSAs were unique to individual tumors, aeTSAs were shared by a large proportion of HGSCs. Taking into account the frequency of aeTSA expression and HLA allele frequencies, we calculated that, in Caucasians, the median number of aeTSAs per tumor would be five. We conclude that, in view of their number and the fact that they are shared by many tumors, aeTSAs may be the most attractive targets for HGSC immunotherapy.

Supplementary figures and legends

Objective Prolonged opioid use is common following traumatic injuries. Although preventive strategies have been recommended, the evidence supporting their use is low. The objectives of this study were to select interdisciplinary strategies to prevent long-term, detrimental opioid use in trauma patients for further evaluation and to identify implementation considerations. Design A consensus study using the nominal group technique. Setting Four trauma systems in Canada. Subjects Participants included expert clinicians and decision makers, and people with lived experience. Methods Participants had to discuss the relevance and implementation of 15 strategies and then rank them using a 7-point Likert scale. Implementation considerations were identified through a synthesis of discussions. Results A total of 41 expert stakeholders formed the nominal groups. Overall, eight strategies were favored: 1) using multimodal approach for pain management, 2) professional follow-up in physical health, 3) assessment of risk factors for opioid misuse, 4) physical stimulation, 5) downward adjustment of opioids based on patient recovery, 6) educational intervention for patients, 7) training offered to professionals on how to prescribe opioids, and 8) optimizing communication between professionals working in different settings. Discussions with expert stakeholders revealed the rationale for the selected strategies and identified issues to consider when implementing them. Conclusion This stakeholder consensus study identified, for further scientific study, a set of interdisciplinary strategies to promote appropriate opioid use following traumatic injuries. These strategies could ultimately decrease the burden associated with long-term opioid use.

Protecting freshwater biodiversity is considered an ultimate challenge but depends on reliable surveys of species distribution and abundance which eDNA metabarcoding (environmental DNA metabarcoding) may offer. To do so, a better understanding of the sources of temporal variation among species eDNA abundance and of data transformation in eDNA metabarcoding studies is needed. Here, we show that transformation based on relative abundance is critical to suitable analyses of eDNA metabarcoding data and that Hellinger transformation performed slightly better than other methods. Furthermore, we show that site localities significantly explain eDNA metabarcoding variation, while no variation is explained by time of sampling. This indicates that species communities vary more spatially than temporally within a dendritic system composed of small rivers. We then further documented the community structure in the St. Charles River (Québec City, Canada) and six of its tributaries. This revealed the existence of eight species communities explaining 82.1% of eDNA read variation within this river network. Moreover, variation in environmental variables among sites explained 53.0% of eDNA reads, while sampling events and temporal environmental variation explained no eDNA metabarcoding variation. Altogether, this supports the claim that eDNA metabarcoding is a powerful tool to document and monitor fish communities in watersheds composed of small river dendritic systems.

Objective Up to 50% of patients develop high-impact chronic pain after an acute care experience and many psychological variables have been identified in this process. We conducted a systematic review and meta-analysis of randomized controlled trials to assess the effect of psychological interventions within 3 months after pain onset. Methods We searched databases for articles published until July 2019. We used standardized mean differences with 95% confidence internals to assess treatment effect. Results Eighteen trials were found eligible; 11 of which were included in the meta-analyses. Trials were mainly performed in back pain patients in the middle to late adulthood. Regarding pain intensity, the effect of psychological interventions compared to standard treatments was non-significant at 3 months, 6 months and 12 months. We found a moderate significant effect size in favor of psychological interventions compared to standard treatments with regard to disability at 12 months and a small significant effect with regard to coping with pain at 3 months when compared to information. Most of the meta-analyses' findings were associated with a low level of evidence. Discussion This systematic review and meta-analysis showed no significant effect of psychological interventions on pain intensity. A positive and significant trend related to these interventions was shown on disability and coping with pain, when compared to standard treatment and information, respectively. However, these findings must be interpretated with caution considering the limited sample of trials. More rigorous RCTs performed in patients with a high-risk psychological profile are required to elucidate the efficacy of psychological interventions in preventing chronic pain.

To evaluate how Canadian clinicians involved in trauma patient care and prescribing opioids perceive the use and effectiveness of strategies to prevent long-term opioid therapy following trauma. Barriers and facilitators to the implementation of these strategies were also assessed.We conducted a web-based cross-sectional survey. Potential participants were identified by trauma program managers and directors of the targeted departments in three Canadian provinces. We designed our questionnaire using standard health survey research methods. The questionnaire was administered between April 2021 and November 2021.Our response rate was 47% (350/744), and 52% (181/350) of participants completed the entire survey. Most respondents (71%, 129/181) worked in teaching hospitals. Multimodal analgesia (93%, 240/257), nonsteroidal anti-inflammatory agents (77%, 198/257), and physical stimulation (75%, 193/257) were the strategies perceived to be the most frequently used. Several preventive strategies were perceived to be very effective by over 80% of respondents. Of these, some that were reported as not being frequently used were perceived to be among the most effective ones, including guidelines or protocols, assessing risk factors for opioid misuse, physical health follow-up by a professional, training for clinicians, patient education, and prescription monitoring systems. Staff shortages, time constraints, and organizational practices were identified as the main barriers to the implementation of the highest ranked preventive strategies.Several strategies to prevent long-term opioid therapy following trauma are perceived as being effective by those prescribing opioids in this population. Some of these strategies appear to be commonly used in everyday practice and others less so. Future research should focus on which preventive strategies should be given higher priority for implementation before assessing their effectiveness.RéSUMé: OBJECTIF: Évaluer comment les cliniciens canadiens impliqués dans les soins aux patients traumatisés et prescrivant des opioïdes perçoivent l’utilisation et l’efficacité des stratégies visant à prévenir le traitement prolongé par opioïde après un traumatisme. Les obstacles et facilitateurs de la mise en œuvre de ces stratégies ont aussi été analysés. MéTHODES: Nous avons réalisé une enquête transversale via le Web. Les participants potentiels ont été identifiés par les gestionnaires et directeurs de programmes de traumatologie des départements ciblés dans trois provinces canadiennes. Nous avons conçu notre questionnaire en utilisant la méthodologie de recherche usuelle des enquêtes de santé. Le questionnaire a été administré entre avril 2021 et novembre 2021. RéSULTATS: Notre taux de réponse a été de 47 % (350/744) et 52 % (181/350) des participants ont complété l’enquête dans sa totalité. La majorité des personnes interrogées (71 %, 129/181) travaillait dans des hôpitaux universitaires. L’analgésie multimodale (93 %, 240/257), les anti-inflammatoires non stéroïdiens (77 %, 198/257) et la stimulation physique (75 %, 193/257) étaient les stratégies perçues comme étant le plus fréquemment utilisées. Plusieurs stratégies préventives étaient perçues comme étant très efficaces par plus de 80 % des répondants. Parmi celles-ci, certaines étaient signalées comme n’étant pas utilisées très souvent, mais perçues comme étant les plus efficaces, notamment les lignes directrices et protocoles évaluant les facteurs de risque d’utilisation abusive des opioïdes, le suivi de la santé physique par un professionnel, la formation des cliniciens, l’éducation des patients et les systèmes de suivi des prescriptions. La pénurie de personnels, les contraintes de temps et les pratiques de l’établissement ont été identifiées comme étant les principaux obstacles à la mise en place des stratégies préventives classées parmi les premières. CONCLUSIONS: Plusieurs stratégies de prévention du traitement par opioïdes à long terme après un traumatisme sont perçues comme efficaces par ceux qui les prescrivent à cette population de patients. Certaines de ces stratégies apparaissent comme couramment utilisées dans la pratique quotidienne et d’autres moins souvent. La recherche future devrait se concentrer sur la détermination des stratégies préventives auxquelles il faudrait accorder la plus grande priorité de mise en œuvre avant d’évaluer leur efficacité.

Agricultural practices such as manure applications could contribute to the spread of antibiotic resistance genes (ARGs) within the environment. Our objective was to assess the impact of certain fertilization methods (mineral or manure) and tillage practices (reduced or conventional) on the presence of ARGs and bacteria in soil and drainage water under wheat and grain corn crops. Targeted ARGs tet(T), sul1, and blaCTX-M-1 in liquid hog manure, soil, and water samples were quantified by qPCR. Conventional PCR was used to detect mcr-1 and mcr-2. ARGs in control plots were detected despite the absence of manure, representing an environmental reservoir of resistant microorganisms. The manure application rate higher than 39 m3/ha increased tet(T) and sul1 gene concentrations in soil for more than 180 days. Tillage practices had no impact on ARG concentrations in soil and water samples. The blaCTX-M-1 gene was only detected in seven water samples in 2016, but no link was established with the treatments. The mcr-1 and mcr-2 genes were not detected in all tested samples. This study demonstrated that tet(T) and sul1 gene concentrations increased in soil after liquid hog manure application as well as in drainage water in the next weeks.

Background Endogenous retroelements (EREs) constitute about 42% of the human genome and have been implicated in common human diseases such as autoimmunity and cancer. The dominant paradigm holds that EREs are expressed in embryonic stem cells (ESCs) and germline cells but are repressed in differentiated somatic cells. Despite evidence that some EREs can be expressed at the RNA and protein levels in specific contexts, a system-level evaluation of their expression in human tissues is lacking. Methods Using RNA sequencing data, we analyzed ERE expression in 32 human tissues and cell types, including medullary thymic epithelial cells (mTECs). A tissue specificity index was computed to identify tissue-restricted ERE families. We also analyzed the transcriptome of mTECs in wild-type and autoimmune regulator (AIRE)-deficient mice. Finally, we developed a proteogenomic workflow combining RNA sequencing and mass spectrometry (MS) in order to evaluate whether EREs might be translated and generate MHC I-associated peptides (MAP) in B-lymphoblastoid cell lines (B-LCL) from 16 individuals. Results We report that all human tissues express EREs, but the breadth and magnitude of ERE expression are very heterogeneous from one tissue to another. ERE expression was particularly high in two MHC I-deficient tissues (ESCs and testis) and one MHC I-expressing tissue, mTECs. In mutant mice, we report that the exceptional expression of EREs in mTECs was AIRE-independent. MS analyses identified 103 non-redundant ERE-derived MAPs (ereMAPs) in B-LCLs. These ereMAPs preferentially derived from sense translation of intronic EREs. Notably, detailed analyses of their amino acid composition revealed that ERE-derived MAPs presented homology to viral MAPs. Conclusions This study shows that ERE expression in somatic tissues is more pervasive and heterogeneous than anticipated. The high and diversified expression of EREs in mTECs and their ability to generate MAPs suggest that EREs may play an important role in the establishment of self-tolerance. The viral-like properties of ERE-derived MAPs suggest that those not expressed in mTECs can be highly immunogenic.

High-grade serous ovarian cancer (HGSC), the principal cause of death from gynecologic malignancies in the world, has not significantly benefited from advances in cancer immunotherapy. Although HGSC infiltration by lymphocytes correlates with superior survival, the nature of antigens that can elicit anti-HGSC immune responses is unknown. The goal of this study was to establish the global landscape of HGSC tumor-specific antigens (TSA) using a mass spectrometry pipeline that interrogated all reading frames of all genomic regions. In 23 HGSC tumors, we identified 103 TSAs. Classic TSA discovery approaches focusing only on mutated exonic sequences would have uncovered only three of these TSAs. Other mutated TSAs resulted from out-of-frame exonic translation (n = 2) or from noncoding sequences (n = 7). One group of TSAs (n = 91) derived from aberrantly expressed unmutated genomic sequences, which were not expressed in normal tissues. These aberrantly expressed TSAs (aeTSA) originated primarily from nonexonic sequences, in particular intronic (29%) and intergenic (22%) sequences. Their expression was regulated at the transcriptional level by variations in gene copy number and DNA methylation. Although mutated TSAs were unique to individual tumors, aeTSAs were shared by a large proportion of HGSCs. Taking into account the frequency of aeTSA expression and HLA allele frequencies, we calculated that, in Caucasians, the median number of aeTSAs per tumor would be five. We conclude that, in view of their number and the fact that they are shared by many tumors, aeTSAs may be the most attractive targets for HGSC immunotherapy.

Sonia Grzelak,1,2 Mélanie Bérubé,1,2 Marc-Aurèle Gagnon,1 Caroline Côté,1,2 Valérie Turcotte,3 Stéphane Pelet,4 Étienne Belzile4 1Population Health and Optimal Practices Research Unit (Trauma - Emergency - Critical Care Medicine), Laval University Research Center (Hôpital de l’Enfant-Jésus), Quebec City, QC, Canada; 2Faculty of Nursing, Laval University, Quebec City, QC, Canada; 3Nursing Department, CIUSSS du Nord-de-l’Île-de-Montréal, Hôpital du Sacré-Coeur de Montréal, Montréal, QC, Canada; 4Department of Orthopedic Surgery, CHU de Québec-Université Laval (Hôpital de l’Enfant-Jésus), Quebec City, QC, CanadaCorrespondence: Sonia GrzelakPopulation Health and Optimal Practices Research Unit, Laval University Research Center (Hôpital de l’Enfant-Jésus), 1401, 18 e rue, Quebec City, QC, G1V 1Z4, Canada, Tel +1 418 649-0252, ext 66600, Fax +1 418-649-5733, Email sonia.grzelak.1@ulaval.caPurpose: To examine 1) pain management strategies within the care trajectory of orthopaedic trauma patients and patients’ perception of their effectiveness, 2) adverse effects (AEs) associated with pharmacological treatments, particularly opioids and cannabis, and 3) patients’ perceptions of strategies that should be applied after an orthopaedic trauma and support that they should obtain from health professionals for their use.Patients and Methods: This study was conducted with orthopaedic trauma patients in a level 1 trauma center. A convergent mixed-methods design was used. Data on pain experience, pain management strategies used and AEs were collected with self-administered questionnaires at hospital discharge (T1) and at 3 months after injury (T2). Patients’ preferences about the pain management strategies used, the required support and AEs were further examined through semi-structured individual interviews at the same time measures. Descriptive statistics and thematic analyses were performed.Results: Seventy-one patients were recruited and 30 individual interviews were undertaken. Pharmacological pain management strategies used at T1 and T2 were mainly opioids (95.8%; 20.8%) and acetaminophen (91.5%; 37.5%). The most frequently applied non-pharmacological strategies were sleep (95.6%) and physical positioning (89.7%) at T1 and massage (46.3%) and relaxation (32.5%) at T2. Findings from quantitative and qualitative analyses highlighted that non-pharmacological strategies, such as comfort, massage, distraction, and physical therapy, were perceived as the most effective by participants. Most common AEs related to opioids were dry mouth (78.8%) and fatigue (66.1%) at T1 and insomnia (30.0%) and fatigue (20.0%) at T2. Dry mouth (28.6%) and drowsiness (14.3%) were the most reported AEs by patients using recreational cannabis. An important need for information at hospital discharge and for a personalized follow-up was identified by participants during interviews.Conclusion: Despite its AEs, we found that opioids are still the leading pain management strategy after an orthopaedic trauma and that more efforts are needed to implement non-pharmacological strategies. Cannabis was taken for recreational purposes but patients also used it for pain relief. Support from health professionals is needed to promote the adequate use of these strategies.Keywords: orthopaedic trauma, pain, pharmacological strategies, non-pharmacological strategies, opioids, cannabis

Background Chronic opioid use has been documented in up to 20% of patients with traumatic injuries. Hence, we developed the Tapering Opioids Prescription Program for high-risk Trauma (TOPP-Trauma) patients. Aims To assess the feasibility and acceptability of TOPP-Trauma, examine the feasibility of the research methods, and describe its potential efficacy in reducing long-term opioid use. Design A two-arm pilot randomized controlled trial. Methods Fifty participants discharged home were assigned to TOPP-Trauma or an educational pamphlet. Feasibility was assessed based on ability to provide the program components. The acceptability was assessed with the Treatment Acceptability and Preference Questionnaire. The feasibility of the research methods was evaluated according to standard parameters. Self-reported morphine equivalent dose (MED) and MEDs supplied by pharmacies were measured at 6 and 12 weeks. Results Eighty percent or more of TOPP-Trauma components were delivered as planned, and the program was deemed highly acceptable. Approximately 10% of screened patients were eligible. Eighty-five percent of eligible patients agreed to participate with 20% attrition rates. TOPP-Trauma participants used less MED/day compared to the control group at 6 and 12 weeks (1.2. vs. 12.2 mg; 0.4. vs 4.0 mg), and pharmacies supplied less than half of cumulative MEDs to those who received the program at 12 weeks, but the differences were not statistically significant. Conclusions Some challenges need to be addressed before testing TOPP-Trauma. These include creating strategies to decrease attrition, offering the program throughout the care continuum to higher risk patients, and evaluating the impacts of reduced opioid use.

Summary: Combining RNA sequencing, ribosome profiling, and mass spectrometry, we elucidate the contribution of non-canonical translation to the proteome and major histocompatibility complex (MHC) class I immunopeptidome. Remarkably, of 14,498 proteins identified in three human B cell lymphomas, 2,503 are non-canonical proteins. Of these, 28% are novel isoforms and 72% are cryptic proteins encoded by ostensibly non-coding regions (60%) or frameshifted canonical genes (12%). Cryptic proteins are translated as efficiently as canonical proteins, have more predicted disordered residues and lower stability, and critically generate MHC-I peptides 5-fold more efficiently per translation event. Translating 5′ “untranslated” regions hinders downstream translation of genes involved in transcription, translation, and antiviral responses. Novel protein isoforms show strong enrichment for signaling pathways deregulated in cancer. Only a small fraction of cryptic proteins detected in the proteome contribute to the MHC-I immunopeptidome, demonstrating the high preferential access of cryptic defective ribosomal products to the class I pathway.

Depression is a mood disorder that affects many people globally. One possible positive factor in the treatment of this disorder is support from relatives and friends of the affected person. However, the former can be helpless in aiding the depressed person due to a lack of awareness on the signs of depression and how it impacts their lives. It is in this context that Equilibrium was developed. Equilibrium is a serious narrative game that aims to raise awareness about depression and the importance of external help in the recovery process of those affected. It does so with a grounded narrative and accessible gameplay, in a format that is easily shareable.

IntroductionGlobally every year, millions of patients sustain traumatic injuries and require acute care surgeries. A high incidence of chronic opioid use (up to 58%) has been documented in these populations with significant negative individual and societal impacts. Despite the importance of this public health issue, optimal strategies to limit the chronic use of opioids after trauma and acute care surgery are not clear. We aim to identify existing strategies to prevent chronic opioid use in these populations.Methods and analysisWe will perform a scoping review of peer-reviewed and non-peer-reviewed literature to identify studies, reviews, recommendations and guidelines on strategies aimed at preventing chronic opioid use in patients after trauma and acute care surgery. We will search MEDLINE, EMBASE, PsycINFO, CINHAL, Cochrane Central Register of Controlled Trials, Web of Science, ProQuest and websites of trauma and acute care surgery, pain, government and professional organisations. Databases will be searched for papers published from 1 January 2005 to a maximum of 6 months before submission of the final manuscript. Two reviewers will independently evaluate studies for eligibility and extract data from included studies using a standardised data abstraction form. Preventive strategies will be classified according to their types and targeted trauma populations and acute care surgery procedures.Ethics and disseminationResearch ethics approval is not required as this study is based on the secondary use of published data. This work will inform research and clinical stakeholders on the required next steps towards the uptake of effective strategies aimed at preventing chronic opioid use in trauma and acute care surgery patients.

IntroductionOrthopaedic injuries affect almost 90% of trauma patients. A previous scoping review and expert consultation survey identified 15 potential low-value intra-hospital practices in the adult orthopaedic trauma population. Limiting the frequency of such practices could reduce adverse events, improve clinical outcomes and free up resources. The aim of this study is to synthesise the evidence on intra-hospital practices for orthopaedic injuries, previously identified as potentially of low value.Methods and analysisWe will search Medline, Excerpta Medica Database (EMBASE), the Cochrane Central Register of Controlled Trials and Epistemonikos to identify systematic reviews, randomised controlled trials (RCTs), quasi-RCTs, cohort studies and case–control studies that evaluate selected practices according to a priori PICOS statements (Population–Intervention–Comparator–Outcome–Study design) . We will evaluate the methodological quality for systematic reviews using the Measurement Tool to Assess Systematic Reviews version 2 (AMSTAR-2). Risk of bias in original studies will be evaluated with the Cochrane revised tool for RCTs (RoB2) and with the risk of bias in non-randomised studies of interventions (ROBINS-I) tool. If for a given practice, more than two original studies on our primary outcome are identified, we will conduct meta-analysis using a random effects model and assess heterogeneity using the I2index. We will assess credibility of evidence (I–IV) based on statistical significance, sample size, heterogeneity and bias as per published criteria.Ethics and disseminationEthics approval is not required as original data will not be collected. Knowledge users from three level I trauma centres are involved in the design and conduct of the study in accordance with an integrated knowledge translation approach. Findings related to the rapid review will be available in May 2020. They will be presented to key stakeholders to inform discussions and raise awareness on low-value injury care. In addition, results will be disseminated in a peer-reviewed journal, at national and international scientific meetings and to healthcare associations.

MHC class I–associated peptides (MAPs) define the immune self for CD8+ T lymphocytes and are key targets of cancer immunosurveillance. Here, the goals of our work were to determine whether the entire set of protein-coding genes could generate MAPs and whether specific features influence the ability of discrete genes to generate MAPs. Using proteogenomics, we have identified 25,270 MAPs isolated from the B lymphocytes of 18 individuals who collectively expressed 27 high-frequency HLA-A,B allotypes. The entire MAP repertoire presented by these 27 allotypes covered only 10% of the exomic sequences expressed in B lymphocytes. Indeed, 41% of expressed protein-coding genes generated no MAPs, while 59% of genes generated up to 64 MAPs, often derived from adjacent regions and presented by different allotypes. We next identified several features of transcripts and proteins associated with efficient MAP production. From these data, we built a logistic regression model that predicts with good accuracy whether a gene generates MAPs. Our results show preferential selection of MAPs from a limited repertoire of proteins with distinctive features. The notion that the MHC class I immunopeptidome presents only a small fraction of the protein-coding genome for monitoring by the immune system has profound implications in autoimmunity and cancer immunology.

Tumor-specific antigens (TSAs) represent ideal targets for cancer immunotherapy, but few have been identified thus far. We therefore developed a proteogenomic approach to enable the high-throughput discovery of TSAs coded by potentially all genomic regions. In two murine cancer cell lines and seven human primary tumors, we identified a total of 40 TSAs, about 90% of which derived from allegedly noncoding regions and would have been missed by standard exome-based approaches. Moreover, most of these TSAs derived from nonmutated yet aberrantly expressed transcripts (such as endogenous retroelements) that could be shared by multiple tumor types. Last, we demonstrated that, in mice, the strength of antitumor responses after TSA vaccination was influenced by two parameters that can be estimated in humans and could serve for TSA prioritization in clinical studies: TSA expression and the frequency of TSA-responsive T cells in the preimmune repertoire. In conclusion, the strategy reported herein could considerably facilitate the identification and prioritization of actionable human TSAs.

Antibiotics are used to fight bacterial infections. However, a selective pressure gave rise to bacteria resistant to antibiotics. This leaves scientists worried about the danger to human and animal health. Some strategies can be borrowed to reduce the use of antibiotics in chicken farms. Much research has been carried out to look for natural agents with similar beneficial effects of growth promoters. The aim of these alternatives is to maintain a low mortality rate, a good level of animal yield while preserving environment and consumer health. Among these, the most popular are probiotics, prebiotics, enzymes, organic acids, immunostimulants, bacteriocins, bacteriophages, phytogenic feed additives, phytoncides, nanoparticles and essential oils. Keywords: Broiler chicken, Antibiotic, Probiotic, Prebiotic, Enzyme, Essential oil

Freshwater eels Anguilla spp. are declining worldwide, and a major challenge is understanding why these panmictic species show contrasting patterns of intraspecific phenotypic variation and recruitment. We present results on studies of the American Eel A. rostrata to understand and discriminate the effects of origin and plasticity on growth and sex determination. We considered two separate growth and one length-at-age data sets. The first growth data set originated from a 34-month rearing experiment starting from the glass eel life stage to test the effects of origin, salinity, and density on growth and sex determination. The second growth data set originated from a shorter rearing experiment of 18 months starting at the yellow eel stage (around 3 years old) and compared transplanted individuals in Lake Ontario (LO) with natural migrants to the LO area. The third data set compared transplanted individuals in LO sampled by electrofishing with naturally migrating individuals. Sex ratios were identic...

A two-year field experiment was conducted in order to evaluate the persistence of generic Escherichia coli in strawberry after irrigation with naturally E. coli -contaminated surface water. Sixteen experimental plots representing actual field conditions were set, including two methods of irrigation (overhead and subsurface drip) and two mulch types (straw and plastic). Two irrigations were performed each year with water having an E. coli content varying between 460 and 2242 CFU per 100 ml. Strawberries were harvested before irrigation and 1, 4 and 24 h following irrigation. E. coli counts could not be determined in any of the 256 strawberry samples. Enrichment procedure revealed more positive samples under straw mulch (6.4%) compared to plastic mulch (4.3%), but this difference was not statistically significant ( P = 0.3991). Higher strawberry contamination was also observed in overhead irrigation treatments (8.6%) compared to drip irrigation (2.1%) ( P = 0.0674). The risk to detect E. coli in overhead-irrigated strawberries was 4.5-fold higher than in strawberries under drip irrigation. Four hours following irrigation, the risk to detect E. coli in fruits was 4.0-fold lower than the risk observed 1 h after irrigation. Increasing the delay to 24 h led to a 7.4-fold lower risk. In actual conditions that may be encountered in strawberry productions, this study showed a limited persistence of E. coli in strawberries following irrigation.

Significant technological advances in both affinity chromatography and mass spectrometry have facilitated the identification of peptides associated with the major histocompatibility complex class I (MHC I) molecules, and enabled a greater understanding of the dynamic nature of the immunopeptidome of normal and neoplastic cells. While the isolation of MHC I-associated peptides (MIPs) typically used mild acid elution (MAE) or immunoprecipitation (IP), limited information currently exists regarding their respective analytical merits. Here, a comparison of these approaches for the isolation of two different B-cell lymphoblast cell models is presented, and it is reported on the recovery, reproducibility, scalability, and complementarity of identification from each method. Both approaches yielded reproducible datasets for peptide extracts obtained from 2 to 100 million cells, with 2016 to 5093 MIPs, respectively. The IP typically provides up to 6.4-fold increase in MIPs compared to the MAE. The comprehensiveness of these immunopeptidome analyses is extended using personalized genomic database of B-cell lymphoblasts, and it is discovered that 0.4% of their respective MIP repertoire harbored nonsynonymous single nucleotide variations (also known as minor histocompatibility antigens, MiHAs).

We performed population genetic analyses on the American eel (Anguilla rostrata) with three main objectives. First, we conducted the most comprehensive analysis of neutral genetic population structure to date to revisit the null hypothesis of panmixia in this species. Second, we used this data to provide the first estimates of contemporary effective population size (Ne ) and to document temporal variation in effective number of breeders (Nb ) in American eel. Third, we tested for statistical associations between temporal variation in the North Atlantic Oscillation (NAO), the effective number of breeders and two indices of recruit abundance. A total of 2142 eels from 32 sampling locations were genotyped with 18 microsatellite loci. All measures of differentiation were essentially zero, and no evidence for significant spatial or temporal genetic differentiation was found. The panmixia hypothesis should thus be accepted for this species. Nb estimates varied by a factor of 23 among 12 cohorts, from 473 to 10,999. The effective population size Ne was estimated at 10,532 (95% CI, 9312-11,752). This study also showed that genetically based demographic indices, namely Nb and allelic richness (Ar), can be used as surrogates for the abundance of breeders and recruits, which were both shown to be positively influenced by variation during high (positive) NAO phases. Thus, long-term genetic monitoring of American glass eels at several sites along the North American Atlantic coast would represent a powerful and efficient complement to census monitoring to track demographic fluctuations and better understand their causes.

MHC I–associated peptides (MIPs) play an essential role in normal homeostasis and diverse pathologic conditions. MIPs derive mainly from defective ribosomal products (DRiPs), a subset of nascent proteins that fail to achieve a proper conformation and the physical nature of which remains elusive. In the present study, we used high-throughput proteomic and transcriptomic methods to unravel the structure and biogenesis of MIPs presented by HLA-A and HLA-B molecules on human EBV-infected B lymphocytes from 4 patients. We found that although HLA-different subjects present distinctive MIPs derived from different proteins, these MIPs originate from proteins that are functionally interconnected and implicated in similar biologic pathways. Secondly, the MIP repertoire of human B cells showed no bias toward conserved versus polymorphic genomic sequences, were derived preferentially from abundant transcripts, and conveyed to the cell surface a cell-type–specific signature. Finally, we discovered that MIPs derive preferentially from transcripts bearing miRNA response elements. Furthermore, whereas MIPs of HLA-disparate subjects are coded by different sets of transcripts, these transcripts are regulated by mostly similar miRNAs. Our data support an emerging model in which the generation of MIPs by a transcript depends on its abundance and DRiP rate, which is regulated to a large extent by miRNAs.

This study evaluated the presence of pathogenic human and zoonotic viruses on irrigated, field-grown strawberries. Norovirus genogroup I, rotavirus, and swine hepatitis E virus genogroup 3 were detected on strawberries, and irrigation water is suspected as the contamination origin.

During their larval leptocephalus phase, newly hatched American eels undergo an extensive oceanic migration from the Sargasso Sea toward coastal and freshwater habitats. Their subsequent metamorphosis into glass eel is accompanied by drastic morphological and physiological changes preceding settlement over a wide geographic range. The main objective of this study was to compare RNA/DNA ratios and condition factor among glass eels in order to test the null hypothesis of no difference in physiological status and metabolic activity of glass eels at the outcome of their oceanic migration. This was achieved by analyzing glass eel samples collected at the mouth of 17 tributaries covering a latitudinal gradient across the species distribution range from Florida to Gaspesie (Quebec). Our main observations were (i) a latitudinal increase in mean total length; (ii) a latitudinal variation in mean RNA/DNA ratios, which was best explained by a quadratic model reaching its minimum in the central range of sampling locations; and (iii) a latitudinal variation in Fulton's condition factor, which was best explained by a quadratic model reaching its maximum in the central range of sampling locations. Below we discuss the possible links between latitudinal variation in glass eel physiological status and variable energetic and environmental constraints to oceanic migration as a function of latitudinal distribution.

Our understanding of the genetic basis of local adaptation has recently benefited from the increased power to identify functional variants associated with environmental variables at the genome scale. However, it often remains challenging to determine whether locally adaptive alleles are actively maintained at intermediate frequencies by spatially varying selection. Here, we evaluate the extent to which this particular type of balancing selection explains the retention of adaptive genetic variation in the extreme situation of perfect panmixia, using the American eel (Anguilla rostrata) as a model. We first conducted a genome scan between two samples from opposite ends of a latitudinal environmental gradient using 454 sequencing of individually tagged cDNA libraries. Candidate SNPs were then genotyped in 992 individuals from 16 sampling sites at different life stages of the same cohort (including larvae from the Sargasso Sea, glass eels, and 1-year-old individuals) as well as in glass eels of the following cohort. Evidence for spatially varying selection was found at 13 loci showing correlations between allele frequencies and environmental variables across the entire species range. Simulations under a multiple-niche Levene’s model using estimated relative fitness values among genotypes rarely predicted a stable polymorphic equilibrium at these loci. Our results suggest that some genetic-by-environment interactions detected in our study arise during the progress toward fixation of a globally advantageous allele with spatially variable effects on fitness.

Variation in gene regulation may be involved in the differences observed for life history traits within species. American eel (Anguilla rostrata) is well known to harbor distinct ecotypes within a single panmictic population. We examined the expression of genes involved in the regulation of appetite as well as lipid and glycogen among glass eels migrating to different locations on the Canadian east coast and captured at two different periods of upstream migration. Gene expression levels of three reference and five candidate genes were analyzed by real-time PCR with Taqman probes in recently captured wild glass eels. All gene transcripts were detected in glass eels. Of the five candidate genes, bile salt activated and triacylglycerol lipases were respectively 7.65 and 3.25 times more expressed in glass eels from the St. Lawrence estuary than in those from Nova Scotia, and there was no effect related to the two-week difference in capture date. These two genes explained 82.41% of the dissimilarity between the two rivers. In contrast, glycogen phosphorylase, ghrelin, and leptin receptor genes showed no significant differences in gene transcription. These results confirmed at the molecular level an observation that was recently made at the phenotypic level that glass eels from the St. Lawrence estuary have a greater capacity to use lipid reserves to sustain their metabolic needs. These observations add to the body of evidence supporting the hypothesis that regional phenotypic variation observed in American eel is determined early in life and that part of this variation is likely under genetic control.

Cryptosporidium and Giardia (oo)cyst concentrations are frequently used for assessing drinking water safety. The widely used USEPA Method 1623 provides total counts of (oo)cysts, but may not be accurate for human health risk characterization, since it does not provide infectivity information. The total counts and infectious fraction of Cryptosporidium oocysts and the total counts of Giardia cysts were assessed in major fecal pollution sources. Fresh calf and cow feces, their manure, and the discharge point were sampled in a small rural sub-watershed (n = 20, 21, 10, 10). Median concentrations for total (oo)cysts were higher in calves (333 oocysts g−1; 111 cysts g−1) than in cows (52 oocysts g−1; 7 cysts g−1). Infectious oocysts were found in 17 (7%) of the samples and none were found in manure or at the discharge point. Urban sources were sampled in the influent and effluent (n = 19, 18) of two wastewater treatment plants. Peak concentrations were 533 oocysts L−1 and 9,010 cysts L−1 for influents and 89 oocysts L−1 and 472 cysts L−1 for effluents. Infectious oocyst fractions varied from below the detection limit to 7–22% in the effluent and influent respectively. These infectious fractions are significantly lower than those currently used for quantitative microbial risk assessment estimates.

Pre-clinical studies have shown that injection of allogeneic T cells primed against a single minor histocompatibility antigen (MiHA) could cure hematologic cancers (HC) without causing any toxicity to the host. However, translation of this approach in humans has been hampered by the paucity of molecularly defined human MiHAs. Using a novel proteogenomic approach, we have analyzed cells from 13 volunteers and discovered a vast repertoire of MiHAs presented by the most common HLA haplotype in European Americans: HLA-A*02:01;B*44:03. Notably, out of >6000 MiHAs, we have identified a set of 39 MiHAs that share optimal features for immunotherapy of HCs. These 'optimal MiHAs' are coded by common alleles of genes that are preferentially expressed in hematopoietic cells. Bioinformatic modeling based on MiHA allelic frequencies showed that the 39 optimal MiHAs would enable MiHA-targeted immunotherapy of practically all HLA-A*02:01;B*44:03 patients. Further extension of this strategy to a few additional HLA haplotypes would allow treatment of almost all patients.

In 2011 and 2012, trials consisting of experimental plots were carried out to evaluate the presence of pathogenic (Listeria monocytogenes, Salmonella) and prevalence of indicator (Escherichia coli) microorganisms in broccoli fertilized with liquid hog manure or mineral fertilizers and irrigated zero, one, or two times with E. coli-contaminated water. In 2011, results showed that E. coli contamination in broccoli heads was affected by the interval between irrigation and sampling (P = 0.0236), with a significant decrease between the first and third day following irrigation (P = 0.0064). In 2012, irrigation frequency significantly increased E. coli prevalence in broccoli samples (P = 0.0499). In 2012, E. coli counts in the soil were significantly influenced by the type of fertilizer applied, as plots receiving liquid hog manure showed higher bacterial counts (P = 0.0006). L. monocytogenes was recovered in one broccoli sample, but geno-serogrouping differentiated the isolate from those recovered in manure and irrigation water. The L. monocytogenes serogroup IIA, pulsotype 188 strain was found in six soil samples and in irrigation water applied 5 days before soil sampling. This study highlights the link between E. coli levels in irrigation water, irrigation frequency, and interval between irrigation and harvest on produce contamination. It also demonstrates that L. monocytogenes introduced into the soil following irrigation can persist for up to 5 days.

The fate of antimicrobial active compound residues in the environment, and especially antibiotics used in swine husbandry are of particular interest for their potential toxicity and contribution to antibiotic resistance. The presence of relatively high concentrations of bioactive compounds has been reported in agricultural areas but few information is available on their degradation products. Veterinary antibiotics reach terrestrial environments through many routes, including application of swine manure to soils. The objectives of this project were first, to develop an analytical method able to quantify and identify veterinary antibiotics and their degradation products in manure, soil and water samples; and second, to study the distribution of these target compounds in soils and drainage waters. A brief evaluation of their potential toxicity in the environment was also made. In order to achieve these objectives, liquid chromatography coupled to high-resolution mass spectrometry was used for its ability to quantify contaminants with sensitivity and selectivity, and its capacity to identify degradation products. Samples of manure, soil and water came from a long-term experimental site where swine manure containing veterinary antibiotics has been applied for many years. In this study, tetracycline antibiotics were found at several hundred μg L(-1) in the swine manure slurry used for fertilization, several hundred of ng L(-1) in drainage waters and several ng g(-1) in soils, while degradation products were sometimes found at concentrations higher than the parent compounds.

Restriction of T cell responses to a few epitopes (immunodominance) is a central feature of immune responses. We analyzed the entire transcriptome of effector CD8 T cells specific for a dominant (H7a) and a cryptic (HY) mouse Ag and performed a longitudinal analysis of selected T cell differentiation markers. We found that Ag specificity had a relatively modest influence on the repertoire of genes that are transcriptionally modulated by the CD8 T cell differentiation program. Although the differentiation programs of anti-H7a and anti-HY T cells were similar, they did not progress simultaneously. The expansion peak of anti-H7a T cells was reached on day 10 while that of anti-HY T cells was attained on days 15–20. Between days 10 and 20, anti-H7a T cells were in the contraction phase and anti-HY T cells in the expansion phase. Furthermore, expansion and development of effector function were well-synchronized in anti-H7a T cells but were disconnected in anti-HY T cells. We propose that, by leading to selective expansion of the fittest CD8 T cells, immunodominance may be beneficial to the host. Inhibition of the T cell response to cryptic Ag would ensure that host resources (APC, cytokines) for which T cells compete are devoted to T cells with the best effector potential. One implication is that favoring expansion of the fittest effector T cells in general may be more important than increasing the diversity of the T cell repertoire.

Pathogenic microorganisms in manure represents a potential hazard to human and animal. Few data describing the microbiological content of liquid hog manure intended for spreading are available in the scientific literature. The objective of this project was to verify the presence and estimate the persistence of Escherichia coli , Salmonella spp . , Yersinia enterocolitica and Cryptosporidium spp. in liquid hog manure from storages under conditions typical of commercial swine production in Quebec. E. coli content of liquid hog manure taken during the spreading period in the 32 hog operations varied between 0 and 5.52 log 10 CFU/g with a 95% confidence interval of 3.16–4.23 log 10 CFU/g. Salmonella spp., Y. enterocolitica and Cryptosporidium spp. were detected in 37%, 9% and 3% samples, respectively. Under the conditions of this experiment, the predicted number of days required to obtain a 90% reduction of E. coli populations varied between 15 and 26 days, while the predicted storage period necessary to reach undetectable levels was 54–114 days. The maximal persistence of Salmonella observed in this study was 88 days. A 73-day storage time was insufficient for the elimination of Y. enterocolitica .

The objective of this study was to evaluate the efficiency of a low temperature anaerobic treatment to reduce viable populations of indicator microorganisms (total coliforms, Escherichia coli) and the presence of selected pathogens (Salmonella, Yersinia enterocolitica, Cryptosporidium and Giardia) in swine slurries from different sources. Experiments were carried out in 40 l Sequencing Batch Reactors (SBRs). Experimental results indicated that anaerobic digestion of swine manure slurry at 20 � C for 20 days in an intermittently fed SBR: (1) reduced indigenous populations of total coliforms by 97.94–100%; (2) reduced indigenous populations of E. coli by 99.67–100%; (3) resulted in undetectable levels of indigenous strains of Salmonella, Cryptosporidium, and Giardia .I t can be considered as a promising method for reducing indigenous indicator and pathogenic microorganisms populations in liquid swine manure slurries. � 2005 Elsevier Ltd. All rights reserved.

The STT3 subunit of the oligosaccharyltransferase complex plays a critical role in the N-glycosylation process. From Arabidopsis thaliana to Homo sapiens, two functional STT3 isoforms have been identified, STT3-A and STT3-B. We report that the last transmembrane (TM) segment of STT3-B corresponds to a topogenic determinant that is sufficient for proper integration and orientation of STT3-B C-terminal domain. Notably, the last TM segment of STT3-A and -B isoforms present major differences in amino acid sequence and predicted 3D structure. We also identified a bipartite nuclear targeting sequence in the C-terminal tail of STT3-B that is absent in STT3-A. The latter sequence is sufficient to induce nucleolar localization of a reporter protein. Our results show that STT3-A and -B display two structural differences that may have a drastic influence on their function and might account for the remarkable evolutionary conservation of the two STT3 paralogs.