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Abstract Aim Illicitly manufactured fentanyl and its analogs are the primary drivers of opioid overdose deaths in the United States (U.S.). People who use drugs may be exposed to fentanyl or its analogs intentionally or unintentionally. This study sought to identify strategies used by rural people who use drugs to reduce harms associated with unintentional fentanyl exposure. Methods This analysis focused on 349 semi-structured qualitative interviews across 10 states and 58 rural counties in the U.S conducted between 2018 and 2020. Interview guides were collaboratively standardized across sites and included questions about drug use history (including drugs currently used, frequency of use, mode of administration) and questions specific to fentanyl. Deductive coding was used to code all data, then inductive coding of overdose and fentanyl codes was conducted by an interdisciplinary writing team. Results Participants described being concerned that fentanyl had saturated the drug market, in both stimulant and opioid supplies. Participants utilized strategies including: (1) avoiding drugs that were perceived to contain fentanyl, (2) buying drugs from trusted sources, (3) using fentanyl test strips, 4) using small doses and non-injection routes, (5) using with other people, (6) tasting, smelling, and looking at drugs before use, and (7) carrying and using naloxone. Most people who used drugs used a combination of these strategies as there was an overwhelming fear of fatal overdose. Conclusion People who use drugs living in rural areas of the U.S. are aware that fentanyl is in their drug supply and use several strategies to prevent associated harms, including fatal overdose. Increasing access to harm reduction tools (e.g., fentanyl test strips, naloxone) and services (e.g., community drug checking, syringe services programs, overdose prevention centers) should be prioritized to address the polysubstance-involved overdose crisis. These efforts should target persons who use opioids and other drugs that may contain fentanyl.

Abstract Background Patient-reported outcome and experience measures (PROMs and PREMs, respectively) are evidence-based, standardized questionnaires that can be used to capture patients’ perspectives of their health and health care. While substantial investments have been made in the implementation of PROMs and PREMs, their use remains fragmented and limited in many settings. Analysis of multi-level barriers and enablers to the implementation of PROMs and PREMs has been hampered by the lack of use of state-of-the-art implementation science frameworks. This umbrella review aims to consolidate available evidence from existing quantitative, qualitative, and mixed-methods systematic and scoping reviews covering factors that influence the implementation of PROMs and PREMs in healthcare settings. Methods An umbrella review of systematic and scoping reviews will be conducted following the guidelines of the Joanna Briggs Institute (JBI). Qualitative, quantitative, and mixed methods reviews of studies focusing on the implementation of PROMs and/or PREMs in all healthcare settings will be considered for inclusion. Eight bibliographical databases will be searched. All review steps will be conducted by two reviewers independently. Included reviews will be appraised and data will be extracted in four steps: (1) assessing the methodological quality of reviews using the JBI Critical Appraisal Checklist; (2) extracting data from included reviews; (3) theory-based coding of barriers and enablers using the Consolidated Framework for Implementation Research (CFIR) 2.0; and (4) identifying the barriers and enablers best supported by reviews using the Grading of Recommendations Assessment, Development and Evaluation-Confidence in the Evidence from Reviews of Qualitative research (GRADE-CERQual) approach. Findings will be presented in diagrammatic and tabular forms in a manner that aligns with the objective and scope of this umbrella review, along with a narrative summary. Discussion This umbrella review of quantitative, qualitative, and mixed-methods systematic and scoping reviews will inform policymakers, researchers, managers, and clinicians regarding which factors hamper or enable the adoption and sustained use of PROMs and PREMs in healthcare settings, and the level of confidence in the evidence supporting these factors. Findings will orient the selection and adaptation of implementation strategies tailored to the factors identified. Systematic review registration PROSPERO CRD42023421845.

Abstract Background Self-directed mobility during childhood can influence development, social participation, and independent living later in life. For children who experience challenges with walking, manual wheelchairs (MWCs) provide a means for self-directed mobility. An effective MWC skills training program exists for adults, but controlled trials have not yet been documented in children and adolescents. This paper outlines the protocol for a multi-centre randomized wait-list controlled trial. The primary objective is to test the hypothesis that children and adolescents who receive MWC skills training will have higher MWC skills capacity compared to children and adolescents in the control group who receive usual care. The secondary objectives are to explore the influence of MWC skills training in children and adolescents (MWC use self-efficacy and satisfaction with participation in meaningful activities), and parents (perceived MWC skills); and to measure retention three months later. Methods A multi-centre, parallel-group, single-blind randomized wait-list controlled trial will be conducted. A sample of 60 children and adolescents who use MWCs will be recruited in rehabilitation centres, specialized schools, and the communities of three Canadian cities. Participants will be randomized (1:1) to the experimental (Wheelchair Skills Training Program [WSTP]) or wait-list control group (usual care). Performance-based and self-report measures will be completed at baseline (T1), three months (post-intervention, T2), and three months post-intervention (T3). The primary outcome will be MWC skills capacity post-intervention. Secondary outcomes will be MWC use self-efficacy and satisfaction with participation of the child/adolescent, and parent-perceived MWC skills. The WSTP will consist of 12 sessions, 45–60 min each, delivered 1–2 times per week by trained personnel with health professions education. Training will be customized according to the child’s baseline skills and participation goals that require the use of the MWC. The wait-list control group will receive usual care for 3 months and then receive the WSTP after completing T2 evaluations. Data will be analysed using ANCOVA (controlling for baseline scores). Discussion MWC skills training may be one way to improve self-directed mobility and related outcomes for children and adolescents. The results of this multi-centre randomized wait-list controlled trial will allow for the effectiveness of the intervention to be evaluated in a variety of clinical contexts and geographical regions. Trial registration ClinicalTrials.gov: NCT05564247, Version October 3, 2022.

Abstract Background Mild traumatic brain injury (mTBI) is an increasing public health problem, because of its persistent symptoms and several functional consequences. Understanding the prognosis of a condition is an important component of clinical decision-making and can help to guide the prevention of persistent symptoms following mTBI. The prognosis of mTBI has stimulated several empirical primary research papers and many systematic reviews leading to the identification of a wide range of factors. We aim to synthesize these factors to get a better understanding of their breadth and scope. Methods We conducted an overview of systematic reviews. We searched in databases systematic reviews synthesizing evidence about the prognosis of persistent symptoms after mTBI in the adult population. Two reviewers independently screened all references and selected eligible reviews based on eligibility criteria. They extracted relevant information using an extraction grid. They also rated independently the risk of bias using the ROBIS tool. We synthesized evidence into a comprehensive conceptual map to facilitate the understanding of prognostic factors that have an impact on persistent post-concussion symptoms. Results From the 3857 references retrieved in a database search, we included 25 systematic reviews integrating the results of 312 primary articles published between 1957 and 2019. We examined 35 prognostic factors from the systematics reviews. No single prognostic factor demonstrated convincing and conclusive results. However, age, sex, and multiple concussions showed an affirmatory association with persistent post-concussion outcomes in systematic reviews. Conclusion We highlighted the need for a comprehensive picture of prognostic factors related to persistent post-concussion symptoms. We believe that these prognostic factors would guide clinical decisions and research related to prevention and intervention regarding persistent post-concussion symptoms. Systematic review registration PROSPERO CRD42020176676

Abstract Background Cysteine-dense peptides (CDPs) are an attractive pharmaceutical scaffold that display extreme biochemical properties, low immunogenicity, and the ability to bind targets with high affinity and selectivity. While many CDPs have potential and confirmed therapeutic uses, synthesis of CDPs is a challenge. Recent advances have made the recombinant expression of CDPs a viable alternative to chemical synthesis. Moreover, identifying CDPs that can be expressed in mammalian cells is crucial in predicting their compatibility with gene therapy and mRNA therapy. Currently, we lack the ability to identify CDPs that will express recombinantly in mammalian cells without labour intensive experimentation. To address this, we developed CysPresso, a novel machine learning model that predicts recombinant expression of CDPs based on primary sequence. Results We tested various protein representations generated by deep learning algorithms (SeqVec, proteInfer, AlphaFold2) for their suitability in predicting CDP expression and found that AlphaFold2 representations possessed the best predictive features. We then optimized the model by concatenation of AlphaFold2 representations, time series transformation with random convolutional kernels, and dataset partitioning. Conclusion Our novel model, CysPresso, is the first to successfully predict recombinant CDP expression in mammalian cells and is particularly well suited for predicting recombinant expression of knottin peptides. When preprocessing the deep learning protein representation for supervised machine learning, we found that random convolutional kernel transformation preserves more pertinent information relevant for predicting expressibility than embedding averaging. Our study showcases the applicability of deep learning-based protein representations, such as those provided by AlphaFold2, in tasks beyond structure prediction.

Abstract Background This article outlines the protocol for a trial to test the effectiveness of a nature-based intervention called Open Sky School to reduce mental health problems among elementary school children. Experimental studies show that contact with nature (e.g. walks in parks) improve mental health. A growing number of teachers have been applying outdoor education within the regular school curriculum and evidence suggests that such teaching methods could improve students’ mental health but a randomized controlled trial has never been conducted. Methods A two-arm clustered randomized controlled trial will be conducted in elementary schools across Québec, Canada. Following informed consent by teachers, parents and students, schools will be randomly assigned 1:1 to the intervention or the control group with a total of 2500 5-6th grade students and 100 teachers expected to participate. The intervention will take place outdoors in a green-space (2 h per week for 12 weeks) and include a toolkit of 30 activities to foster well-being (e.g. mindfulness) and academic competencies (e.g. mathematics). Questionnaires will be administered to teachers and students before, immediately after and 3 months after the intervention. The primary outcome will be reductions of mental health problems in children from pre-to-post test (Social Behavior Questionnaire: self and teacher reports). Secondary outcomes include depression, positive and negative affect, nature connectedness, and pro-environmental behaviors among children. We will explore, immediate benefits on teacher’s well-being and positive and negative affect and sustained benefits among students at 3 months follow-up. For the primary outcome, we will explore moderators including child’s sex, child’s disability status, the green-space of neighbourhoods, the school’s socio-economic position and teacher’s experience. Discussion In conducting the first randomized controlled trial of the Open Sky School, our results could provide empirical evidence on the effectiveness of nature-based interventions in reducing mental health problems among elementary school children. Trial registration This study was registered with clinicaltrials.gov: NCT05662436 on December 22, 2022.

Abstract Background Chronic health conditions associated with long-term drug use may pose additional risks to people who use drugs (PWUD) when coupled with COVID-19 infection. Despite this, PWUD, especially those living in rural areas, may be less likely to seek out health services. Previous research has highlighted the increased disease burden of COVID-19 among PWUD. Our manuscript supplements this literature by exploring unique attitudes of PWUD living in rural areas toward the pandemic, COVID-19 vaccination, and the role of harm reduction (HR) organizations in raising health awareness among PWUD. Methods Semi-structured interviews were conducted with 20 PWUD living in rural southern Illinois. Audio recordings were professionally transcribed. A preliminary codebook was created based on interview domains. Two trained coders conducted iterative coding of the transcripts, and new codes were added through line-by-line coding and thematic grouping. Results Twenty participants (45% female, mean age of 38) completed interviews between June and November 2021. Participants reported negative impacts of the pandemic on mental health, financial wellbeing, and drug quality. However, the health impacts of COVID-19 were often described as less concerning than its impacts on these other aspects of life. Many expressed doubt in the severity of COVID-19 infection. Among the 16 unvaccinated participants who reported receiving most of their information from the internet or word of mouth, uncertainty about vaccine contents and distrust of healthcare and government institutions engendered wariness of the vaccination. Distrust of healthcare providers was related to past stigmatization and judgement, but did not extend to the local HR organization, which was unanimously endorsed as a positive institution. Among participants who did not access services directly from the HR organization, secondary distribution of HR supplies by other PWUD was a universally cited form of health maintenance. Participants expressed interest in low-threshold healthcare, including COVID-19 vaccination, should it be offered in the local HR organization’s office and mobile units. Conclusion COVID-19 and related public health measures have affected this community in numerous ways. Integrating healthcare services into harm reduction infrastructures and mobilizing secondary distributors of supplies may promote greater engagement with vaccination programs and other healthcare services. Trial number NCT04427202.

Abstract Background Cardiopulmonary resuscitation (CPR) decreases lung volume below the functional residual capacity and can generate intrathoracic airway closure. Conversely, large insufflations can induce thoracic distension and jeopardize circulation. The capnogram (CO2 signal) obtained during continuous chest compressions can reflect intrathoracic airway closure, and we hypothesized here that it can also indicate thoracic distension. Objectives To test whether a specific capnogram may identify thoracic distension during CPR and to assess the impact of thoracic distension on gas exchange and hemodynamics. Methods (1) In out-of-hospital cardiac arrest patients, we identified on capnograms three patterns: intrathoracic airway closure, thoracic distension or regular pattern. An algorithm was designed to identify them automatically. (2) To link CO2 patterns with ventilation, we conducted three experiments: (i) reproducing the CO2 patterns in human cadavers, (ii) assessing the influence of tidal volume and respiratory mechanics on thoracic distension using a mechanical lung model and (iii) exploring the impact of thoracic distension patterns on different circulation parameters during CPR on a pig model. Measurements and main results (1) Clinical data: 202 capnograms were collected. Intrathoracic airway closure was present in 35%, thoracic distension in 22% and regular pattern in 43%. (2) Experiments: (i) Higher insufflated volumes reproduced thoracic distension CO2 patterns in 5 cadavers. (ii) In the mechanical lung model, thoracic distension patterns were associated with higher volumes and longer time constants. (iii) In six pigs during CPR with various tidal volumes, a CO2 pattern of thoracic distension, but not tidal volume per se, was associated with a significant decrease in blood pressure and cerebral perfusion. Conclusions During CPR, capnograms reflecting intrathoracic airway closure, thoracic distension or regular pattern can be identified. In the animal experiment, a thoracic distension pattern on the capnogram is associated with a negative impact of ventilation on blood pressure and cerebral perfusion during CPR, not predicted by tidal volume per se.

Abstract Background Multiple mini-interviews (MMI) are used to assess non-academic attributes for selection in medicine and other healthcare professions. It remains unclear if different MMI station formats (discussions, role-plays, collaboration) assess different dimensions. Methods Based on station formats of the 2018 and 2019 Integrated French MMI (IFMMI), which comprised five discussions, three role-plays and two collaboration stations, the authors performed confirmatory factor analysis (CFA) using the lavaan 0.6-5 R package and compared a one-factor solution to a three-factor solution for scores of the 2018 (n = 1438) and 2019 (n = 1440) cohorts of the IFMMI across three medical schools in Quebec, Canada. Results The three-factor solution was retained, with discussions, role-plays and collaboration stations all loading adequately with their scores. Furthermore, all three factors had moderate-to-high covariance (range 0.44 to 0.64). The model fit was also excellent with a Comparative fit index (CFI) of 0.983 (good if > 0.9), a Tucker Lewis index of 0.976 (good if > 0.95), a Standardized Root Mean Square Residual of 0.021 (good if < .08) and a Root Mean Square Error of 0.023 (good if

Abstract Background Children’s ability to engage in meaningful activities is positively influenced by their ability to move independently. Preliminary evidence in children suggests that wheelchair skills training improves wheelchair skills, which are important for independent mobility. The Wheelchair Skills Training Program is a standardized program to teach wheelchair skills. However, it is underutilized in pediatric rehabilitation settings. To increase its utilization, 3 pediatric-specific Wheelchair Skills Training Program resources related to indoor skills were developed (i.e., a storybook, four instructional posters, and a training workbook). This study aimed to describe occupational therapists’ (OTs) and pediatric manual wheelchair users’ (PMWUs) perceived satisfaction with the storybook, instructional posters and training workbook, and to explore their perceptions regarding the usability, relevance, and feasibility of these resources in pediatric rehabilitation settings. Methods A descriptive qualitative design was used. Convenience samples of OTs and PMWUs were recruited in a rehabilitation center and affiliated schools. A focus group with OTs and semi-structured interviews with PMWUs were conducted by videoconference to obtain participants’ feedback on the resource prototypes and suggestions for improvement. Data were deductively analyzed using the Framework method. Results Eight OTs and 5 PMWUs expressed general satisfaction with the resources, describing them as usable, relevant, and feasible to integrate into wheelchair skills training with novice wheelchair users and younger children. All OTs and 3 PMWUs expressed the desire to use the resources for wheelchair skills training. Two PMWUs perceived the resources were not relevant to them because they already mastered the skills. The participants suggested minor modifications for improving the resources (e.g., more action in the story, increased precision of illustrations related to the characters’ position in the wheelchair). Conclusion OTs and PMWUs were satisfied with the resources, perceiving them to be applicable for training wheelchair skills among young children and novice wheelchair users. The resources represent a concrete solution to facilitate the use of the Wheelchair Skills Training Program in pediatric rehabilitation settings. Additional resources are needed to better reach older and more experienced PMWUs (i.e., of intermediate and advanced skill levels).

Abstract Background The discovery of the CRISPR-Cas9 system and its applicability in mammalian embryos has revolutionized the way we generate genetically engineered animal models. To date, models harbouring conditional alleles (i.e. two loxP sites flanking an exon or a critical DNA sequence of interest) are amongst the most widely requested project type that are challenging to generate as they require simultaneous cleavage of the genome using two guides in order to properly integrate the repair template. An approach, using embryo sequential electroporation has been reported in the literature to successfully introduce loxP sites on the same allele. Here, we describe a modification of this sequential electroporation procedure that demonstrated the production of conditional allele mouse models for eight different genes via one of two possible strategies: either by consecutive sequential electroporation (strategy A) or non-consecutive sequential electroporation (strategy B). This latest strategy originated from using the by-product produced when using consecutive sequential electroporation (i.e. mice with a single targeted loxP site) to complete the project. Results By using strategy A, we demonstrated successful generation of conditional allele models for three different genes (Icam1, Lox, and Sar1b), with targeting efficiencies varying between 5 and 13%. By using strategy B, we generated five conditional allele models (Loxl1, Pard6a, Pard6g, Clcf1, and Mapkapk5), with targeting efficiencies varying between 3 and 25%. Conclusion Our modified electroporation-based approach, involving one of the two alternative strategies, allowed the production of conditional allele models for eight different genes via two different possible paths. This reproducible method will serve as another reliable approach in addition to other well-established methodologies in the literature for conditional allele mouse model generation.

Abstract Background Drug overdose rates in the United States have been steadily increasing, particularly in rural areas. The COVID-19 pandemic and associated mitigation strategies may have increased overdose risk for people who use drugs by impacting social, community, and structural factors. Methods The study included a quantitative survey focused on COVID-19 administered to 50 people who use drugs and semi-structured qualitative interviews with 17 people who use drugs, 12 of whom also participated in the quantitative survey. Descriptive statistics were run for the quantitative data. Qualitative coding was line-by-line then grouped thematically. Quantitative and qualitative data were integrated during analysis. Results Findings demonstrate how COVID-19 disruptions at the structural and community level affected outcomes related to mental health and drug use at the individual level. Themes that emerged from the qualitative interviews were (1) lack of employment opportunities, (2) food and housing insecurity, (3) community stigma impacting health service use, (4) mental health strains, and (5) drug market disruptions. Structural and community changes increased anxiety, depression, and loneliness on the individual level, as well as changes in drug use patterns, all of which are likely to increase overdose risk. Conclusion The COVID-19 pandemic, and mitigation strategies aimed at curbing infection, disrupted communities and lives of people who use drugs. These disruptions altered individual drug use and mental health outcomes, which could increase risk for overdose. We recommend addressing structural and community factors, including developing multi-level interventions, to combat overdose. Trial registration Clinicaltrails.gov: NCT04427202. Registered June 11, 2020: https://clinicaltrials.gov/ct2/show/NCT04427202?term=pho+mai&draw=2&rank=3

Abstract Background The tall wheatgrass species Thinopyrum elongatum carries a strong fusarium head blight (FHB) resistance locus located on the long arm of chromosome 7 (7EL) as well as resistance to leaf and stem rusts, all diseases with a significant impact on wheat production. Towards understanding the contribution of Th. elongatum 7EL to improvement of disease resistance in wheat, the genomic sequence of the 7EL fragment present in the wheat Chinese Spring (CS) telosomic addition line CS-7EL was determined and the contribution and impact of 7EL on the rachis transcriptome during FHB infection was compared between CS and CS-7EL. Results We assembled the Th. elongatum 7EL chromosome arm using a reference-guided approach. Combining this assembly with the available reference sequence for CS hexaploid wheat provided a reliable reference for interrogating the transcriptomic differences in response to infection conferred by the 7EL fragment. Comparison of the transcriptomes of rachis tissues from CS and CS-7EL showed expression of Th. elongatum transcripts as well as modulation of wheat transcript expression profiles in the CS-7EL line. Expression profiles at 4 days after infection with Fusarium graminearum, the causal agent of FHB, showed an increased in expression of genes associated with an effective defense response, in particular glucan endo-1,3-beta-glucosidases and chitinases, in the FHB-resistant line CS-7EL while there was a larger increase in differential expression for genes associated with the level of fungal infection in the FHB-susceptible line CS. One hundred and seven 7EL transcripts were expressed in the smallest 7EL region defined to carry FHB resistance. Conclusion 7EL contributed to CS-7EL transcriptome by direct expression and through alteration of wheat transcript profiles. FHB resistance in CS-7EL was associated with transcriptome changes suggesting a more effective defense response. A list of candidate genes for the FHB resistance locus on 7EL has been established.

Abstract Background We analyzed the association between substance use (SU) and condomless sex (CS) among HIV-negative adults reporting heterosexual sex in the Seek, Test, Treat, and Retain (STTR) consortium. We describe the impact of SU as well as person/partner and context-related factors on CS, identifying combinations of factors that indicate the highest likelihood of CS. Methods We analyzed data from four US-based STTR studies to examine the effect of SU on CS using two SU exposures: 1) recent SU (within 3 months) and 2) SU before/during sex. Behavioral data were collected via 1:1 or self-administered computerized interviews. Adjusted individual-study, multivariable relative risk regression was used to examine the relationship between CS and SU. We also examined interactions with type of sex and partner HIV status. Pooled effect estimates were calculated using traditional fixed-effects meta-analysis. We analyzed data for recent SU (n = 6781; 82% men, median age = 33 years) and SU before/during sex (n = 2915; 69% men, median age = 40 years). Results For both exposure classifications, any SU other than cannabis increased the likelihood of CS relative to non-SU (8–16%, p-values

Abstract Background Concerns have been raised that the COVID-19 pandemic could increase risk for adverse mental health outcomes, especially in young adults, a vulnerable age group. We investigated changes in depression and anxiety symptoms (overall and severe) from before to during the pandemic, as well as whether these changes are linked to COVID-19-related stressors and pre-existing vulnerabilities in young adults followed in the context of a population-based cohort. Method Participants (n = 1039) from the Quebec Longitudinal Study of Child Development reported on their depression (Centre for Epidemiological Studies Depression Scale, short form) and anxiety (General Anxiety Disorder-7 Scale) symptoms and completed a COVID-19 questionnaire during the first wave of the COVID-19 pandemic in the summer of 2020 (age 22 years). Assessments at age 20 (2018) were used to estimate pre-pandemic depression and anxiety symptom severity. Results While mean levels of depression and anxiety symptoms did not change from before to during the first wave of the COVID-19 pandemic (e.g., the mean of depressive symptoms was 9.30 in 2018 and 9.59 in 2020), we observed a slight increase in rates of severe depression (scores ≥ 21) from before (6.1%) to during (8.2%) the pandemic. Most COVID-19-related variables (e.g., loss of education/occupation, frequent news-seeking) – except living alone – and most pre-existing vulnerabilities (e.g., low SES, low social support) were not associated with changes in depression or anxiety symptoms. However, results varied as a function of pre-pandemic levels of depression and anxiety: depression and anxiety symptoms increased among adults with the lowest levels of symptoms before the pandemic, while they decreased among those with the highest levels of symptoms, possibly reflecting a regression to the mean. Conclusions Depression and anxiety symptoms in young adults from Québec in Summer 2020 were mostly comparable to symptoms reported in 2018. Most COVID-19-related stressors and pre-existing vulnerabilities were not associated with changes in symptoms, except living alone and pre-existing symptoms of depression and anxiety. However, the increased rate of severe depression warrants further investigation.

Abstract Background There is an increased level of senescent cells and toll-like teceptor-1, -2, -4, and -6 (TLR) expression in degenerating intervertebral discs (IVDs) from back pain patients. However, it is currently not known if the increase in expression of TLRs is related to the senescent cells or if it is a more general increase on all cells. It is also not known if TLR activation in IVD cells will induce cell senescence. Methods Cells from non-degenerate human IVD were obtained from spine donors and cells from degenerate IVDs came from patients undergoing surgery for low back pain. Gene expression of TLR-1,2,4,6, senescence and senescence-associated secretory phenotype (SASP) markers was evaluated by RT-qPCR in isolated cells. Matrix synthesis was verified with safranin-O staining and Dimethyl-Methylene Blue Assay (DMMB) confirmed proteoglycan content. Protein expression of p16 INK4a , SASP factors, and TLR-2 was evaluated by immunocytochemistry (ICC) and/or by enzyme-linked immunosorbent assay (ELISA). Results An increase in senescent cells was found following 48-h induction with a TLR-2/6 agonist in cells from both non-degenerate and degenerating human IVDs. Higher levels of SASP factors, TLR-2 gene expression, and protein expression were found following 48-h induction with TLR-2/6 agonist. Treatment with o-vanillin reduced the number of senescent cells, and increased matrix synthesis in IVD cells from back pain patients. Treatment with o-vanillin after induction with TLR-2/6 agonist reduced gene and protein expression of SASP factors and TLR-2. Co-localized staining of p16 INK4a and TLR-2 demonstrated that senescent cells have a high TLR-2 expression. Conclusions Taken together our data demonstrate that activation of TLR-2/6 induce senescence and increase TLR-2 and SASP expression in cells from non-degenerate IVDs of organ donors without degeneration and back pain and in cells from degenerating human IVD of patients with disc degeneration and back pain. The senescent cells showed high TLR-2 expression suggesting a link between TLR activation and cell senescence in human IVD cells. The reduction in senescence, SASP, and TLR-2 expression suggest o-vanillin as a potential disease-modifying drug for patients with disc degeneration and back pain.

Abstract Background Medical societies and funding agencies strongly recommend that patients be included as partners in research publications and grant applications. Although this “top-down” approach is certainly efficient at forcing this new and desirable type of collaboration, our past experience demonstrated that it often results in an ambiguous relationship as not yet well integrated into the cultures of either patients’ or the researchers’. The question our group raised from this observation was: “How to generate a cultural shift toward a fruitful and long-lasting collaboration between patients and researchers? A “bottom-up” approach was key to our stakeholders. The overall objective was to build a trusting and bidirectional-ecosystem between patients and researchers. The specific objectives were to document: 1) the steps that led to the development of the first patient-partner strategic committee within a research center in the Province of Québec; 2) the committee’s achievements after 3 years. Methods Eighteen volunteer members, 12 patient-partners and 6 clinician/institutional representatives, were invited to represent the six research themes of the Centre de recherche du CHU de Sherbrooke (CRCHUS) (Quebec, Canada). Information on the services offered by Committee was disseminated internally and to external partners. Committee members satisfaction was evaluated. Results From May 2017 to April 2020, members attended 29 scheduled and 6 ad hoc meetings and contributed to activities requiring over 1000 h of volunteer time in 2018–2019 and 1907 h in the 2019–2020 period. The Committee’s implication spanned governance, expertise, and knowledge transfer in research. Participation in these activities increased annually at local, provincial, national and international levels. The Patient-Partner Committee collaborated with various local (n = 7), provincial (n = 6) and national (n = 4) partners. Member satisfaction with the Committee’s mandate and format was 100%. Conclusions The CRCHUS co-constructed a Patient-Partner Strategic Committee which resulted in meaningful bilateral, trusting and fruitful collaborations between patients, researchers and partners. The “bottom-up” approach - envisioned and implemented by the Committee, where the expertise and the needs of patients complemented those of researchers, foundations, networks and decision-makers - is key to the success of a cultural shift. The CRCHUS Committee created a hub to develop the relevant intrinsic potential aimed at changing the socio-cultural environment of science.

Abstract Background A recent literature review emphasized the importance of assessing dual-task (DT) abilities with tasks that are representative of community ambulation. Assessing DT ability in real-life activities using standardized protocols remains difficult. Virtual reality (VR) may represent an interesting alternative enabling the exposure to different scenarios simulating community walking. To better understand dual-task abilities in everyday life activities, the aims of this study were (1) to assess locomotor and cognitive dual-task cost (DTC) during representative daily living activities, using VR, in healthy adults; and 2) to explore the influence of the nature and complexity of locomotor and cognitive tasks on DTC. Methods Fifteen healthy young adults (24.9 ± 2.7 years old, 8 women) were recruited to walk in a virtual 100 m shopping mall corridor, while remembering a 5-item list (DT condition), using an omnidirectional platform and a VR headset. Two levels of difficulty were proposed for the locomotor task (with vs. without virtual agent avoidance) and for the cognitive task (with vs. without items modification). These tasks were also performed in single task (ST) condition. Locomotor and cognitive DTC were measured by comparing performances in ST and DT conditions. Locomotor performance was characterized using walking speed, walking fluidity, and minimal distance between the participant and the virtual agent during avoidance. Cognitive performance was assessed with the number of items correctly recalled. Presence of DTC were determined with one-sample Wilcoxon signed-rank tests. To explore the influence of the tasks’ complexity and nature on DTC, a nonparametric two-way repeated measure ANOVA was performed. Results No locomotor interference was measured for any of the outcomes. A cognitive DTC of 6.67% was measured (p = .017) while participants performed simultaneously both complex locomotor and cognitive tasks. A significant interaction between locomotor task complexity and cognitive task nature (p = .002) was identified on cognitive DTC. Conclusions In challenging locomotor and cognitive conditions, healthy young adults present DTC in cognitive accuracy, which was influenced by the locomotor task complexity task and the cognitive task nature. A similar VR-based protocol might be used to investigate DT abilities in older adults and individuals with a stroke.

Abstract Background Adolescent idiopathic scoliosis (AIS) has been associated with diminished postural stability and a greater prevalence of back pain. Currently, the literature is lacking information on the effect of spinal fusion on both postural stability and its association with back pain. Our objectives were to evaluate the postsurgical effect of spinal morphological changes on static standing balance and assess the influence of these alterations on reported pain throughout the perioperative period. Methods Twenty consecutive AIS patients schedule to undergo spinal fusion surgery were recruited and followed prospectively at the Shriners Hospitals for Children-Canada. Data was collected at the preoperative, 6 weeks and 6 months postoperative visits. Spinal morphology data was collected through 3D reconstructed simultaneous standing biplanar radiographs using the SterEOS software. Postural balance was assessed through Moticon© sensor insoles and analyzed through their software. The data was simultaneously collected as part of the Global Biomechanical and morphological Assessment. Pain was evaluated through self-reported questionnaires. Results Morphological curve parameters were significantly reduced after surgery. Balance parameters did not change significantly throughout the perioperative period with the exception of the Center of Pressure of the left foot medial/lateral transient shift (P = 0.017) at 6 weeks. Of note, preoperative balance parameters were associated with the degree of right thoracic Cobb angles (P = 0.029 R = 0.528). Pain scores significantly improved 6 weeks and 6 months after the surgery. Pain intensity diminished in the thoracic and lumbar spine but worsen in the neck region at the 6 weeks and 6 months postoperative time points (P = 0.044). Greater residual Cobb angle difference between Mid thoracic and Thoracolumbar/Lumbar curves was associated with greater pain severity at 6 weeks postop (P

Abstract Background Drought stress is one of the major factors limiting wheat production globally. Improving drought tolerance is important for agriculture sustainability. Although various morphological, physiological and biochemical responses associated with drought tolerance have been documented, the molecular mechanisms and regulatory genes that are needed to improve drought tolerance in crops require further investigation. We have used a novel 4-component version (for overexpression) and a 3-component version (for underexpression) of a barley stripe mosaic virus-based (BSMV) system for functional characterization of the C2H2-type zinc finger protein TaZFP1B in wheat. These expression systems avoid the need to produce transgenic plant lines and greatly speed up functional gene characterization. Results We show that overexpression of TaZFP1B stimulates plant growth and up-regulates different oxidative stress-responsive genes under well-watered conditions. Plants that overexpress TaZFP1B are more drought tolerant at critical periods of the plant’s life cycle. Furthermore, RNA-Seq analysis revealed that plants overexpressing TaZFP1B reprogram their transcriptome, resulting in physiological and physical modifications that help wheat to grow and survive under drought stress. In contrast, plants transformed to underexpress TaZFP1B are significantly less tolerant to drought and growth is negatively affected. Conclusions This study clearly shows that the two versions of the BSMV system can be used for fast and efficient functional characterization of genes in crops. The extent of transcriptome reprogramming in plants that overexpress TaZFP1B indicates that the encoded transcription factor is a key regulator of drought tolerance in wheat.

Abstract Background Preschoolers regularly display disruptive behaviors in child care settings because they have not yet developed the social skills necessary to interact prosocially with others. Disruptive behaviors interfere with daily routines and can lead to conflict with peers and educators. We investigated the impact of a social skills training program led by childcare educators on children’s social behaviors and tested whether the impact varied according to the child’s sex and family socio-economic status. Methods Nineteen public Child Care Centers (CCC, n = 361 children) located in low socio-economic neighborhoods of Montreal, Canada, were randomized into one of two conditions: 1) intervention (n = 10 CCC; 185 children) or 2) wait list control (n = 9 CCC; 176 children). Educators rated children’s behaviors (i.e., disruptive and prosocial behaviors) before and after the intervention. Hierarchical linear mixed models were used to account for the nested structure of the data. Results At pre-intervention, no differences in disruptive and prosocial behaviors were observed between the experimental conditions. At post-intervention, we found a significant sex by intervention interaction (β intervention by sex = − 1.19, p = 0.04) indicating that girls in the intervention condition exhibited lower levels of disruptive behaviors compared to girls in the control condition (f2 effect size = − 0.15). There was no effect of the intervention for boys. Conclusions Girls may benefit more than boys from social skills training offered in the child care context. Studies with larger sample sizes and greater intervention intensity are needed to confirm the results. Trial registration Current clinical trial number is ISRCTN84339956 (Retrospectively registered in March 2017). No amendment to initial protocol.

Abstract Background Emergency departments (EDs) in rural and remote areas face challenges in delivering accessible, high quality and efficient services. The objective of this pilot study was to test the feasibility and relevance of the selected approach and to explore challenges and solutions to improve delivery of care in selected EDs. Methods We conducted an exploratory multiple case study in two rural EDs in Québec, Canada. A survey filled out by the head nurse for each ED provided a descriptive statistical portrait. Semi-structured interviews were conducted with ED health professionals, decision-makers and citizens (n = 68) and analyzed inductively and thematically. Results The two EDs differed with regards to number of annual visits, inter-facility transfers and wait time. Stakeholders stressed the influence of context on ED challenges and solutions, related to: 1) governance and management (e.g. lack of representation, poor efficiency, ill-adapted standards); 2) health services organization (e.g. limited access to primary healthcare and long-term care, challenges with transfers); 3) resources (e.g. lack of infrastructure, limited access to specialists, difficult staff recruitment/retention); 4) and professional practice (e.g. isolation, large scope, maintaining competencies with low case volumes, need for continuing education, teamwork and protocols). There was a general agreement between stakeholder groups. Conclusions Our findings show the feasibility and relevance of mobilizing stakeholders to identify context-specific challenges and solutions. It confirms the importance of undertaking a larger study to improve the delivery of care in rural EDs.

Abstract Background Fusarium head blight (FHB) is a major disease of cereal crops, caused by the fungal pathogen Fusarium graminearum and related species. Breeding wheat for FHB resistance contributes to increase yields and grain quality and to reduce the use of fungicides. The identification of genes and markers for FHB resistance in different wheat genotypes has nevertheless proven challenging. Results In this study, early infection by F. graminearum was analyzed in a doubled haploid population derived from the cross of the moderately resistant wheat genotypes Wuhan 1 and Nyubai. Three quantitative trait loci (QTL) were identified: 1AL was associated with lower deoxynivalenol content, and 4BS and 5A were associated with reduced F. graminearum infection at 2 days post inoculation. Early resistance alleles were inherited from Wuhan 1 for QTL 1AL and 4BS and inherited from Nyubai for the 5A QTL. Cis and trans expression QTL (eQTL) were identified using RNA-seq data from infected head samples. Hotspots for trans eQTL were identified in the vicinity of the 1AL and 4BS QTL peaks. Among differentially expressed genes with cis eQTL within the QTL support intervals, nine genes had higher expression associated with FHB early resistance, and four genes had higher expression associated with FHB early susceptibility. Conclusions Our analysis of genotype and gene expression data of wheat infected by F. graminearum identified three QTL associated with FHB early resistance, and linked genes with eQTL and differential expression patterns to those QTL. These findings may have applications in breeding wheat for early resistance to FHB.

Abstract Background Targeted genome editing using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has been applied in a large number of plant species. Using a gene-specific single guide RNA (sgRNA) and the CRISPR/Cas9 system, small editing events such as deletions of few bases can be obtained. However larger deletions are required for some applications. In addition, identification and characterization of edited events can be challenging in plants with complex genomes, such as wheat. Results In this study, we used the CRISPR/Cas9 system and developed a protocol that yielded high number of large deletions employing a pair of co-expressed sgRNA to target the same gene. The protocol was validated by targeting three genes, TaABCC6, TaNFXL1 and TansLTP9.4 in a wheat protoplast assay. Deletions of sequences located between the two sgRNA in each gene were the most frequent editing events observed for two of the three genes. A comparative assessment of editing frequencies between a codon-optimized Cas9 for expression in algae, crCas9, and a plant codon-optimized Cas9, pcoCas9, showed more consistent results with the vector expressing pcoCas9. Editing of TaNFXL1 by co-expression of sgRNA pair was investigated in transgenic wheat plants. Given the ploidy of bread wheat, a rapid, robust and inexpensive genotyping protocol was also adapted for hexaploid genomes and shown to be a useful tool to identify homoeolog-specific editing events in wheat. Conclusions Co-expressed pairs of sgRNA targeting single genes in conjunction with the CRISPR/Cas9 system produced large deletions in wheat. In addition, a genotyping protocol to identify editing events in homoeologs of TaNFXL1 was successfully adapted.

Abstract Background CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as “two-donor floxing” method). Results We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach. Conclusion We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis, an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.

Abstract Background Fusarium head blight (FHB) is a problem of great concern in small grain cereals, especially wheat. A quantitative trait locus (QTL) for FHB resistance (FHB_SFI) located on the long arm of chromosome 2D in the spring wheat genotype Wuhan 1 is a resistance locus which has potential to improve the FHB resistance of bread wheat since it confers effective resistance to wheat breeding lines. Recently, differentially expressed genes (DEG) have been identified by comparing near isogenic lines (NIL) carrying the susceptible and resistant alleles for the 2DL QTL, using RNA-Seq. In the present study, we aimed to identify candidate genes located within the genetic interval for the 2DL QTL for FHB resistance, as assessed by single floret inoculation (FHB_SFI), and possibly contributing to it. Results Combining previous and additional bioinformatics analyses, 26 DEG that were located on chromosome arm 2DL were selected for further characterization of their expression profile by RT-qPCR. Seven of those DEG showed a consistent differential expression profile between either three pairs of near isogenic lines or other genotypes carrying the R and S alleles for the 2DL QTL for FHB resistance. UN25696, which was identified in previous expression work using microarray was also confirmed to have a differential expression pattern. Those eight candidate genes were further characterized in 85 lines of a double haploid mapping population derived from the cross Wuhan 1/Nyubai, the population where the 2DL QTL was originally identified. The expression QTL for gene Traes_2DL_179570792 overlapped completely with the mapping interval for the 2DL QTL for FHB_SFI while the expression QTL for UN25696 mapped near the QTL, but did not overlap with it. None of the other genes had a significant eQTL on chromosome 2DL. Higher expression of Traes_2DL_179570792 and UN25696 was associated with the resistant allele at that locus. Conclusions Of the 26 DEG from the 2DL chromosome further characterized in this study, only two had an expression QTL located in or near the interval for the 2DL QTL. Traes_2DL_179570792 is the first expression marker identified as associated with the 2DL QTL.

Abstract Background Mentalizing, the mental capacity to understand oneself and others in terms of mental states, has been found to be reduced in some mental disorders such as Borderline Personality Disorder (BPD). Some studies have suggested that Eating Disorders (EDs) may also be associated with impairments in mentalizing, but studies have not always yielded consistent results. This is the first study to systematically investigate mentalizing impairments in patients with Bulimia Nervosa (BN) compared with controls. In addition, we investigated whether impairments in mentalizing were related to BPD features, rather than BN per se, given the high comorbidity between BPD and BN. Methods Patients with BN (n = 53) and healthy controls (HCs; n = 87) completed a battery of measures assessing mentalizing including the Reflective Function Questionnaires (RFQ), the Object Relations Inventory (ORI; Differentiation-Relatedness Scales) and the Reading The Mind in The Eyes Test (RMET). Results Patients with BN scored significantly lower than HCs on all tests of mentalizing, with moderate to large between-group effect sizes. These differences were partially accounted for by BPD features as assessed with the Zanarini Rating Scale for Borderline Personality Disorder (ZAN-BPD), and partially by bulimic symptoms measured with the Eating Disorder Examination Questionnaire (EDE-Q). Conclusions Patients with BN have significantly lower levels of mentalizing as assessed with a broad range of tests compared to HCs. These differences were related to both bulimic symptoms and BPD features. Although further research in larger samples is needed, if replicated, these findings suggest that poor mentalizing may be a significant factor in BN patients and should be addressed in treatment, regardless of the presence of BPD features.

Abstract Background Phytohormones are key regulators of plant growth, development, and signalling networks involved in responses to diverse biotic and abiotic stresses. Transcriptional reference maps of hormone responses have been reported for several model plant species such as Arabidopsis thaliana, Oryza sativa, and Brachypodium distachyon. However, because of species differences and the complexity of the wheat genome, these transcriptome data are not appropriate reference material for wheat studies. Results We comprehensively analysed the transcriptomic responses in wheat spikes to seven phytohormones, including indole acetic acid (IAA), gibberellic acid (GA), abscisic acid (ABA), ethylene (ET), cytokinin (CK), salicylic acid (SA), and methyl jasmonic acid (MeJA). A total of 3386 genes were differentially expressed at 24 h after the hormone treatments. Furthermore, 22.7% of these genes exhibited overlapping transcriptional responses for at least two hormones, implying there is crosstalk among phytohormones. We subsequently identified genes with expression levels that were significantly and differentially induced by a specific phytohormone (i.e., hormone-specific responses). The data for these hormone-responsive genes were then compared with the transcriptome data for wheat spikes exposed to biotic (Fusarium head blight) and abiotic (water deficit) stresses. Conclusion Our data were used to develop a transcriptional reference map of hormone responses in wheat spikes.

Abstract Background Refinement of parameters defining prostate cancer (PC) prognosis are urgently needed to identify patients with indolent versus aggressive disease. The Canadian Prostate Cancer Biomaker Network (CPCBN) consists of researchers from four Canadian provinces to create a validation cohort to address issues dealing with PC diagnosis and management. Methods A total of 1512 radical prostatectomy (RP) specimens from five different biorepositories affiliated with teaching hospitals were selected to constitute the cohort. Tumoral and adjacent benign tissues were arrayed on tissue microarrays (TMAs). A patient clinical database was developed and includes data on diagnosis, treatment and clinical outcome. Results Mean age at diagnosis of patients in the cohort was 61 years. Of these patients, 31% had a low grade (≤6) Gleason score (GS), 55% had GS 7 (40% of 3 + 4 and 15% of 4 + 3) and 14% had high GS (≥8) PC. The median follow-up of the cohort was 113 months. A total of 34% had a biochemical relapse, 4% developed bone metastasis and 3% of patients died from PC while 9% died of other causes. Pathological review of the TMAs confirmed the presence of tumor and benign tissue cores for > 94% of patients. Immunohistochemistry and FISH analyses, performed on a small set of specimens, showed high quality results and no biorepository-specific bias. Conclusions The CPCBN RP cohort is representative of real world PC disease observed in the Canadian population. The frequency of biochemical relapse and bone metastasis as events allows for a precise assessment of the prognostic value of biomarkers. This resource is available, in a step-wise manner, for researchers who intend to validate prognostic biomarkers in PC. Combining multiple biomarkers with clinical and pathologic parameters that are predictive of outcome will aid in clinical decision-making for patients treated for PC.

Abstract Background Fusarium head blight (FHB) of wheat in North America is caused mostly by the fungal pathogen Fusarium graminearum (Fg). Upon exposure to Fg, wheat initiates a series of cellular responses involving massive transcriptional reprogramming. In this study, we analyzed transcriptomics data of four wheat genotypes (Nyubai, Wuhan 1, HC374, and Shaw), at 2 and 4 days post inoculation (dpi) with Fg, using RNA-seq technology. Results A total of 37,772 differentially expressed genes (DEGs) were identified, 28,961 from wheat and 8811 from the pathogen. The susceptible genotype Shaw exhibited the highest number of host and pathogen DEGs, including 2270 DEGs associating with FHB susceptibility. Protein serine/threonine kinases and LRR-RK were associated with susceptibility at 2 dpi, while several ethylene-responsive, WRKY, Myb, bZIP and NAC-domain containing transcription factors were associated with susceptibility at 4 dpi. In the three resistant genotypes, 220 DEGs were associated with resistance. Glutathione S-transferase (GST), membrane proteins and distinct LRR-RKs were associated with FHB resistance across the three genotypes. Genes with unique, high up-regulation by Fg in Wuhan 1 were mostly transiently expressed at 2 dpi, while many defense-associated genes were up-regulated at both 2 and 4 dpi in Nyubai; the majority of unique genes up-regulated in HC374 were detected at 4 dpi only. In the pathogen, most genes showed increased expression between 2 and 4 dpi in all genotypes, with stronger levels in the susceptible host; however two pectate lyases and a hydrolase were expressed higher at 2 dpi, and acetyltransferase activity was highly enriched at 4 dpi. Conclusions There was an early up-regulation of LRR-RKs, different between susceptible and resistant genotypes; subsequently, distinct sets of genes associated with defense response were up-regulated. Differences in expression profiles among the resistant genotypes indicate genotype-specific defense mechanisms. This study also shows a greater resemblance in transcriptomics of HC374 to Nyubai, consistent with their sharing of two FHB resistance QTLs on 3BS and 5AS, compared to Wuhan 1 which carries one QTL on 2DL in common with HC374.

Abstract Background Over the last decade, active surveillance has proven to be a safe approach for patients with low-risk prostate cancer. Although active surveillance presents several advantages for both patients and the health care system, all eligible patients do not adopt this approach. Our goal was to evaluate the factors that influence physicians to recommend active surveillance and the barriers that impact adherence to this approach. Methods Focus groups (n = 5) were held with physicians who provided care for men with low-risk prostate cancer and had engaged in conversations with men and their families about active surveillance. The experience of health care professionals (HCPs) was captured to understand their decisions in proposing active surveillance and to reveal the barriers and facilitators that affect the adherence to this approach. A content analysis was performed on the verbatim transcripts from the sessions. Results Although physicians agreed that active surveillance is a suitable approach for low-risk prostate cancer patients, they were concerned about the rapidly evolving and non-standardized guidelines for patient follow-up. They pointed out the need for additional tools to appropriately identify proper patients for whom active surveillance is the best option. Urologists and radiation-oncologists were keen to collaborate with each other, but the role of general practitioner remained controversial once patients were referred to a specialist. Conclusions Integration of more reliable tools and/or markers in addition to more specific guidelines for patient follow-up would increase the confidence of both patients and physicians in the choice of active surveillance.

Abstract Background The Fos-related antigen 1 (FRA-1) transcription factor promotes tumor cell growth, invasion and metastasis. Phosphorylation of FRA-1 increases protein stability and function. We identify a novel signaling axis that leads to increased phosphorylation of FRA-1, increased extracellular matrix (ECM)-induced breast cancer cell invasion and is prognostic of poor outcome in patients with breast cancer. Methods While characterizing five breast cancer cell lines derived from primary human breast tumors, we identified BRC-31 as a novel basal-like cell model that expresses elevated FRA-1 levels. We interrogated the functional contribution of FRA-1 and an upstream signaling axis in breast cancer cell invasion. We extended this analysis to determine the prognostic significance of this signaling axis in samples derived from patients with breast cancer. Results BRC-31 cells display elevated focal adhesion kinase (FAK), SRC and extracellular signal-regulated (ERK2) phosphorylation relative to luminal breast cancer models. Inhibition of this signaling axis, with pharmacological inhibitors, reduces the phosphorylation and stabilization of FRA-1. Elevated integrin αVβ3 and uPAR expression in these cells suggested that integrin receptors might activate this FAK-SRC-ERK2 signaling. Transient knockdown of urokinase/plasminogen activator urokinase receptor (uPAR) in basal-like breast cancer cells grown on vitronectin reduces FRA-1 phosphorylation and stabilization; and uPAR and FRA-1 are required for vitronectin-induced cell invasion. In clinical samples, a molecular component signature consisting of vitronectin-uPAR-uPA-FRA-1 predicts poor overall survival in patients with breast cancer and correlates with an FRA-1 transcriptional signature. Conclusions We have identified a novel signaling axis that leads to phosphorylation and enhanced activity of FRA-1, a transcription factor that is emerging as an important modulator of breast cancer progression and metastasis.

Abstract Background We report an unusual case of a synchronous rectal and metachronous vaginal metastatic renal cell carcinoma. Case presentation A 78-year-old woman presented with hematochezia and a colonoscopy revealed a metastatic clear-cell renal cell carcinoma rectal polyp biopsy-proven. Abdominal computed tomography identified a 9.0-cm left renal mass with renal vein thrombosis, for which she underwent a laparoscopic radical nephrectomy. Histopathological examination confirmed a pT3a clear-cell renal cell carcinoma. Seven months later, the patient presented with vaginal bleeding. Physical examination revealed a vaginal polypoid mass and biopsy confirmed a clear-cell renal cell carcinoma metastasis. Conclusions This case represents unusual manifestations of metastatic renal cell carcinoma and is a reminder of the wide spectrum of clinical course of this disease.

Abstract Background In prostate cancer, men diagnosed with low risk disease may be monitored through an active surveillance. This research explored the perspectives of men with prostate cancer regarding their decision-making process for active surveillance to identify factors that influence their decision and assist health professionals in having conversations about this option. Methods Focus group interviews (n = 7) were held in several Canadian cities with men (N = 52) diagnosed with prostate cancer and eligible for active surveillance. The men’s viewpoints were captured regarding their understanding of active surveillance, the factors that influenced their decision, and their experience with the approach. A content and theme analysis was performed on the verbatim transcripts from the sessions. Results Patients described their concerns of living with their disease without intervention, but were reassured by the close monitoring under AS while avoiding harmful side effects associated with treatments. Conversations with their doctor and how AS was described were cited as key influences in their decision, in addition to availability of information on treatment options, distrust in the health system, personality, experiences and opinions of others, and personal perspectives on quality of life. Conclusions Men require a thorough explanation on AS as a safe and valid option, as well as guidance towards supportive resources in their decision-making.

Abstract Background Most preschoolers growing up in western industrialized countries receive child care services (CCS) during the day, while their parents are at work. Meta-analytic data suggest that CCS represent a stressful experience for preschoolers. This may be because preschoolers have not yet developed the social skills necessary to cope with the new and rapidly fluctuating social contexts of CCS. We tested the effectiveness of a child care-based social skill training program aiming to improve children’s social behaviors and reduce the stress they experience. Method and design We used a cluster randomized control trial (cRCT) to compare children’s social behaviors and stress levels in pre- and post-intervention according to whether they received a social skill training intervention or not. Nineteen (n = 19) public CCS (n = 362, 3-years-old preschoolers) of underprivileged neighborhoods (Montreal, Canada) were randomized to one of two conditions: 1) social skills training (n = 10 CCS); or 2) waiting list control group (n = 9 CCS). Educators in the intervention group conducted bi-weekly social skills training sessions over a period of 8 months. The intervention covered four topics: making social contacts, problem solving, emotional self-regulation, as well as emotional expression and recognition. Main outcome measures included preschoolers’ disruptive (e.g. aggression, opposition, conflicts) and prosocial behaviors (e.g. sharing toys, helping another child), and stress levels assessed by salivary cortisol sampling at pre and post intervention assessments. Educators’ practices will be tested as potential mediators of the expected changes in behaviors and neuroendocrine stress. Discussion To our knowledge, this is the first cRCT to test the effectiveness of a child care based social skill training program on the reduction of disruptive behaviors and levels of stress. Significant challenges include the degree of adherence to the intervention protocol as well educators and preschoolers’ turnover. Trial registration Current clinical trial number is ISRCTN84339956 (Ongoing study, Retrospectively registered on March 2017) No amendment to initial protocol.

Abstract Background The STTR treatment cascade provides a framework for research aimed at improving the delivery of services, care and outcomes of PLWH. The development of effective approaches to increase HIV diagnoses and engage PLWH in subsequent steps of the treatment cascade could lead to earlier and sustained ART treatment resulting in viral suppression. There is an unmet need for research applying the treatment cascade to improve outcomes for those with criminal justice involvement. Methods The Seek, Test, Treat, and Retain (STTR) criminal justice (CJ) cohort combines data from 11 studies across the HIV treatment cascade that focused on persons involved in the criminal justice system, often but not exclusively for reasons related to substance use. The studies were conducted in a variety of CJ settings and collected information across 11 pre-selected domains: demographic characteristics, CJ involvement, HIV risk behaviors, HIV and/or Hepatitis C infections, laboratory measures of CD4 T-cell count (CD4) and HIV RNA viral load (VL), mental illness, health related quality of life (QoL), socioeconomic status, health care access, substance use, and social support. Results The STTR CJ cohort includes data on 11,070 individuals with and without HIV infection who range in age from 18 to 77 years, with a median age at baseline of 37 years. The cohort reflects racial, ethnic and gender distributions in the U.S. CJ system, and 64% of participants are African-American, 12% are Hispanic and 83% are men. Cohort members reported a wide range of HIV risk behaviors including history of injection drug use and, among those who reported on pre-incarceration sexual behaviors, the prevalence of unprotected sexual intercourse ranged across studies from 4% to 79%. Across all studies, 53% percent of the STTR CJ cohort reported recent polysubstance use. Conclusions The STTR CJ cohort is comprised of participants from a wide range of CJ settings including jail, prison, and community supervision who report considerable diversity in their characteristics and behavioral practices. We have developed harmonized measures, where feasible, to improve the integration of these studies together to answer questions that cannot otherwise be addressed.

In the original publication [1], the copyright line was incorrectly published as “© The Author(s) 2019”. The corrected copyright line should read as “© Her Majesty the Queen in Right of Canada as represented by the Minister of Agriculture and Agri-Food Canada, 2019”. The original article has been corrected.

Following publication of the original article [1], we have been notified that some important information was omitted by the authors in the Copyright note. The Copyright note should read as below.

Abstract Intravenous immunoglobulin (IVIg) is a blood-derived product, used for the treatment of immunodeficiency and autoimmune diseases. Since a range of immunotherapies have recently been proposed as a therapeutic strategy for Parkinson’s disease (PD), we investigated the effects of an IVIg treatment in a neurotoxin-induced animal model of PD. Mice received four injections of MPTP (15 mg/kg) at 2-hour intervals followed by a 14-day IVIg treatment, which induced key immune-related changes such as increased regulatory T-cell population and decreased CD4+/CD8+ ratio. The MPTP treatment induced significant 80% and 84% decreases of striatal dopamine concentrations (P P P

Abstract Non-necrotizing epithelioid granulomas have been described in association with many primary tumors. In such cases, they are designated as sarcoid-like reaction. Although it is more seen in carcinomas than in sarcomas, it is very rarely reported in renal carcinoma. Here, we describe a rare association of prominent peritumoral sarcoid-like reaction without intratumoral granulomas and conventional clear cell renal carcinoma in a 62-year-old-male, without clinical or laboratory finding of sarcoidosis. At 30 months follow-up, he had no recurrence. Virtual Slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/4054525336657922

Abstract Background Mounting evidence supports a significant role of inflammation in Parkinson's disease (PD) pathophysiology, with several inflammatory pathways being suggested as playing a role in the dopaminergic degeneration seen in humans and animal models of the disease. These include tumor necrosis factor, prostaglandins and oxidative-related stress components. However, the role of innate immunity has not been established in PD. Methods Based on the fact that the myeloid differentiation primary response gene (88) (MyD88) is the most common adaptor protein implicated in toll-like receptor (TLR) signaling, critical in the innate immune response, we undertook a study to investigate the potential contribution of this specific pathway to MPTP-induced brain dopaminergic degeneration using MyD88 knock out mice (MyD88-/-), following our observations that the MyD88-dependent pathway was critical for MPTP dopaminergic toxicity in the enteric nervous system. Post-mortem analyses assessing nigrostriatal dopaminergic degeneration and inflammation were performed using HPLC, western blots, autoradiography and immunofluorescence. Results Our results demonstrate that MyD88-/- mice are as vulnerable to MPTP-induced dopamine and DOPAC striatal depletion as wild type mice. Furthermore, MyD88-/- mice show similar striatal dopamine transporter and tyrosine hydroxylase loss, as well as dopaminergic cell loss in the substantia nigra pars compacta in response to MPTP. To evaluate the extent of the inflammatory response created by the MPTP regimen utilized, we further performed bioluminescence imaging using TLR2-luc/gfp transgenic mice and microglial density analysis, which revealed a modest brain microglial response following MPTP. This was accompanied by a significant astrocytic reaction in the striatum, which was of similar magnitude both in wild type and MyD88-/- mice. Conclusions Our results suggest that subacute MPTP-induced dopaminergic degeneration observed in the central nervous system is MyD88-independent, in contrast to our recent observations that this pathway, in the same cohort of animals, is critical in the loss of dopaminergic neurons in the enteric nervous system.

Abstract Background Class III Homeodomain Leucine Zipper (HD-Zip III) proteins have been implicated in the regulation of cambium identity, as well as primary and secondary vascular differentiation and patterning in herbaceous plants. They have been proposed to regulate wood formation but relatively little evidence is available to validate such a role. We characterised and compared HD-Zip III gene family in an angiosperm tree, Populus spp. (poplar), and the gymnosperm Picea glauca (white spruce), representing two highly evolutionarily divergent groups. Results Full-length cDNA sequences were isolated from poplar and white spruce. Phylogenetic reconstruction indicated that some of the gymnosperm sequences were derived from lineages that diverged earlier than angiosperm sequences, and seem to have been lost in angiosperm lineages. Transcript accumulation profiles were assessed by RT-qPCR on tissue panels from both species and in poplar trees in response to an inhibitor of polar auxin transport. The overall transcript profiles HD-Zip III complexes in white spruce and poplar exhibited substantial differences, reflecting their evolutionary history. Furthermore, two poplar sequences homologous to HD-Zip III genes involved in xylem development in Arabidopsis and Zinnia were over-expressed in poplar plants. PtaHB1 over-expression produced noticeable effects on petiole and primary shoot fibre development, suggesting that PtaHB1 is involved in primary xylem development. We also obtained evidence indicating that expression of PtaHB1 affected the transcriptome by altering the accumulation of 48 distinct transcripts, many of which are predicted to be involved in growth and cell wall synthesis. Most of them were down-regulated, as was the case for several of the poplar HD-Zip III sequences. No visible physiological effect of over-expression was observed on PtaHB7 transgenic trees, suggesting that PtaHB1 and PtaHB7 likely have distinct roles in tree development, which is in agreement with the functions that have been assigned to close homologs in herbaceous plants. Conclusions This study provides an overview of HD-zip III genes related to woody plant development and identifies sequences putatively involved in secondary vascular growth in angiosperms and in gymnosperms. These gene sequences are candidate regulators of wood formation and could be a source of molecular markers for tree breeding related to wood properties.

Abstract Background We previously identified that Ran protein, a member of the Ras GTPase family, is highly expressed in high grade and high stage serous epithelial ovarian cancers, and that its overexpression is associated with a poor prognosis. Ran is known to contribute to both nucleocytoplasmic transport and cell cycle progression, but its role in ovarian cancer is not well defined. Results Using a lentivirus-based tetracycline-inducible shRNA approach, we show that downregulation of Ran expression in aggressive ovarian cancer cell lines affects cellular proliferation by inducing a caspase-3 associated apoptosis. Using a xenograft tumor assay, we demonstrate that depletion of Ran results in decreased tumorigenesis, and eventual tumor formation is associated with tumor cells that express Ran protein. Conclusion Our results suggest a role for Ran in ovarian cancer cell survival and tumorigenicity and suggest that this critical GTPase may be suitable as a therapeutic target.

Abstract Background Global gene expression profiling by DNA microarrays is an invaluable tool in biological research. However, existing labeling methods are time consuming and costly and therefore often limit the scale of microarray experiments and sample throughput. Here we introduce a new, fast, inexpensive method for direct random-primed fluorescent labeling of eukaryotic cDNA for gene expression analysis and compare the results obtained on the NimbleGen microarray platform with two other widely-used labeling methods, namely the NimbleGen-recommended double-stranded cDNA protocol and the indirect (aminoallyl) method. Results Two total RNA samples were labeled with each method and hybridized to NimbleGen expression arrays. Although all methods tested here provided similar global results and biological conclusions, the new direct random-primed cDNA labeling method provided slightly better correlation between replicates compared to the other methods and thus increased ability to find statistically significant differentially expressed genes. Conclusion The new direct random-primed cDNA labeling method introduced here is suitable for gene expression microarrays and provides a rapid, inexpensive alternative to existing methods. Using NimbleGen microarrays, the method produced excellent results comparable to those obtained with other methods. However, the simplicity and cost-effectiveness of the new method allows for increased sample throughput in microarray experiments and makes the process amenable to automation with a relatively simple liquid handling system.

Abstract Background The unique properties of wheat flour primarily depend on gluten, which is the most important source of protein for human being. γ-Gliadins have been considered to be the most ancient of the wheat gluten family. The complex family structure of γ-gliadins complicates the determination of their function. Moreover, γ-gliadins contain several sets of celiac disease epitopes. However, no systematic research has been conducted yet. Results A total of 170 γ-gliadin genes were isolated from common wheat and its closely related species, among which 138 sequences are putatively functional. The ORF lengths of these sequences range from 678 to 1089 bp, and the repetitive region is mainly responsible for the size heterogeneity of γ-gliadins. The repeat motif P(Q/L/S/T/I/V/R/A)F(S/Y/V/Q/I/C/L)P(R/L/S/T/H/C/Y)Q1–2(P(S/L/T/A/F/H)QQ)1–2is repeated from 7 to 22 times. Sequence polymorphism and linkage disequilibrium analyses show that γ-gliadins are highly diverse. Phylogenic analyses indicate that there is no obvious discrimination between Sitopsis and Ae. tauschii at the Gli-1 loci, compared with diploid wheat. According to the number and placement of cysteine residues, we defined nine cysteine patterns and 17 subgroups. Alternatively, we classified γ-gliadins into two types based on the length of repetitive domain. Amino acid composition analyses indicate that there is a wide range of essential amino acids in γ-gliadins, and those γ-gliadins from subgroup SG-10 and SG-12 and γ-gliadins with a short repetitive domain are more nutritional. A screening of toxic epitopes shows that γ-gliadins with a pattern of C9 and γ-gliadins with a short repetitive domain almost lack any epitopes. Conclusion γ-Gliadin sequences in wheat and closely related Aegilops species are diverse. Each group/subgroup contributes differently to nutritional quality and epitope content. It is suggested that the genes with a short repetitive domain are more nutritional and valuable. Therefore, it is possible to breed wheat varieties, the γ-gliadins of which are less, even non-toxic and more nutritional.

Abstract Background Infection with HIV-1 has been shown to alter expression of a large array of host cell genes. However, previous studies aimed at investigating the putative HIV-1-induced modulation of host gene expression have been mostly performed in established human cell lines. To better approximate natural conditions, we monitored gene expression changes in a cell population highly enriched in human primary CD4+ T lymphocytes exposed to HIV-1 using commercial oligonucleotide microarrays from Affymetrix. Results We report here that HIV-1 influences expression of genes related to many important biological processes such as DNA repair, cellular cycle, RNA metabolism and apoptosis. Notably, expression of the p53 tumor suppressor and genes involved in p53 homeostasis such as GADD34 were up-regulated by HIV-1 at the mRNA level. This observation is distinct from the previously reported p53 phosphorylation and stabilization at the protein level, which precedes HIV-1-induced apoptosis. We present evidence that the HIV-1-mediated increase in p53 gene expression is associated with virus-mediated induction of type-I interferon (i.e. IFN-α and IFN-β). Conclusion These observations have important implications for our understanding of HIV-1 pathogenesis, particularly in respect to the virus-induced depletion of CD4+ T cells.

Abstract Background Increasing energy costs and environmental concerns have motivated engineering microbes for the production of "second generation" biofuels that have better properties than ethanol. Results and conclusion Saccharomyces cerevisiae was engineered with an n-butanol biosynthetic pathway, in which isozymes from a number of different organisms (S. cerevisiae, Escherichia coli, Clostridium beijerinckii, and Ralstonia eutropha) were substituted for the Clostridial enzymes and their effect on n-butanol production was compared. By choosing the appropriate isozymes, we were able to improve production of n-butanol ten-fold to 2.5 mg/L. The most productive strains harbored the C. beijerinckii 3-hydroxybutyryl-CoA dehydrogenase, which uses NADH as a co-factor, rather than the R. eutropha isozyme, which uses NADPH, and the acetoacetyl-CoA transferase from S. cerevisiae or E. coli rather than that from R. eutropha. Surprisingly, expression of the genes encoding the butyryl-CoA dehydrogenase from C. beijerinckii (bcd and etfAB) did not improve butanol production significantly as previously reported in E. coli. Using metabolite analysis, we were able to determine which steps in the n-butanol biosynthetic pathway were the most problematic and ripe for future improvement.

Abstract Background Serous epithelial ovarian tumors can be subdivided into benign (BOV), low malignant potential (LMP) or borderline and invasive (TOV) tumors. Although the molecular characteristics of serous BOV, LMP and low grade (LG) TOV tumors has been initiated, definitive immunohistochemical markers to distinguish between these tumor types have not been defined. Methods In the present study, we used a tissue array composed of 27 BOVs, 78 LMPs and 23 LG TOVs to evaluate the protein expression of a subset of selected candidates identified in our previous studies (Ape1, Set, Ran, Ccne1 and Trail) or known to be implicated in epithelial ovarian cancer disease (p21, Ccnb1, Ckd1). Results Statistically significant difference in protein expression was observed for Ccnb1 when BOV tumors were compared to LMP tumors (p = 0.003). When BOV were compared to LG TOV tumors, Trail was significantly expressed at a higher level in malignant tumors (p = 0.01). Expression of p21 was significantly lower in LG tumors when compared with either BOVs (p = 0.03) or LMPs (p = 0.001). We also observed that expression of p21 was higher in LMP tumors with no (p = 0.02) or non-invasive (p = 0.01) implants compared to the LMP associated with invasive implants. Conclusion This study represents an extensive analyse of the benign and highly differentiated ovarian disease from an immunohistochemical perspective.

Abstract Background Due to the global occurrence of multi-drug-resistant malarial parasites (Plasmodium falciparum), the anti-malarial drug most effective against malaria is artemisinin, a natural product (sesquiterpene lactone endoperoxide) extracted from sweet wormwood (Artemisia annua). However, artemisinin is in short supply and unaffordable to most malaria patients. Artemisinin can be semi-synthesized from its precursor artemisinic acid, which can be synthesized from simple sugars using microorganisms genetically engineered with genes from A. annua. In order to develop an industrially competent yeast strain, detailed analyses of microbial physiology and development of gene expression strategies are required. Results Three plant genes coding for amorphadiene synthase, amorphadiene oxidase (AMO or CYP71AV1), and cytochrome P450 reductase, which in concert divert carbon flux from farnesyl diphosphate to artemisinic acid, were expressed from a single plasmid. The artemisinic acid production in the engineered yeast reached 250 μg mL-1 in shake-flask cultures and 1 g L-1 in bio-reactors with the use of Leu2d selection marker and appropriate medium formulation. When plasmid stability was measured, the yeast strain synthesizing amorphadiene alone maintained the plasmid in 84% of the cells, whereas the yeast strain synthesizing artemisinic acid showed poor plasmid stability. Inactivation of AMO by a point-mutation restored the high plasmid stability, indicating that the low plasmid stability is not caused by production of the AMO protein but by artemisinic acid synthesis or accumulation. Semi-quantitative reverse-transcriptase (RT)-PCR and quantitative real time-PCR consistently showed that pleiotropic drug resistance (PDR) genes, belonging to the family of ATP-Binding Cassette (ABC) transporter, were massively induced in the yeast strain producing artemisinic acid, relative to the yeast strain producing the hydrocarbon amorphadiene alone. Global transcriptional analysis by yeast microarray further demonstrated that the induction of drug-resistant genes such as ABC transporters and major facilitator superfamily (MSF) genes is the primary cellular stress-response; in addition, oxidative and osmotic stress responses were observed in the engineered yeast. Conclusion The data presented here suggest that the engineered yeast producing artemisinic acid suffers oxidative and drug-associated stresses. The use of plant-derived transporters and optimizing AMO activity may improve the yield of artemisinic acid production in the engineered yeast.