Spatially resolved whole transcriptome profiling in human and mouse tissue using Digital Spatial Profiling.

Emerging spatial profiling technology has enabled high-plex molecular profiling in biological tissues, preserving the spatial and morphological context of gene expression. Here, we describe expanding the chemistry for the Digital Spatial Profiling platform to quantify whole transcriptomes in human and mouse tissues using a wide range of spatial profiling strategies and sample types. We designed multiplexed in situ hybridization probes targeting the protein-coding genes of the human and mouse transcriptomes, referred to as the human or mouse Whole Transcriptome Atlas (WTA). Human and mouse WTAs were validated in cell lines for concordance with orthogonal gene expression profiling methods in regions ranging from ∼10-500 cells. By benchmarking against bulk RNA-seq and fluorescence in situ hybridization, we show robust transcript detection down to ∼100 transcripts per region. To assess the performance of WTA across tissue and sample types, we applied WTA to biological questions in cancer, molecular pathology, and developmental biology. Spatial profiling with WTA detected expected gene expression differences between tumor and tumor microenvironment, identified disease-specific gene expression heterogeneity in histological structures of the human kidney, and comprehensively mapped transcriptional programs in anatomical substructures of nine organs in the developing mouse embryo. Digital Spatial Profiling technology with the WTA assays provides a flexible method for spatial whole transcriptome profiling applicable to diverse tissue types and biological contexts.

Multiplex digital spatial profiling of proteins and RNA in fixed tissue.

Digital Spatial Profiling (DSP) is a method for highly multiplex spatial profiling of proteins or RNAs suitable for use on formalin-fixed, paraffin-embedded (FFPE) samples. The approach relies on (1) multiplexed readout of proteins or RNAs using oligonucleotide tags; (2) oligonucleotide tags attached to affinity reagents (antibodies or RNA probes) through a photocleavable (PC) linker; and (3) photocleaving light projected onto the tissue sample to release PC oligonucleotides in any spatial pattern across a region of interest (ROI) covering 1 to ~5,000 cells. DSP is capable of single-cell sensitivity within an ROI using the antibody readout, with RNA detection feasible down to ~600 individual mRNA transcripts. We show spatial profiling of up to 44 proteins and 96 genes (928 RNA probes) in lymphoid, colorectal tumor and autoimmune tissues by using the nCounter system and 1,412 genes (4,998 RNA probes) by using next-generation sequencing (NGS). DSP may be used to profile not only proteins and RNAs in biobanked samples but also immune markers in patient samples, with potential prognostic and predictive potential for clinical decision-making.

Recall of pain and discomfort during oral procedures experienced by intubated critically ill patients in the intensive care unit: A qualitative elicitation study.

Background: Intubated and mechanically ventilated patients in the intensive care unit (ICU) may experience pain during routine oral procedures such as oral suctioning and tooth brushing. Despite the importance of pain prevention and management, little is known about patients' experiences of procedural oral pain. Aims: The aim of this study was to explore patients' recollections and recommendations for pain and discomfort during routine oral procedures. Methods: A qualitative descriptive design was used. Adult patients were recruited from a mixed medical-surgical-trauma ICU in an academic hospital in Toronto, Canada. Participants were interviewed using object elicitation methods within 7 days of discharge from the ICU. Data were analyzed using directed content analysis methods. Results: We recruited 33 participants who were primarily male (23, 70%), with an average age of 54 (SD = 18) years, admitted with a medical (13, 39%), trauma (11, 33%), or surgical (9, 27%) diagnosis and dentate (27, 82%). Most participants described oral procedures as painful, discomforting, and emotionally distressing. Identified sources of pain included dry, inflamed oral tissues and procedural technique. Procedural pain behaviors were perceived to be frequently misinterpreted by clinicians as agitation, with consequences including physical restraint and unrelieved suffering. Participants advocated for greater frequency of oral care to prevent oral health deterioration, anticipatory procedural guidance, and structured pain assessment to mitigate the dehumanizing experience of unmanaged pain. Conclusions: Patients described routine oral care procedures as painful and recalled suboptimal management of such pain. Procedural oral pain is an important target for practice improvement.

Contexte: Les patients intubés et ventilés mécaniquement dans l'unité de soins intensifs (USI) peuvent ressentir de la douleur lors des procédures buccales de routine telles que l'aspiration et le brossage de dents. Malgré l'importance de la prévention et de la prise en charge de la douleur, on sait peu de choses sur l'expérience des patients en matière de douleur buccale liée aux procédures.Objectifs: Cette étude a pour but d'étudier les souvenirs et les recommandations des patients concernant la douleur et l'inconfort lors des procédures buccales de routine.Méthodes: Un modèle descriptif qualitatif a été utilisé. Les patients adultes ont été recrutés dans une unité mixte de soins intensifs médico-chirurgicaux et de traumatologie d'un hôpital universitaire de Toronto, au Canada. Les participants ont été interrogés à l'aide de méthodes d'élicitation des objets dans les sept jours suivant leur sortie de l'unité de soins intensifs. Les données ont été analysées à l'aide de méthodes d'analyse de contenu dirigée.Résultats: Nous avons recruté 33 participants qui étaient principalement des hommes (23, 70 %), âgés de 54 ans (É.-T. 18), admis avec un diagnostic médical (13,3 9 %), traumatique (11, 33 %) ou chirurgical (9, 27 %) et dentés (27, 82 %). La plupart des participants ont décrit les procédures orales comme étant douloureuses, inconfortables et émotionnellement pénibles. Les sources de douleur répertoriées comprenaient les tissus buccaux secs et enflammés et la technique d'intervention. Les comportements douloureux liés aux procédures ont été perçus comme étant souvent interprétés à tort par les cliniciens comme de l'agitation, avec pour conséquences une contrainte physique et une souffrance non soulagée. Les participants ont plaidé pour une plus grande fréquence des soins bucco-dentaires afin de prévenir la détérioration de la santé bucco-dentaire, pour des conseils procéduraux anticipés et pour une évaluation structurée de la douleur afin d'atténuer l'expérience déshumanisante de la douleur non prise en charge.Conclusions: Les patients ont décrit les soins bucco-dentaires de routine comme étant douloureuses et se sont souvenus de la prise en charge sous-optimale de cette douleur. La douleur buccale procédurale est un objectif important pour l'amélioration de la pratique.

Structural and functional characterization of three Type B and C chloramphenicol acetyltransferases from Vibrio species.

Chloramphenicol acetyltransferases (CATs) were among the first antibiotic resistance enzymes identified and have long been studied as model enzymes for examining plasmid-mediated antibiotic resistance. These enzymes acetylate the antibiotic chloramphenicol, which renders it incapable of inhibiting bacterial protein synthesis. CATs can be classified into different types: Type A CATs are known to be important for antibiotic resistance to chloramphenicol and fusidic acid. Type B CATs are often called xenobiotic acetyltransferases and adopt a similar structural fold to streptogramin acetyltransferases, which are known to be critical for streptogramin antibiotic resistance. Type C CATs have recently been identified and can also acetylate chloramphenicol, but their roles in antibiotic resistance are largely unknown. Here, we structurally and kinetically characterized three Vibrio CAT proteins from a nonpathogenic species (Aliivibrio fisheri) and two important human pathogens (Vibrio cholerae and Vibrio vulnificus). We found all three proteins, including one in a superintegron (V. cholerae), acetylated chloramphenicol, but did not acetylate aminoglycosides or dalfopristin. We also determined the 3D crystal structures of these CATs alone and in complex with crystal violet and taurocholate. These compounds are known inhibitors of Type A CATs, but have not been explored in Type B and Type C CATs. Based on sequence, structure, and kinetic analysis, we concluded that the V. cholerae and V. vulnificus CATs belong to the Type B class and the A. fisheri CAT belongs to the Type C class. Ultimately, our results provide a framework for studying the evolution of antibiotic resistance gene acquisition and chloramphenicol acetylation in Vibrio and other species.

School and classroom effects on Daily Physical Activity (DPA) policy implementation fidelity in Ontario classrooms: a multi-level analysis.

BACKGROUND: This paper examines school and classroom effects on Daily Physical Activity (DPA) policy implementation in classrooms in Ontario, Canada. In 2005 the Ontario Ministry of Education mandated a policy requiring school boards to "ensure that all elementary students, including students with special needs, have a minimum of twenty minutes of sustained MVPA each school day during instructional time". Based on an adaptation of Chaudoir's conceptual framework, this paper contributes to understanding the extent to which school factors (as reported by administrators) and classroom factors (as reported by teachers) are associated with policy implementation fidelity at the classroom level. METHODS: Cross-sectional online surveys were conducted in 2014 with elementary school administrators and teachers, based on representative random samples of schools and classrooms. A measure assessing implementation fidelity was developed from the six required components of the policy and for this paper fidelity at the classroom level is treated as the outcome variable. Several school- and classroom-level measures were also included in the surveys and a number of these were selected for inclusion here. Data from the two surveys were merged and selected variables were included in the multi-level analysis. Two-level logistic regression models were conducted to account for nesting of classrooms within schools and a series of models were conducted to identify factors associated with implementation fidelity. RESULTS: The analytic sample for this study included 170 school administrators and 307 classroom teachers from corresponding schools. Findings from the multi-level logistic regression analyses indicated that only classroom/teacher-level factors were significantly associated with implementation fidelity at the classroom level. None of the school/administrator predictors were significantly related to fidelity. The most parsimonious model included five significant classroom/teacher predictors: teachers' perception of DPA as realistic and achievable; confidence (self-efficacy); scheduling DPA in timetables; lack of space; and lack of time. CONCLUSIONS: Findings from the study indicate the theoretical and practical importance of addressing classroom and teacher factors since they are most proximal to implementation fidelity to the policy. Several of these factors also reflect complex structural and organizational contexts, indicating that a systems approach to understanding and supporting DPA implementation fidelity is warranted.

The Development of Practice Guidelines for a Palliative Care Multidisciplinary Case Conference.

CONTEXT: In Ontario, we identified that few hospitals have developed multi-disciplinary case conferences or forums for discussion of patients with palliative care issues. OBJECTIVE: We describe the process of creating a province-wide standards document for palliative care multidisciplinary case conferences (pMCCs). METHODS: A provincial survey and a multidisciplinary cancer conference symposium identified pMCCs as a priority. A literature search focusing on pMCCs and their implementation was completed as well as a current state assessment (survey and interviews) to understand challenges with existing pMCCs in Ontario. A working group was then assembled to draft a recommendation report that was finalized by an expert panel. RESULTS: A total of 22 articles were identified and 10 were used by the working group to create a framework for the pMCC guideline. The current state assessment identified substantial variability in pMCC structure and function. The expert panel made recommendations about meeting format (multidisciplinary discussion encouraged), frequency (at least every two weeks), type of cases to present, attendees (palliative care, nursing, primary care, social work, and community nursing), provider roles and responsibilities, and institutional requirements (pMCC coordinator, meeting room and videoconference capability). All patients (not just those with cancer) with palliative care needs were to be discussed at the pMCC, and pMCCs should serve as a crucial link between the hospital and community. CONCLUSION: We have described the process of creating the first pMCC guideline. A key component of this guideline is that pMCCs should serve as a link between the hospital and community.

A Review of Implementation Outcome Measures of School-Based Physical Activity Interventions.

BACKGROUND: Measuring the implementation of school-based physical activity (PA) interventions is an important prerequisite in assessing their impact. Prior to conducting a study to assess the implementation of the daily physical activity (DPA) policy in Ontario, Canada, a literature review was conducted to identify existing survey instruments to measure 5 implementation outcomes: adoption, fidelity, implementation cost, reach, and sustainability. METHODS: A search for survey instruments to assess these implementation outcomes at the teacher and school administrator levels was conducted in 7 bibliographic databases, as well as the gray literature. Each survey instrument was coded as assessing 1 of the 5 implementation outcomes if it included at least 1 item measuring the construct. RESULTS: Twenty-three survey instruments were identified. None of the instruments were specifically developed to measure the implementation outcomes. Fidelity was the most common implementation outcome measured, followed by adoption. The least common implementation outcome measured was sustainability. Thirty-five percent of survey instruments assessed were previously tested for validity and 26% were previously tested for reliability. CONCLUSIONS: Based on this review, a gap in available instruments to measure implementation outcomes of school-based PA programs was identified. An adapted theoretical framework, presented here, has potential application in future implementation studies.

Evaluation of Daily Physical Activity (DPA) policy implementation in Ontario: surveys of elementary school administrators and teachers.

BACKGROUND: School-based structured opportunities for physical activity can provide health-related benefits to children and youth, and contribute to international guidelines recommending 60 min of moderate-to-vigorous physical activity (MVPA) per day. In 2005, the Ministry of Education in Ontario, Canada, released the Daily Physical Activity (DPA) policy requiring school boards to "ensure that all elementary students, including students with special needs, have a minimum of twenty minutes of sustained MVPA each school day during instructional time". This paper reports on the first provincial study evaluating implementation fidelity to the DPA policy in Ontario elementary schools and classrooms. Using an adapted conceptual framework, the study also examined associations between implementation of DPA and a number of predictors in each of these respective settings. METHODS: Separate cross-sectional online surveys were conducted in 2014 with Ontario elementary school administrators and classroom teachers, based on a representative random sample of schools and classrooms. An implementation fidelity score was developed based on six required components of the DPA policy. Other survey items measured potential predictors of implementation at the school and classroom levels. Descriptive analyses included frequency distributions of implementation fidelity and predictor variables. Bivariate analyses examining associations between implementation and predictors included binary logistic regression for school level data and generalized linear mixed models for classroom level data, in order to adjust for school-level clustering effects. RESULTS: Among administrators, 61.4 % reported implementation fidelity to the policy at the school level, while 50.0 % of teachers reported fidelity at the classroom level. Several factors were found to be significantly associated with implementation fidelity in both school and classroom settings including: awareness of policy requirements; scheduling; monitoring; use of resources and supports; perception that the policy is realistic and achievable; and specific barriers to implementation. CONCLUSIONS: Findings from the surveys indicate incomplete policy implementation and a number of factors significantly associated with implementation fidelity. The results indicate a number of important implications for policy, practice and further research, including the need for additional research to monitor implementation and its predictors, and assess the impacts of study recommendations and subsequent outcomes of a reinvigorated DPA moving forward.

Understanding key influencers' attitudes and beliefs about healthy public policy change for obesity prevention.

OBJECTIVE: As overweight and obesity is a risk factor for chronic diseases, the development of environmental and healthy public policy interventions across multiple sectors has been identified as a key strategy to address this issue. METHODS: In 2009, a survey was developed to assess the attitudes and beliefs regarding health promotion principles, and the priority and acceptability of policy actions to prevent obesity and chronic diseases, among key policy influencers in Alberta and Manitoba, Canada. Surveys were mailed to 1,765 key influencers from five settings: provincial government, municipal government, school boards, print media companies, and workplaces with greater than 500 employees. A total of 236 surveys were completed with a response rate of 15.0%. RESULTS: Findings indicate nearly unanimous influencer support for individual-focused policy approaches and high support for some environmental policies. Restrictive environmental and economic policies received weakest support. Obesity was comparable to smoking with respect to perceptions as a societal responsibility versus a personal responsibility, boding well for the potential of environmental policy interventions for obesity prevention. CONCLUSIONS: This level of influencer support provides a platform for more evidence to be brokered to policy influencers about the effectiveness of environmental policy approaches to obesity prevention.

Annotating N termini for the human proteome project: N termini and Nα-acetylation status differentiate stable cleaved protein species from degradation remnants in the human erythrocyte proteome.

A goal of the Chromosome-centric Human Proteome Project is to identify all human protein species. With 3844 proteins annotated as "missing", this is challenging. Moreover, proteolytic processing generates new protein species with characteristic neo-N termini that are frequently accompanied by altered half-lives, function, interactions, and location. Enucleated and largely void of internal membranes and organelles, erythrocytes are simple yet proteomically challenging cells due to the high hemoglobin content and wide dynamic range of protein concentrations that impedes protein identification. Using the N-terminomics procedure TAILS, we identified 1369 human erythrocyte natural and neo-N-termini and 1234 proteins. Multiple semitryptic N-terminal peptides exhibited improved mass spectrometric identification properties versus the intact tryptic peptide enabling identification of 281 novel erythrocyte proteins and six missing proteins identified for the first time in the human proteome. With an improved bioinformatics workflow, we developed a new classification system and the Terminus Cluster Score. Thereby we described a new stabilizing N-end rule for processed protein termini, which discriminates novel protein species from degradation remnants, and identified protein domain hot spots susceptible to cleavage. Strikingly, 68% of the N-termini were within genome-encoded protein sequences, revealing alternative translation initiation sites, pervasive endoproteolytic processing, and stabilization of protein fragments in vivo. The mass spectrometry proteomics data have been deposited to ProteomeXchange with the data set identifier .

The role of inbreeding depression and mating system in the evolution of heterostyly.

We investigated the role of morph-based differences in the expression of inbreeding depression in loss of the mid-styled morph from populations of tristylous Oxalis alpina. The extent of self-compatibility (SC) of reproductive morphs, the degree of self-fertilization, and the magnitude of inbreeding depression were investigated in three populations of O. alpina differing in their tristylous incompatibility relationships. All three populations exhibited significant inbreeding depression. In two populations with highly modified tristylous incompatibility, manifested as increased reciprocal compatibility between short- and long-styled morphs, substantial SC and self-fertilization of mid-styled morphs were detected, and expected to result in expression of inbreeding depression in the progeny of mid-styled morphs in the natural populations. In contrast, significant self-fertility of the mid-styled morph was absent from the population with typical tristylous incompatibility, and no self-fertilization could be detected. Although self-fertilization and expression of inbreeding depression should result in selection against the mid-styled morph in the later stages of the transition from tristyly to distyly, in O. alpina selection against the mid-styled morph in the early phases of the evolution of distyly is likely due to genic selection against mid-alleles associated with modified tristylous incompatibility, rather than expression of inbreeding depression.

Synthesis and evaluation of self-calibrating ratiometric viscosity sensors.

We describe the design, synthesis and fluorescent profile of a family of self-calibrating dyes that provide ratiometric measurements of fluid viscosity. The design is based on covalently linking a primary fluorophore (reference) that displays a viscosity-independent fluorescence emission with a secondary fluorophore (sensor) that exhibits a viscosity-sensitive fluorescence emission. Characterization of fluorescent properties was made with separate excitation of the units and through Resonance Energy Transfer from the reference to the sensor dye. The chemical structures of both fluorophores and the linker length have been evaluated in order to optimize the overall brightness and sensitivity of the viscosity measurements. We also present an application of such ratiometric dyes for the detection of membrane viscosity changes in a liposome model.