Socioeconomic and immigration status and COVID-19 testing in Toronto, Ontario: retrospective cross-sectional study.

BACKGROUND: Preliminary evidence suggests that individuals living in lower income neighbourhoods are at higher risk of COVID-19 infection. The relationship between sociodemographic characteristics and COVID-19 risk warrants further study. METHODS: We explored the association between COVID-19 test positivity and patients' socio-demographic variables, using neighborhood sociodemographic data collected retrospectively from two COVID-19 Assessment Centres in Toronto, ON. RESULTS: Eighty-three thousand four hundred forty three COVID-19 tests completed between April 5-September 30, 2020, were analyzed. Individuals living in neighbourhoods with the lowest income or highest concentration of immigrants were 3.4 (95% CI: 2.7 to 4.9) and 2.5 (95% CI: 1.8 to 3.7) times more likely to test positive for COVID-19 than those in highest income or lowest immigrant neighbourhoods, respectively. Testing was higher among individuals from higher income neighbourhoods, at lowest COVID-19 risk, compared with those from low-income neighbourhoods. CONCLUSIONS: Targeted efforts are needed to improve testing availability in high-risk regions. These same strategies may also ensure equitable COVID-19 vaccine delivery.

Structural insights into the recognition of cisplatin and AAF-dG lesion by Rad14 (XPA).

Nucleotide excision repair (NER) is responsible for the removal of a large variety of structurally diverse DNA lesions. Mutations of the involved proteins cause the xeroderma pigmentosum (XP) cancer predisposition syndrome. Although the general mechanism of the NER process is well studied, the function of the XPA protein, which is of central importance for successful NER, has remained enigmatic. It is known, that XPA binds kinked DNA structures and that it interacts also with DNA duplexes containing certain lesions, but the mechanism of interactions is unknown. Here we present two crystal structures of the DNA binding domain (DBD) of the yeast XPA homolog Rad14 bound to DNA with either a cisplatin lesion (1,2-GG) or an acetylaminofluorene adduct (AAF-dG). In the structures, we see that two Rad14 molecules bind to the duplex, which induces DNA melting of the duplex remote from the lesion. Each monomer interrogates the duplex with a ß-hairpin, which creates a 13mer duplex recognition motif additionally characterized by a sharp 70° DNA kink at the position of the lesion. Although the 1,2-GG lesion stabilizes the kink due to the covalent fixation of the crosslinked dG bases at a 90° angle, the AAF-dG fully intercalates into the duplex to stabilize the kinked structure.

Tet oxidizes thymine to 5-hydroxymethyluracil in mouse embryonic stem cell DNA.

Ten eleven translocation (Tet) enzymes oxidize the epigenetically important DNA base 5-methylcytosine (mC) stepwise to 5-hydroxymethylcytosine (hmC), 5-formylcytosine and 5-carboxycytosine. It is currently unknown whether Tet-induced oxidation is limited to cytosine-derived nucleobases or whether other nucleobases are oxidized as well. We synthesized isotopologs of all major oxidized pyrimidine and purine bases and performed quantitative MS to show that Tet-induced oxidation is not limited to mC but that thymine is also a substrate that gives 5-hydroxymethyluracil (hmU) in mouse embryonic stem cells (mESCs). Using MS-based isotope tracing, we show that deamination of hmC does not contribute to the steady-state levels of hmU in mESCs. Protein pull-down experiments in combination with peptide tracing identifies hmU as a base that influences binding of chromatin remodeling proteins and transcription factors, suggesting that hmU has a specific function in stem cells besides triggering DNA repair.

Identification of short terminal motifs enriched by antibodies using peptide mass fingerprinting.

MOTIVATION: Mass spectrometry-based protein profiling has become a key technology in biomedical research and biomarker discovery. Sample preparation strategies that reduce the complexity of tryptic digests by immunoaffinity substantially increase throughput and sensitivity in proteomic mass spectrometry. The scarce availability of peptide-specific capture antibodies limits these approaches. Recently antibodies directed against short terminal motifs were found to enrich subsets of peptides with identical terminal sequences. This approach holds the promise of a significant gain in efficiency. TXP (Triple X Proteomics) and context-independent motif specific/global proteome survey binders are variants of this concept. Principally the binding motifs of such antibodies have to be elucidated after generating these antibodies. This entails a substantial effort in the lab, as it requires synthetic peptide libraries and numerous mass spectrometry experiments. RESULTS: We present an algorithm for predicting the antibody-binding motif in a mass spectrum obtained from a tryptic digest of a common cell line after immunoprecipitation. The epitope prediction, based on peptide mass fingerprinting, reveals the most enriched terminal epitopes. The tool provides a P-value for each potential epitope, estimated by sampling random spectra from a peptide database. The second algorithm combines the predicted sequences to more complex binding motifs. A comparison with library screenings shows that the predictions made by the novel methods are reliable and reproducible indicators of the binding properties of an antibody.

Orchestrating the biosynthesis of an unnatural pyrrolysine amino Acid for its direct incorporation into proteins inside living cells.

We here report the construction of an E. coli expression system able to manufacture an unnatural amino acid by artificial biosynthesis. This can be orchestrated with incorporation into protein by amber stop codon suppression inside a living cell. In our case an alkyne-bearing pyrrolysine amino acid was biosynthesized and incorporated site-specifically allowing orthogonal double protein labeling.

Combining ultracentrifugation and peptide termini group-specific immunoprecipitation for multiplex plasma protein analysis.

Blood plasma is a valuable source of potential biomarkers. However, its complexity and the huge dynamic concentration range of its constituents complicate its analysis. To tackle this problem, an immunoprecipitation strategy was employed using antibodies directed against short terminal epitope tags (triple X proteomics antibodies), which allow the enrichment of groups of signature peptides derived from trypsin-digested plasma. Isolated signature peptides are subsequently detected using MALDI-TOF/TOF mass spectrometry. Sensitivity of the immunoaffinity approach was, however, compromised by the presence of contaminant peaks derived from the peptides of nontargeted high abundant proteins. A closer analysis of the enrichment strategy revealed nonspecific peptide binding to the solid phase affinity matrix as the major source of the contaminating peptides. We therefore implemented a sucrose density gradient ultracentrifugation separation step into the procedure. This yielded a 99% depletion of contaminating peptides from a sucrose fraction containing 70% of the peptide-antibody complexes and enabled the detection of the previously undetected low abundance protein filamin-A. Assessment of this novel approach using 15 different triple X proteomics antibodies demonstrated a more consistent detection of a greater number of targeted peptides and a significant reduction in the intensity of nonspecific peptides. Ultracentrifugation coupled with immunoaffinity MS approaches presents a powerful tool for multiplexed plasma protein analysis without the requirement for demanding liquid chromatography separation techniques.

Identification and quantification of a new family of peptide endocannabinoids (Pepcans) showing negative allosteric modulation at CB1 receptors.

The α-hemoglobin-derived dodecapeptide RVD-hemopressin (RVDPVNFKLLSH) has been proposed to be an endogenous agonist for the cannabinoid receptor type 1 (CB(1)). To study this peptide, we have raised mAbs against its C-terminal part. Using an immunoaffinity mass spectrometry approach, a whole family of N-terminally extended peptides in addition to RVD-Hpα were identified in rodent brain extracts and human and mouse plasma. We designated these peptides Pepcan-12 (RVDPVNFKLLSH) to Pepcan-23 (SALSDLHAHKLRVDPVNFKLLSH), referring to peptide length. The most abundant Pepcans found in the brain were tested for CB(1) receptor binding. In the classical radioligand displacement assay, Pepcan-12 was the most efficacious ligand but only partially displaced both [(3)H]CP55,940 and [(3)H]WIN55,212-2. The data were fitted with the allosteric ternary complex model, revealing a cooperativity factor value α < 1, thus indicating a negative allosteric modulation. Dissociation kinetic studies of [(3)H]CP55,940 in the absence and presence of Pepcan-12 confirmed these results by showing increased dissociation rate constants induced by Pepcan-12. A fluorescently labeled Pepcan-12 analog was synthesized to investigate the binding to CB(1) receptors. Competition binding studies revealed K(i) values of several Pepcans in the nanomolar range. Accordingly, using competitive ELISA, we found low nanomolar concentrations of Pepcans in human plasma and ∼100 pmol/g in mouse brain. Surprisingly, Pepcan-12 exhibited potent negative allosteric modulation of the orthosteric agonist-induced cAMP accumulation, [(35)S]GTPγS binding, and CB(1) receptor internalization. Pepcans are the first endogenous allosteric modulators identified for CB(1) receptors. Given their abundance in the brain, Pepcans could play an important physiological role in modulating endocannabinoid signaling.

Diabetes care among individuals with and without schizophrenia in three Canadian provinces: A retrospective cohort study.

OBJECTIVE: Diabetes is present in approximately 10% of people living with schizophrenia and substantially contributes to early mortality, but some aspects of diabetes care among those with schizophrenia have been inadequately investigated to date. We assessed diabetes care and comorbidity management among people with and without schizophrenia. METHODS: We conducted a cohort study with data obtained from primary care electronic medical records stored in the Diabetes Action Canada (DAC) National Repository from Alberta, Ontario, and Quebec, Canada. The population studied included patients with diabetes, with and without schizophrenia, who had at least 3 primary care visits in a 2 year period between July 2017 and June 2019. Outcomes included glycemia; diabetes complication screening and monitoring; antihyperglycemic and cardioprotective medication prescription; health service use. RESULTS: We identified 69,512 patients with diabetes; 911 (1.3%) of whom also had schizophrenia. Prevalence of high HbA1C (>8.5%) (9083/68601; 13.2% vs. 137/911; 15.0%) and high blood pressure (>130/80 mmHg) (4248/68601; 6.2% vs. 73/911; 8.0%) was similar between the two groups. Half (50.0%) of patients with schizophrenia (n = 455) had 11 or more primary care visits in the past year, compared with 27.8% of people without schizophrenia. (p < 0.0001). Patients with schizophrenia had lower odds of ever having blood pressure recorded (OR = 0.81, 95% CI 0.71-0.94) and fewer of those with chronic kidney disease (CKD) were prescribed renin-angiotensin aldosterone system inhibitors, compared to patients without schizophrenia (10.3% vs 15.8%, p = 0.0005). CONCLUSIONS: Patients with diabetes and schizophrenia achieved similar blood glucose and blood pressure levels to those without schizophrenia, and had more primary care visits. However, they had fewer blood pressure readings and lower prescription of recommended medications among those who also had CKD. These results are both encouraging and represent opportunities for improvement in care.

Primary Care Physicians' Perspectives in Leading Breast Cancer Follow-Up Care.

INTRODUCTION: Limited data exist on the barriers associated with transitioning breast cancer follow-up care to primary care physicians (PCPs). This study aimed to describe the current perspectives of PCPs in managing breast cancer follow-up. METHOD: An online survey was distributed to PCPs in Toronto, ON, Canada. Questions examined PCPs' view of transitioning breast cancer follow-up care to their practices. RESULTS: Of 800 PCPs invited, 126 responded (response rate: 15.7%). The types of practice models amongst respondents included blended capitation (42.9%), blended salary (27%), and fee-for-service (17.5%). Seventy-seven percent of respondents stated they provided follow-up care. Approximately half of the respondents stated they were somewhat comfortable providing follow-up care. PCP-led follow-up care was considered either very (49.2%) or somewhat appropriate (30.2%). When asked about financial remuneration, 43.7% of respondents stated it was somewhat important. The factors that influenced the feasibility of PCP-led follow-up care included receipt of a detailed follow-up care plan provided by the specialist after discharge (81%), the ability to re-refer to specialists rapidly (56.3%), and the ability to obtain regular updates of best practice changes (59.5%). The preferred means of educational updates included E-mail (40.5%), continuing medical education events (30.2%), and electronic medical records (19.8%). When the fee model was taken into consideration there was no significant difference in opinions regarding follow-up care. CONCLUSIONS: Transitioning to a PCP-led model was supported by most of the PCPs who participated in this study. Their perspective on PCP-led follow up care and barriers associated with implementation of this model of care needs to be further explored with future studies that include larger sample size and a more diverse PCP population.

Choosing Wisely: An idea worth sustaining.

OBJECTIVES: To evaluate the sustainability potential of Choosing Wisely (CW) to address unnecessary medical care at Ontario community hospitals. DATA SOURCES/STUDY SETTING: Ontario community hospitals and their affiliated family health teams (FHTs). STUDY DESIGN: A mixed-methods study involving the administration of a validated sustainability survey to CW implementation teams followed by their participation in focus groups. DATA COLLECTION/EXTRACTION METHODS: Survey data were collected using an Excel file with an embedded, automated scoring system. We collated individual survey scores and generated aggregate team scores. We also performed descriptive statistics for quantitative data (frequencies, means). Qualitative data were triangulated with quantitative assessments to support data interpretations using the meta-matrix method. PRINCIPAL FINDINGS: Fifteen CW implementation teams across four Ontario community hospitals and six affiliated primary care FHTs participated. CW priority areas investigated were de-prescribing of proton pump inhibitors (PPIs) and reducing Pre-Op testing and BUN/Urea lab testing. Survey results showed steady improvements in sustainability scores from baseline to final follow-up among most implementation teams: 10% increase for PPI de-prescribing (six FHTs) and 2% increase (three hospital teams); 18% increase in BUN/Urea lab testing (three hospital teams). Regardless of site or CW priority area, common facilitators were fit with existing processes and workflows, leadership support, and optimized team communication; common challenges were lack of awareness and buy-in, leadership engagement or a champion, and lack of fit with existing workflow and culture. All teams identified at least one challenge for which they co-designed and implemented a plan to maximize the sustainability potential of their CW initiative. CONCLUSIONS: Evaluating the sustainability potential of an innovation such as Choosing Wisely is critical to ensuring that they have the best potential for impact. Our work highlights that implementation teams can be empowered to influence implementation efforts and to realize positive outcomes for their health care services and patients.

Health system use among patients with mental health conditions in a community based sample in Toronto, Canada: A retrospective cohort study.

OBJECTIVE: To identify hospital and primary care health service use among people with mental health conditions or addictions in an integrated primary-secondary care database in Toronto, Ontario. METHOD: This was a retrospective cohort study of adults with mental health diagnoses using data from the Health Databank Collaborative (HDC), a primary care-hospital linked database in Toronto. Data were included up to March 31st 2019. Negative binomial and logistic regression were used to evaluate associations between health care utilization and various patient characteristics and mental health diagnoses. RESULTS: 28,482 patients age 18 or older were included. The adjusted odds of at least one mental health diagnosis were higher among younger patients (18-30 years vs. 81+years aOR = 1.87; 95% CI:1.68-2.08) and among female patients (aOR = 1.35; 95% CI: 1.27-1.42). Patients with one or more mental health diagnoses had higher adjusted rates of hospital visits compared to those without any mental health diagnosis including addiction (aRR = 1.74, 95% CI: 1.58-1.91) and anxiety (aRR = 1.28, 95% CI: 1.23-1.32). 14.5% of patients with a psychiatric diagnosis were referred to the hospital for specialized psychiatric services, and 38% of patients referred were eventually seen in consultation. The median wait time from the date of referral to the date of consultation was 133 days. CONCLUSIONS: In this community, individuals with mental health diagnoses accessed primary and hospital-based health care at greater rates than those without mental health diagnoses. Wait times for specialized psychiatric care were long and most patients who were referred did not have a consultation. Information about services for patients with mental health conditions can be used to plan and monitor more integrated care across sectors, and ultimately improve outcomes.

Association between new-onset anosmia and positive SARS-CoV-2 tests among people accessing outpatient testing in Toronto, Ontario: a retrospective cross-sectional study.

BACKGROUND: Reports have suggested that anosmia is strongly associated with SARS-CoV-2 infection, but patients were often asked about this symptom after their diagnosis. This study assessed associations between prospectively reported anosmia and other symptoms related to SARS-CoV-2 infection, and SARS-CoV-2 positivity in community testing centres in Toronto, Ontario. METHODS: We conducted a retrospective cross-sectional study in which data were collected from 2 COVID-19 assessment centres affiliated with 2 hospitals in Toronto, Ontario, from Apr. 5 to Sept. 30, 2020. We included symptomatic profiles of all people who underwent a SARS-CoV-2 test at either clinic within the study period. We used generalized estimating equations to account for repeat visits and to assess associations between anosmia and other symptoms and SARS-CoV-2 positivity. RESULTS: A total of 83 443 SARS-CoV-2 tests were conducted across the 2 sites for 72 692 participants during the study period. Of all tests, 1640 (2.0%) were positive; 837 (51.0%) of people who tested positive were asymptomatic. The adjusted odds ratio for the association between anosmia and test positivity was 5.29 (95% confidence interval [CI] 4.50-6.22), with sensitivity of 0.138 (95% CI 0.121-0.154), specificity of 0.980 (95% CI 0.979-0.981), a positive predictive value of 0.120 (95% CI 0.106-0.135) and a negative predictive value of 0.983 (95% CI 0.982-0.984). INTERPRETATION: Anosmia had high specificity and a positive predictive value of 12% for SARS-CoV-2 infection in this community population with low prevalence of SARS-CoV-2 positivity. The presence of anosmia should increase clinical suspicion of SARS-CoV-2 infection, and our findings suggest that people presenting with this symptom should be tested.

Agreement between hospital and primary care on diagnostic labeling for COPD and heart failure in Toronto, Canada: a cross-sectional observational study.

Patients with chronic obstructive pulmonary disease (COPD) or heart failure (HF) are frequently cared for in hospital and in primary care settings. We studied labeling agreement for COPD and HF for patients seen in both settings in Toronto, Canada. This was a retrospective observational study using linked hospital-primary care electronic data from 70 family physicians. Patients were 20 years of age or more and had at least one visit in both settings between 1 January 2012 and 31 December 2014. We recorded labeling concordance and associations with clinical factors. We used capture-recapture models to estimate the size of the populations. COPD concordance was 34%; the odds ratios (ORs) of concordance increased with aging (OR 1.84 for age 75+ vs. <65, 95% CI 0.92-3.69) and more inpatient admissions (OR 2.89 for 3+ visits vs. 0 visits, 95% CI 1.59-5.26). HF concordance was 33%; the ORs of concordance decreased with aging (OR 0.39 for 75+ vs. <65, 95% CI 0.18-0.86) and increased with more admissions (OR = 2.39; 95% CI 1.33-4.30 for 3+ visits vs. 0 visits). Based on capture-recapture models, 21-24% additional patients with COPD and 18-20% additional patients with HF did not have a label in either setting. The primary care prevalence was estimated as 748 COPD patients and 834 HF patients per 100,000 enrolled adult patients. Agreement levels for COPD and HF were low and labeling was incomplete. Further research is needed to improve labeling for these conditions.

Transfer of care of postsurgical patients from hospital to the community setting: cross-sectional survey of primary care physicians.

BACKGROUND: Postsurgical management is often delegated to family physicians once patients are discharged from hospital, but management of such patients may not be effectively communicated. We examined transfer of care of postsurgical patients by surveying family physicians. METHODS: An electronic, self-administered survey was administered from November 2012 to March 2014 to family physicians affiliated with 4 academic and community hospitals in Toronto, Canada. RESULTS: A total of 109 of 589 (19% response rate) family physicians completed the survey. The majority (76%) did not believe that the current transfer of care process was adequate. Uncertainty regarding management resulted in one or more adverse patient events for over half of respondents (62%). CONCLUSIONS: A discrepancy exists between what family physicians desire to have included in transfer tools and the frequency with which these variables are included. Family physicians believe that the current process of transfer of care for postsurgical patients is inadequate and may contribute to adverse events.

TET3 is recruited by REST for context-specific hydroxymethylation and induction of gene expression.

Ten-eleven translocation hydroxylases (TET1-3) oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). In neurons, increased 5hmC levels within gene bodies correlate positively with gene expression. The mechanisms controlling TET activity and 5hmC levels are poorly understood. In particular, it is not known how the neuronal TET3 isoform lacking a DNA-binding domain is targeted to the DNA. To identify factors binding to TET3, we screened for proteins that co-precipitate with TET3 from mouse retina and identified the transcriptional repressor REST as a highly enriched TET3-specific interactor. REST was able to enhance TET3 hydroxylase activity after co-expression and overexpression of TET3-activated transcription of REST target genes. Moreover, we found that TET3 also interacts with NSD3 and two other H3K36 methyltransferases and is able to induce H3K36 trimethylation. We propose a mechanism for transcriptional activation in neurons that involves REST-guided targeting of TET3 to the DNA for directed 5hmC generation and NSD3-mediated H3K36 trimethylation.

G protein-coupled receptor quantification using peptide group-specific enrichment combined with internal peptide standard reporter calibration.

The G protein-coupled receptor (GPCR) super-family comprises the largest and most diverse group of membrane receptors in eukaryotes. GPCRs are involved in a plethora of physiological functions in all kinds of tissues. Detailed knowledge about GPCR presence and expression levels in tissues can be very helpful for drug development as the majority of drugs are designed to modulate membrane receptors. Furthermore, it is known that many adverse drug effects result from GPCR interactions. However, very few satisfactory methods are currently available for the detection and quantification of GPCRs. The detection is complicated by their three-dimensional structure, their hydrophobic properties, and their localization in the plasma membrane with 7-trans-membrane domains and small cytosolic and extracellular domains. Due to these properties it is very difficult to generate specific antibodies directed against GPCRs for sandwich immunoassays and Western blot. We therefore designed an immunoaffinity- and mass spectrometry-based approach to analyze GPCR-specific signature peptides in tryptic digests in rat tissue lysates. The expression levels of four different GPCRs were determined using chemically labeled synthetic standard peptides. Here, we demonstrate for the first time, that peptide immunoaffinity MS-based methods can render a reliable and quantitative analysis of multi-membrane spanning receptor molecules.