Impact of comorbidities on immediate post-operative complications in oral cavity free flap patients.

PURPOSE: To examine the relationship between comorbidities and the development of immediate post-operative complications in patients undergoing oral cavity composite resection (OCCR) with free flap (FF) reconstruction. MATERIALS AND METHODS: Retrospective analysis was completed on all consecutive OCCRs with FF reconstruction performed at a single quaternary care facility between 1999 and 2020. Comorbidities, immediate post-operative complications, patient demographics, and tumor characteristics were collected. Odds ratios (OR) with 95 % confidence intervals were calculated for associations between comorbidities and immediate post-operative complications. RESULTS: 320 patients who underwent OCCR with FF reconstruction were included. One hundred twenty-one (37.8 %) patients developed a post-operative complication during their initial hospital admission. The most common complications were non-pneumonia cardiopulmonary events (14.1 %), pneumonia (9.4 %), and wound infection (8.4 %). Other complications included flap compromise, bleeding, and fistula. On multivariate analysis, patients without comorbid conditions were less likely to develop a post-operative complication (OR 0.64; 0.41-0.98). Atrial fibrillation (OR 2.94; 1.17-7.39) and cerebrovascular disease (OR 2.28; 1.08-4.84) were associated with increased odds of developing any complications. Furthermore, cerebrovascular disease (OR: 2.33; 1.04-5.39) and peripheral vascular disease (OR: 2.7; 1.2-6.08) were independently associated with pneumonia. CONCLUSION: In this retrospective review of patients undergoing OCCR with FF reconstruction for oral cavity SCC, lack of identifiable comorbidities appeared to be protective for post-operative complications while atrial fibrillation and cerebrovascular disease were associated with increased odds of any complication. Pre-existing vascular disease was also associated with an increased risk of pneumonia.

How Generalizable Are Randomized Controlled Trials (RCTs) in Staphylococcus aureus Bacteremia? A Description of the Mortality Gap Between RCTs and Observational Studies.

In Staphylococcus aureus bacteremia, mortality rates in randomized controlled trials (RCTs) are consistently lower than observational studies. Stringent eligibility criteria and omission of early deaths in RCTs contribute to this mortality gap. Clinicians should acknowledge the possibility of a lower treatment effect when applying RCT results to bedside care.

Somatic mutations in collagens are associated with a distinct tumor environment and overall survival in gastric cancer.

BACKGROUND: Gastric cancer is a heterogeneous disease with poorly understood genetic and microenvironmental factors. Mutations in collagen genes are associated with genetic diseases that compromise tissue integrity, but their role in tumor progression has not been extensively reported. Aberrant collagen expression has been long associated with malignant tumor growth, invasion, chemoresistance, and patient outcomes. We hypothesized that somatic mutations in collagens could functionally alter the tumor extracellular matrix. METHODS: We used publicly available datasets including The Tumor Cancer Genome Atlas (TCGA) to interrogate somatic mutations in collagens in stomach adenocarcinomas. To demonstrate that collagens were significantly mutated above background mutation rates, we used a moderated Kolmogorov-Smirnov test along with combination analysis with a bootstrap approach to define the background accounting for mutation rates. Association between mutations and clinicopathological features was evaluated by Fisher or chi-squared tests. Association with overall survival was assessed by Kaplan-Meier and the Cox-Proportional Hazards Model. Gene Set Enrichment Analysis was used to interrogate pathways. Immunohistochemistry and in situ hybridization tested expression of COL7A1 in stomach tumors. RESULTS: In stomach adenocarcinomas, we identified individual collagen genes and sets of collagen genes harboring somatic mutations at a high frequency compared to background in both microsatellite stable, and microsatellite instable tumors in TCGA. Many of the missense mutations resemble the same types of loss of function mutations in collagenopathies that disrupt tissue formation and destabilize cells providing guidance to interpret the somatic mutations. We identified combinations of somatic mutations in collagens associated with overall survival, with a distinctive tumor microenvironment marked by lower matrisome expression and immune cell signatures. Truncation mutations were strongly associated with improved outcomes suggesting that loss of expression of secreted collagens impact tumor progression and treatment response. Germline collagenopathy variants guided interpretation of impactful somatic mutations on tumors. CONCLUSIONS: These observations highlight that many collagens, expressed in non-physiologically relevant conditions in tumors, harbor impactful somatic mutations in tumors, suggesting new approaches for classification and therapy development in stomach cancer. In sum, these findings demonstrate how classification of tumors by collagen mutations identified strong links between specific genotypes and the tumor environment.

The physical activity and nutrition-related corporate social responsibility initiatives of food and beverage companies in Canada and implications for public health.

BACKGROUND: As diet-related diseases have increased over the past decades, large food companies have come under scrutiny for contributing to this public health crisis. In response, the food industry has implemented Corporate Social Responsibility (CSR) initiatives related to nutrition and physical activity to emphasize their concern for consumers. This study sought to describe the nature and targeted demographic of physical activity and nutrition-related CSR initiatives of large food companies in Canada and to compare companies who participate in the Canadian Children's Food and Beverage Advertising Initiative (CAI), a self-regulatory initiative aimed at reducing unhealthy food advertising to children, with non-participating companies. METHODS: A cross-sectional study was conducted in 2016. Thirty-nine large food companies, including 18 participating in the CAI, were included in the study. The webpages, Facebook pages and corporate reports of these companies were surveyed to identify CSR initiatives related to nutrition and physical activity. Initiatives were then classified by type (as either philanthropic, education-oriented, research-oriented or other) and by targeted demographic (i.e. targeted at children under 18 years or the general population). Differences between CAI and non-CAI companies were tested using chi-square and Mann-Whitney U tests. RESULTS: Overall, 63 CSR initiatives were identified; 39 were nutrition-related while 24 were physical activity-related. Most (70%) initiatives were considered philanthropic activities, followed by education-oriented (20%), research-oriented (8%) and other (2%). Almost half (47%; n = 29) of initiatives targeted children. Examples of child-targeted initiatives included support of school milk programs (n = 2), the sponsorship of children's sports programs (n = 2) and the development of educational resources for teachers (n = 1). There were no statistically significant differences in the number of CSR initiatives per company (CAI: Mdn = 1, IQR = 3; non-CAI: Mdn = 0, IQR = 2; p = .183) or the proportion of child-targeted initiatives (CAI: 42%; non-CAI: 54%; p = .343) between CAI and non-CAI companies. CONCLUSION: Food companies, including many that largely sell and market unhealthy products, are heavily involved in physical activity and nutrition-related initiatives in Canada, many of which are targeted to children. Government policies aimed at protecting children from unhealthy food marketing should consider including CSR initiatives that expose children to food company branding.

Development of High-Performance Chemical Isotope Labeling LC-MS for Profiling the Carbonyl Submetabolome.

Metabolites containing a carbonyl group represent several important classes of molecules including various forms of ketones and aldehydes such as steroids and sugars. We report a high-performance chemical isotope labeling (CIL) LC-MS method for profiling the carbonyl submetabolome with high coverage and high accuracy and precision of relative quantification. This method is based on the use of dansylhydrazine (DnsHz) labeling of carbonyl metabolites to change their chemical and physical properties to such an extent that the labeled metabolites can be efficiently separated by reversed phase LC and ionized by electrospray ionization MS. In the analysis of six standards representing different carbonyl classes, acetaldehyde could be ionized only after labeling and MS signals were significantly increased for other 5 standards with an enhancement factor ranging from ∼15-fold for androsterone to ∼940-fold for 2-butanone. Differential 12C- and 13C-DnsHz labeling was developed for quantifying metabolic differences in comparative samples where individual samples were separately labeled with 12C-labeling and spiked with a 13C-labeled pooled sample, followed by LC-MS analysis, peak pair picking, and peak intensity ratio measurement. In the replicate analysis of a 1:1 12C-/13C-labeled human urine mixture (n = 6), an average of 2030 ± 39 pairs per run were detected with 1737 pairs in common, indicating the possibility of detecting a large number of carbonyl metabolites as well as high reproducibility of peak pair detection. The average RSD of the peak pair ratios was 7.6%, and 95.6% of the pairs had a RSD value of less than 20%, demonstrating high precision for peak ratio measurement. In addition, the ratios of most peak pairs were close to the expected value of 1.0 (e.g., 95.5% of them had ratios of between 0.67 and 1.5), showing the high accuracy of the method. For metabolite identification, a library of DnsHz-labeled standards was constructed, including 78 carbonyl metabolites with each containing MS, retention time (RT), and MS/MS information. This library and an online search program for labeled carbonyl metabolite identification based on MS, RT, and MS/MS matches have been implemented in a freely available Website, www.mycompoundid.org . Using this library, out of the 1737 peak pairs detected in urine, 33 metabolites were positively identified. In addition, 1333 peak pairs could be matched to the metabolome databases with most of them belonging to the carbonyl metabolites. These results show that 12C-/13C-DnsHz labeling LC-MS is a useful tool for profiling the carbonyl submetabolome of complex samples with high coverage.

Development of isotope labeling liquid chromatography mass spectrometry for mouse urine metabolomics: quantitative metabolomic study of transgenic mice related to Alzheimer's disease.

Because of a limited volume of urine that can be collected from a mouse, it is very difficult to apply the common strategy of using multiple analytical techniques to analyze the metabolites to increase the metabolome coverage for mouse urine metabolomics. We report an enabling method based on differential isotope labeling liquid chromatography mass spectrometry (LC-MS) for relative quantification of over 950 putative metabolites using 20 µL of urine as the starting material. The workflow involves aliquoting 10 µL of an individual urine sample for ¹²C-dansylation labeling that target amines and phenols. Another 10 µL of aliquot was taken from each sample to generate a pooled sample that was subjected to ¹³C-dansylation labeling. The ¹²C-labeled individual sample was mixed with an equal volume of the ¹³C-labeled pooled sample. The mixture was then analyzed by LC-MS to generate information on metabolite concentration differences among different individual samples. The interday repeatability for the LC-MS runs was assessed, and the median relative standard deviation over 4 days was 5.0%. This workflow was then applied to a metabolomic biomarker discovery study using urine samples obtained from the TgCRND8 mouse model of early onset familial Alzheimer's disease (FAD) throughout the course of their pathological deposition of beta amyloid (Aß). It was showed that there was a distinct metabolomic separation between the AD prone mice and the wild type (control) group. As early as 15-17 weeks of age (presymptomatic), metabolomic differences were observed between the two groups, and after the age of 25 weeks the metabolomic alterations became more pronounced. The metabolomic changes at different ages corroborated well with the phenotype changes in this transgenic mice model. Several useful candidate biomarkers including methionine, desaminotyrosine, taurine, N1-acetylspermidine, and 5-hydroxyindoleacetic acid were identified. Some of them were found in previous metabolomics studies in human cerebrospinal fluid or blood samples. This work illustrates the utility of this isotope labeling LC-MS method for biomarker discovery using mouse urine metabolomics.

Determination of sulfadoxine and pyrimethamine in microvolume human plasma using ultra high performance liquid chromatography-tandam mass spectrometry.

To support the pharmacokinetic study of sulfadoxine (SD) and pyrimethamine (PM) in pregnant women and children, sensitive methods with small sample volume are desirable. Here we report a method to determine SD and PM with microvolume plasma samples: 5 µL plasma samples were cleaned up by protein precipitation with acetonitrile. The deuterated analytes were used as the internal standards. The samples after cleanup were injected onto an ACE Excel SuperC18 column (50 × 2.1 mm, 1.7 µm, Hichrom Limited) connected to a Waters I class UPLC coupled with a Sciex Triple Quad 6500+ Mass Spectrometer and eluted with water and acetonitrile both containing 0.1% formic acid in a gradient mode at 0.8mL/min. Detection utilized ESI+ as the ion source and MRM as the quantification mode. The precursor-to-product ion transitions m/z 311→245 for SD and 249→233 for PM were selected for quantification. The ion transitions for the corresponding internal standards were 315→249 for SD-d4 and 254→235 for PM-d3. The simplest linear regression weighted by 1/x was used for the calibration curves. The calibration ranges were 1-200 µg/mL SD and 2 - 1000ng/mL PM. The mean (± standard deviation) recoveries were 94.3±3.2% (SD) and 97.0±1.5% (PM). The validated method was applied to analysis of 1719 clinical samples, demonstrating the method is suitable for the pharmacokinetic study with samples collected up to day 28 post-dose.

5-Diethylamino-naphthalene-1-sulfonyl chloride (DensCl): a novel triplex isotope labeling reagent for quantitative metabolome analysis by liquid chromatography mass spectrometry.

We describe a new set of isotope reagents, (12)C4-, (12)C2(13)C2-, and (13)C4-5-diethylamino-naphthalene-1-sulfonyl chloride (DensCl), in combination with liquid chromatography Fourier-transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS), for improved analysis of the amine- and phenol-containing submetabolome. The synthesis of the reagents is reported, and an optimized derivatization protocol for labeling amines and phenols is described. To demonstrate the utility of the triplex reagents for metabolome profiling of biological samples, urine samples collected daily from a healthy volunteer over a period of 14 days were analyzed. The overall workflow is straightforward, including differential isotope labeling of individual samples and a pooled sample that serves a global internal standard, mixing of the isotope differentially labeled samples and LC-MS analysis for relative metabolome quantification. Comparing to the dansyl chloride (DnsCl) duplex isotope reagents, the new triplex DensCl reagents offer the advantages of improved metabolite detectability due to enhanced sensitivity (i.e., about 1000 peak pairs detected by DensCl labeling vs about 600 peak pairs detected by DnsCl labeling) and analysis speed (i.e., simultaneous analysis of two comparative samples by DensCl vs only one comparative sample analyzed by DnsCl).

Tumor collagens predict genetic features and patient outcomes.

The extracellular matrix (ECM) is a critical determinant of tumor fate that reflects the output from myriad cell types in the tumor. Collagens constitute the principal components of the tumor ECM. The changing collagen composition in tumors along with their impact on patient outcomes and possible biomarkers remains largely unknown. The RNA expression of the 43 collagen genes from solid tumors in The Cancer Genome Atlas (TCGA) was clustered to classify tumors. PanCancer analysis revealed how collagens by themselves can identify the tissue of origin. Clustering by collagens in each cancer type demonstrated strong associations with survival, specific immunoenvironments, somatic gene mutations, copy number variations, and aneuploidy. We developed a machine learning classifier that predicts aneuploidy, and chromosome arm copy number alteration (CNA) status based on collagen expression alone with high accuracy in many cancer types with somatic mutations, suggesting a strong relationship between the collagen ECM context and specific molecular alterations. These findings have broad implications in defining the relationship between cancer-related genetic defects and the tumor microenvironment to improve prognosis and therapeutic targeting for patient care, opening new avenues of investigation to define tumor ecosystems.

POINT: Pipeline for Offline Conversion and Integration of Geocodes and Neighborhood Data.

OBJECTIVES: Geocoding, the process of converting addresses into precise geographic coordinates, allows researchers and health systems to obtain neighborhood-level estimates of social determinants of health. This information supports opportunities to personalize care and interventions for individual patients based on the environments where they live. We developed an integrated offline geocoding pipeline to streamline the process of obtaining address-based variables, which can be integrated into existing data processing pipelines. METHODS: POINT is a web-based, containerized, application for geocoding addresses that can be deployed offline and made available to multiple users across an organization. Our application supports use through both a graphical user interface and application programming interface to query geographic variables, by census tract, without exposing sensitive patient data. We evaluated our application's performance using two datasets: one consisting of 1 million nationally representative addresses sampled from Open Addresses, and the other consisting of 3,096 previously geocoded patient addresses. RESULTS: A total of 99.4 and 99.8% of addresses in the Open Addresses and patient addresses datasets, respectively, were geocoded successfully. Census tract assignment was concordant with reference in greater than 90% of addresses for both datasets. Among successful geocodes, median (interquartile range) distances from reference coordinates were 52.5 (26.5-119.4) and 14.5 (10.9-24.6) m for the two datasets. CONCLUSION: POINT successfully geocodes more addresses and yields similar accuracy to existing solutions, including the U.S. Census Bureau's official geocoder. Addresses are considered protected health information and cannot be shared with common online geocoding services. POINT is an offline solution that enables scalability to multiple users and integrates downstream mapping to neighborhood-level variables with a pipeline that allows users to incorporate additional datasets as they become available. As health systems and researchers continue to explore and improve health equity, it is essential to quickly and accurately obtain neighborhood variables in a Health Insurance Portability and Accountability Act (HIPAA)-compliant way.

Vitamin K-dependent carboxylation regulates Ca2+ flux and adaptation to metabolic stress in ß cells.

Vitamin K is a micronutrient necessary for γ-carboxylation of glutamic acids. This post-translational modification occurs in the endoplasmic reticulum (ER) and affects secreted proteins. Recent clinical studies implicate vitamin K in the pathophysiology of diabetes, but the underlying molecular mechanism remains unknown. Here, we show that mouse ß cells lacking γ-carboxylation fail to adapt their insulin secretion in the context of age-related insulin resistance or diet-induced ß cell stress. In human islets, γ-carboxylase expression positively correlates with improved insulin secretion in response to glucose. We identify endoplasmic reticulum Gla protein (ERGP) as a γ-carboxylated ER-resident Ca2+-binding protein expressed in ß cells. Mechanistically, γ-carboxylation of ERGP protects cells against Ca2+ overfilling by diminishing STIM1 and Orai1 interaction and restraining store-operated Ca2+ entry. These results reveal a critical role of vitamin K-dependent carboxylation in regulation of Ca2+ flux in ß cells and in their capacity to adapt to metabolic stress.

Comparison of Preprint Postings of Randomized Clinical Trials on COVID-19 and Corresponding Published Journal Articles: A Systematic Review.

Importance: Randomized clinical trials (RCTs) on COVID-19 are increasingly being posted as preprints before publication in a scientific, peer-reviewed journal. Objective: To assess time to journal publication for COVID-19 RCT preprints and to compare differences between pairs of preprints and corresponding journal articles. Evidence Review: This systematic review used a meta-epidemiologic approach to conduct a literature search using the World Health Organization COVID-19 database and Embase to identify preprints published between January 1 and December 31, 2021. This review included RCTs with human participants and research questions regarding the treatment or prevention of COVID-19. For each preprint, a literature search was done to locate the corresponding journal article. Two independent reviewers read the full text, extracted data, and assessed risk of bias using the Cochrane Risk of Bias 2 tool. Time to publication was analyzed using a Cox proportional hazards regression model. Differences between preprint and journal article pairs in terms of outcomes, analyses, results, or conclusions were described. Statistical analysis was performed on October 17, 2022. Findings: This study included 152 preprints. As of October 1, 2022, 119 of 152 preprints (78.3%) had been published in journals. The median time to publication was 186 days (range, 17-407 days). In a multivariable model, larger sample size and low risk of bias were associated with journal publication. With a sample size of less than 200 as the reference, sample sizes of 201 to 1000 and greater than 1000 had hazard ratios (HRs) of 1.23 (95% CI, 0.80-1.91) and 2.19 (95% CI, 1.36-3.53) for publication, respectively. With high risk of bias as the reference, medium-risk articles with some concerns for bias had an HR of 1.77 (95% CI, 1.02-3.09); those with a low risk of bias had an HR of 3.01 (95% CI, 1.71-5.30). Of the 119 published preprints, there were differences in terms of outcomes, analyses, results, or conclusions in 65 studies (54.6%). The main conclusion in the preprint contradicted the conclusion in the journal article for 2 studies (1.7%). Conclusions and Relevance: These findings suggest that there is a substantial time lag from preprint posting to journal publication. Preprints with smaller sample sizes and high risk of bias were less likely to be published. Finally, although differences in terms of outcomes, analyses, results, or conclusions were observed for preprint and journal article pairs in most studies, the main conclusion remained consistent for the majority of studies.

Staphylococcus aureus bacteremia mortality across country income groups: A secondary analysis of a systematic review.

OBJECTIVES: Staphylococcus aureus bacteremia (SAB) is a common infection worldwide. We compared SAB mortality in low- and middle-income countries (LMIC) versus high-income countries (HIC) in a meta-analysis. METHODS: We searched MEDLINE, Embase, and Cochrane Database of Systematic Reviews from 1991-2021 and included observational, single-country studies on patients with positive blood cultures for S. aureus. The main outcome was the proportion of patients with SAB who died in the hospital. A generalized linear mixed random-effects model was used to pool estimates, and a meta-regression was used to adjust for study-level characteristics. RESULTS: A total of 332 studies involving 517,671 patients in 39 countries were included. No study was conducted in a low-income country. Only 33 (10%) studies were performed in middle-income countries (MIC), which described 6,216 patients. The pooled in-hospital mortality was 32.4% (95% confidence interval [CI] 27.2%-38.2%, T2 = 0.3063) in MIC and 22.3% (95% CI 20.1%-24.6%, T2 = 0.3257) in HIC. In a meta-regression model, MIC had higher in-hospital mortality (adjusted odds ratio 1.37, 95% CI 1.11-1.71; P = 0.0042) than HIC. CONCLUSION: In SAB studies, LMIC are poorly represented. In-hospital mortality was significantly higher in MIC than in HIC. Research should be conducted in LMIC to characterize differences in care processes driving the mortality gap.

Staphylococcus aureus bacteraemia mortality: a systematic review and meta-analysis.

BACKGROUND: Precise estimates of mortality in Staphylococcus aureus bacteraemia (SAB) are important to convey prognosis and guide the design of interventional studies. OBJECTIVES: We performed a systematic review and meta-analysis to estimate all-cause mortality in SAB and explore mortality change over time. DATA SOURCES: The MEDLINE and Embase databases, as well as the Cochrane Database of Systematic Reviews, were searched from January 1, 1991 to May 7, 2021. STUDY ELIGIBILITY CRITERIA: Human observational studies on patients with S. aureus bloodstream infection were included. PARTICIPANTS: The study analyzed data of patients with a positive blood culture for S. aureus. METHODS: Two independent reviewers extracted study data and assessed risk of bias using the Newcastle-Ottawa Scale. A generalized, linear, mixed random effects model was used to pool estimates. RESULTS: A total of 341 studies were included, describing a total of 536,791 patients. From 2011 onward, the estimated mortality was 10.4% (95% CI, 9.0%-12.1%) at 7 days, 13.3% (95% CI, 11.1%-15.8%) at 2 weeks, 18.1% (95% CI, 16.3%-20.0%) at 1 month, 27.0% (95% CI, 21.5%-33.3%) at 3 months, and 30.2% (95% CI, 22.4%-39.3%) at 1 year. In a meta-regression model of 1-month mortality, methicillin-resistant S. aureus had a higher mortality rate (adjusted OR (aOR): 1.04; 95% CI, 1.02-1.06 per 10% increase in methicillin-resistant S. aureus proportion). Compared with prior to 2001, more recent time periods had a lower mortality rate (aOR: 0.88; 95% CI, 0.75-1.03 for 2001-2010; aOR: 0.82; 95% CI, 0.69-0.97 for 2011 onward). CONCLUSIONS: SAB mortality has decreased over the last 3 decades. However, more than one in four patients will die within 3 months, and continuous improvement in care remains necessary.

What Is the Optimal Follow-up Length for Mortality in Staphylococcus aureus Bacteremia? Observations From a Systematic Review of Attributable Mortality.

Background: Deaths following Staphylococcus aureus bacteremia (SAB) may be related or unrelated to the infection. In SAB therapeutics research, the length of follow-up should be optimized to capture most attributable deaths and minimize nonattributable deaths. We performed a secondary analysis of a systematic review to describe attributable mortality in SAB over time. Methods: We systematically searched Medline, Embase, and Cochrane Database of Systematic Reviews from 1 January 1991 to 7 May 2021 for human observational studies of SAB. To be included in this secondary analysis, the study must have reported attributable mortality. Two reviewers extracted study data and assessed risk of bias independently. Pooling of study estimates was not performed due to heterogeneity in the definition of attributable deaths. Results: Twenty-four observational cohort studies were included. The median proportion of all-cause deaths that were attributable to SAB was 77% (interquartile range [IQR], 72%-89%) at 1 month and 62% (IQR, 58%-75%) at 3 months. At 1 year, this proportion was 57% in 1 study. In 2 studies that described the rate of increase in mortality over time, 2-week follow-up captured 68 of 79 (86%) and 48 of 57 (84%) attributable deaths that occurred by 3 months. By comparison, 1-month follow-up captured 54 of 57 (95%) and 56 of 60 (93%) attributable deaths that occurred by 3 months in 2 studies. Conclusions: The proportion of deaths that are attributable to SAB decreases as follow-up lengthens. Follow-up duration between 1 and 3 months seems optimal if evaluating processes of care that impact SAB mortality. Clinical Trials Registration: PROSPERO CRD42021253891.

Near-infrared fluorescence lifetime pH-sensitive probes.

We report what we believe to be the first near-infrared pH-sensitive fluorescence lifetime molecular probe suitable for biological applications in physiological range. Specifically, we modified a known fluorophore skeleton, hexamethylindotricarbocyanine, with a tertiary amine functionality that was electronically coupled to the fluorophore, to generate a pH-sensitive probe. The pK(a) of the probe depended critically on the location of the amine. Peripheral substitution at the 5-position of the indole ring resulted in a compound with pK(a) ∼ 4.9 as determined by emission spectroscopy. In contrast, substitution at the meso-position shifted the pK(a) to 5.5. The resulting compound, LS482, demonstrated steady-state and fluorescence-lifetime pH-sensitivity. This sensitivity stemmed from distinct lifetimes for protonated (∼1.16 ns in acidic DMSO) and deprotonated (∼1.4 ns in basic DMSO) components. The suitability of the fluorescent dyes for biological applications was demonstrated with a fluorescence-lifetime tomography system. The ability to interrogate cellular processes and subsequently translate the findings in living organisms further augments the potential of these lifetime-based pH probes.

Rational approach to select small peptide molecular probes labeled with fluorescent cyanine dyes for in vivo optical imaging.

We demonstrate that the structure of carbocyanine dyes, which are commonly used to label small peptides for molecular imaging and not the bound peptide, controls the rate of extravasation from blood vessels to tissue. By examining several near-infrared (NIR) carbocyanine fluorophores, we demonstrate a quantitative correlation between the binding of a dye to albumin, a model plasma protein, and the rate of extravasation of the probe into tissue. Binding of the dyes was measured by fluorescence quenching of the tryptophans in albumin and was found to be inversely proportional to the rate of extravasation. The rate of extravasation, determined by kurtosis from longitudinal imaging studies using rodent ear models, provided a basis for quantitative measurements. Structure-activity studies aimed at evaluating a representative library of NIR fluorescent cyanine probes showed that hydrophilic dyes with binding constants several orders of magnitude lower than their hydrophobic counterparts have much faster extravasation rate, establishing a foundation for rational probe design. The correlation provides a guideline for dye selection in optical imaging and a method to verify if a certain dye is optimal for a specific molecular imaging application.

Use of high resolution/accurate mass full scan/data-dependent acquisition for targeted/non-targeted screening in equine doping control.

High-resolution mass spectrometry (HRMS) is a very powerful technology for equine doping control analysis. The more recently developed hybrid type of Orbitrap-based HRMS instrument allows for both targeted and non-targeted screening analyses in a single liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) run. In the present study, an LC-HRMS/MS method was developed and validated to detect prohibited substances in equine sports. The substances were recovered from equine plasma by liquid-liquid extraction (LLE) using methyl tert-butyl ether and were separated on a C18 reversed-phase column using mobile phases of 5 mM ammonium formate and acetonitrile. A 7.5 min LC gradient was employed to elute substances and results indicated that the LC method generated sharp and symmetric chromatographic peaks. An in-house equine doping compound database and a spectral library were built to increase method specificity for substances of interest. Five criteria, i.e. accurate mass, retention time, isotope pattern, selected HRMS/MS fragment ions (compound database) and HRMS/MS spectra (spectral library), were employed for targeted screening. We utilized these criteria to validate targeted detection of 451 substances within our in-house equine doping compound database. By using all five criteria in screening, the false screening positive rate is significantly reduced. A screening strategy and a Microsoft Excel macro were developed to facilitate interpretation and reporting of results. As the simultaneous acquisition of the full scan HRMS data provides the opportunity for retrospective non-targeted analysis, our findings highlight the use of this novel methodology as a simple, rapid, and reliably reproducible strategy to meet the challenge of identifying an increasing number of doping substances that could potentially impact the integrity of the horse racing community.

Thromboembolic events following cardioversion of acute atrial fibrillation and flutter: a systematic review and meta-analysis.

BACKGROUND: Recent studies have presented concerning data on the safety of cardioversion for acute atrial fibrillation and flutter. We conducted this meta-analysis to evaluate the effect of oral anticoagulation use on thromboembolic events post-cardioversion of low-risk acute atrial fibrillation and flutter patients of < 48 h in duration. METHODS: We searched MEDLINE, Embase, and Cochrane from inception through February 6, 2020 for studies reporting thromboembolic events post-cardioversion of acute atrial fibrillation and flutter. Main outcome was thromboembolic events within 30 days post-cardioversion. Primary analysis compared thromboembolic events based on oral anticoagulation use versus no oral anticoagulation use. Secondary analysis was based on baseline thromboembolic risk. We performed meta-analyses where 2 or more studies were available, by applying the DerSimonian-Laird random-effects model. Risk of bias was assessed with the Quality in Prognostic Studies tool. RESULTS: Of 717 titles screened, 20 studies met inclusion criteria. Primary analysis of seven studies with low risk of bias demonstrated insufficient evidence regarding the risk of thromboembolic events associated with oral anticoagulation use (RR = 0.82 where RR < 1 suggests decreased risk with oral anticoagulation use; 95% CI 0.27 to 2.47; I2 = 0%). Secondary analysis of 13 studies revealed increased risk of thromboembolic events with high baseline thromboembolic risk (RR = 2.25 where RR > 1 indicates increased risk with higher CHADS2 or CHA2DS2-VASc scores; 95% CI 1.25 to 4.04; I2 = 0%). CONCLUSION: Primary analysis revealed insufficient evidence regarding the effect of oral anticoagulation use on thromboembolic events post-cardioversion of low-risk acute atrial fibrillation and flutter, though the event rate is low in contemporary practice. Our findings can better inform patient-centered decision-making when considering 4-week oral anticoagulation use for acute atrial fibrillation and flutter patients.

RéSUMé: CONTEXTE: Des études récentes ont présenté des données sur la sécurité de la cardioversion pour la fibrillation auriculaire aiguë et le flutter. Nous avons mené cette méta-analyse pour évaluer l'effet de l'utilisation de l'anticoagulation orale sur les événements thromboemboliques post-cardioversion de patients atteints de fibrillation auriculaire aiguë à faible risque et de flutter de moins de 48 heures. LES MéTHODES: Nous avons recherché dans MEDLINE, Embase et Cochrane depuis le début jusqu'au 6 février 2020 des études faisant état d'événements thromboemboliques après une cardioversion de la fibrillation auriculaire aiguë et du flutter. Le principal résultat a été des événements thromboemboliques dans les 30 jours suivant la cardioversion. L'analyse primaire a comparé les événements thromboemboliques basés sur l'utilisation de l'anticoagulation orale par rapport à l'absence d'anticoagulation orale. L'analyse secondaire était basée sur le risque thromboembolique de base. Nous avons effectué des méta-analyses lorsque deux études ou plus étaient disponibles, en appliquant le modèle à effets aléatoires DerSimonian-Laird. Le risque de biais a été évalué avec l'outil Quality in Prognostic Studies. RéSULTATS: Sur les 717 titres examinés, 20 études ont répondu aux critères d'inclusion. L'analyse primaire de sept études présentant un faible risque de biais a démontré l'insuffisance des preuves concernant le risque d'événements thromboemboliques associés à l'utilisation d'anticoagulation orale (RR = 0,82 où RR < 1 suggère une diminution du risque avec l'utilisation d'anticoagulation orale ; IC 95 % 0,27 à 2,47 ; I2 = 0 %). L'analyse secondaire de 13 études a révélé un risque accru d'événements thromboemboliques avec un risque thromboembolique de base élevé (RR = 2,25 où RR > 1 indique un risque accru avec des scores CHADS2 ou CHA2DS2-VASc plus élevés ; 95 % CI 1,25 à 4,04 ; I2 = 0 %). CONCLUSIONS: L'analyse primaire a révélé des preuves insuffisantes concernant l'effet de l'utilisation de l'anticoagulation orale sur les événements thromboemboliques après une cardioversion de fibrillation auriculaire aiguë à faible risque et de flutter, bien que le taux d'événements soit faible dans la pratique contemporaine. Nos conclusions peuvent mieux éclairer la prise de décision centrée sur le patient lorsqu'il s'agit d'envisager l'utilisation de l'anticoagulation orale pendant 4 semaines pour les patients souffrant de fibrillation auriculaire aiguë et de flutter.

High-performance isotope labeling for profiling carboxylic acid-containing metabolites in biofluids by mass spectrometry.

We have developed a new isotope labeling method, based on the use of isotope-coded p-dimethylaminophenacyl (DmPA) bromide as a reagent, combined with liquid chromatography-mass spectrometry (LC-MS) for high-performance metabolome analysis with a focus on profiling carboxylic acid-containing metabolites. Derivatization is simple, fast (1 h plus 30 min for quenching the reaction), and applicable to a wide range of carboxylic acids with a high yield and little or no side reaction products. This labeling method is demonstrated to be not only effective in introducing an isotope tag for accurate metabolite quantification but also improving the chromatographic retention of the metabolites in reversed-phase (RP) LC, enhancing ESI efficiency by 2-4 orders of magnitude, and facilitating the identification of metabolite peaks in LC-MS. In triplicate experiments of a 1:1 ratio of (13)C-/(12)C-DmPA labeled human urine, we were able to detect 2671, 2546, and 2820 ion pairs from metabolites containing one or more carboxylic acid groups.