Directional Atherectomy of the Common Femoral Artery: Complications and Outcomes.

BACKGROUND: The use of directional atherectomy (DA) with or without drug-coated balloon (DCB) may be considered for the management of common femoral artery (CFA) occlusive disease because of its minimally invasive nature with early mobilization, reduced incision complications, and infection rates. However, it has recognized complications, which may be related to the learning curve. We present our initial experience using DA and suggest changes that may, based on our practice, improve outcomes. METHODS: Retrospective analysis with a prospective data collection from 2 centers to analyze outcomes in all consecutive patients treated during 1 year (n = 25). Patients who underwent CFA DA with/without DCB for CFA >70% stenosis. Primary end points include technical success, primary patency of the CFA, morbidity, and mortality. Secondary end points include change in Rutherford-Becker class, length of stay, and target lesion revascularization rate. RESULTS: Between July 2017 and December 2018, 25 patients underwent CFA DA. Two had an occluded CFA, and 23 had >70% CFA stenosis as determined by ultrasound scan (USS) and/or computed tomography angiogram (CTA) preoperatively. There were no deaths within 30 days. Procedure-related complications included 2 cases of CFA pseudoaneurysm (one of them repaired by open surgery) and 1 CFA perforation (repaired with covered stent). No distal embolization or limb loss occurred. Mean length of stay was 1.9 days. Primary and secondary patency at 3 and 6 months was 100%. At 12 months, it was 96%. CONCLUSIONS: Early results suggest that CFA DA with/without DCB is safe and effective. Previous CTA, focused USS, and/or intravascular USS may be useful to minimize the risk of pseudoaneurysm or perforation by excessive thinning of the media. Experience is required to prevent localized dilatation over time.

The Impact of Changing Race-Specific Equations for Lung Function Tests among Veterans with COPD.

RATIONALE: The American Thoracic Society recommended a single reference equation for spirometry but the impact to patients is not known. OBJECTIVE: To estimate the effect of changing to a single reference equation among Veterans with chronic obstructive pulmonary disease (COPD). METHODS: Cross-sectional study including Veterans aged ≥40 to ≤89 years with COPD and spirometry results from 21 facilities between 2010 - 2019. We collected race/ethnicity data from the electronic health record. We estimated the percentage change in the number of Veterans with lung function meeting clinical thresholds used to determine eligibility for lung resection for cancer, lung volume reduction surgery (LVRS), and lung transplant referral. We estimated the change for each level of VA service connection and financial impact. RESULTS: We identified 44,892 Veterans; Asian (0.5%), Black (11.8%), White (80.8%), and Hispanic (1.8%). When changing to a single reference equation, Asian and Black Veterans had reduced predicted lung function that could result in less surgical lung resection (4.4% and 11.1% respectively), while increasing LVRS (1.7% and 3.8%), and lung transplant evaluation for Black Veterans (1.2%). White Veterans had increased predicted lung function and could experience increased lung resection (8.1%), with less LVRS (3.3%), and lung transplant evaluation (0.9%). Some Asian and Black Veterans could experience an increase in monthly disability payments (+$540.38 and $398.38), while Hispanic White and White Veterans could see a decrease (-$588.79). When aggregated, Hispanic Veterans experienced changes attributable to their racial identity, and because this sample was predominantly Hispanic White, had similar results to White Veterans. CONCLUSIONS: Changing the reference equation could affect access to treatment and disability benefits, depending on race. If adopted, the use of discrete clinical thresholds needs to be reassessed, considering patient-centered outcomes.

Sleep Testing and Mortality in a Propensity-matched Cohort of Patients with Chronic Obstructive Pulmonary Disease.

Rationale: Many advocate the application of propensity-matching methods to real-world data to answer key questions around obstructive sleep apnea (OSA) management. One such question is whether identifying undiagnosed OSA impacts mortality in high-risk populations, such as those with chronic obstructive pulmonary disease (COPD). Objectives: Assess the association of sleep testing with mortality among patients with COPD and a high likelihood of undiagnosed OSA. Methods: We identified patients with COPD and a high likelihood of undiagnosed OSA. We then distinguished those receiving sleep testing within 90 days of index COPD encounters. We calculated propensity scores for testing based on 37 variables and compared long-term mortality in matched groups. In sensitivity analyses, we compared mortality using inverse propensity weighting and instrumental variable methods. We also compared the incidence of nonfatal events including adverse outcomes (hospitalizations and COPD exacerbations) and routine services that are regularly indicated in COPD (influenza vaccination and pulmonary function testing). We compared the incidence of each nonfatal event as a composite outcome with death and separately compared the marginal probability of each nonfatal event independently, with death as a competing risk. Results: Among 135,958 patients, 1,957 (1.4%) received sleep testing. We propensity matched all patients with sleep testing to an equal number without testing, achieving excellent balance on observed confounders, with standardized differences < 0.10. We observed lower mortality risk among patients with sleep testing (incidence rate ratio, 0.88; 95% confidence interval [CI], 0.79-0.99) and similar results using inverse propensity weighting and instrumental variable methods. Contrary to mortality, we found that sleep testing was associated with a similar or greater risk for nonfatal adverse events, including inpatient COPD exacerbations (subhazard ratio, 1.29; 95% CI, 1.02-1.62) and routine services like influenza vaccination (subhazard ratio, 1.26; 95% CI, 1.17-1.36). Conclusions: Our disparate findings can be interpreted in multiple ways. Sleep testing may indeed cause both reduced mortality and greater incidence of nonfatal adverse outcomes and routine services. However, it is also possible that our findings stem from residual confounding by patients' likelihood of accessing care. Given the limitations of propensity-based analyses, we cannot confidently distinguish these two possibilities. This uncertainty highlights the limitations of using propensity-based analyses to guide patient care and policy decisions.

Fluorescence resonance energy transfer-based assay for characterization of hepatitis C virus NS3-4A protease activity in live cells.

The NS3/4A protease from hepatitis C virus (HCV) plays a key role in viral replication. We report a system for monitoring the activity of this enzyme in single living mammalian cells. We constructed a fluorescence resonance energy transfer (FRET) probe that consists of an enhanced cyan fluorescent protein-citrine fusion, with a cleavage site for HCV NS3/4A protease embedded within the linker between them. Expression of the biosensor in mammalian cells resulted in a FRET signal, and cotransfection with the NS3/4A expression vector produced a significant reduction in FRET, indicating that the cleavage site was processed. Western blot and spectrofluorimetry analysis confirmed the physical cleavage of the fusion probe by the NS3/4A protease. As the level of FRET decay was a function of the protease activity, the system allowed testing of NS3/4A protease variants with different catalytic efficiencies. This FRET probe could be adapted for high-throughput screening of new HCV NS3/4 protease inhibitors.

Imaging FRET standards by steady-state fluorescence and lifetime methods.

Imaging fluorescence resonance energy transfer (FRET) between molecules labeled with fluorescent proteins is emerging as a powerful tool to study changes in ions, ligands, and molecular interactions in their physiological cellular environment. Different methods use either steady-state fluorescence properties or lifetime to quantify the FRET rate. In addition, some provide the absolute FRET efficiency whereas others are simply a relative index very much influenced by the actual settings and instrumentation used, which makes the interpretation of a given FRET rate very difficult. The use and exchange of FRET standards in laboratories using these techniques would help to overcome this drawback. We report here the construction and systematic evaluation of FRET standard probes of varying FRET efficiencies. The standards for intramolecular FRET were protein fusions of the cyan and yellow variants of A. victoria green fluorescent protein (ECFP and citrine) joined by short linkers or larger protein spacers, or ECFP tagged with a tetracysteine motif and labeled with the biarsenical fluorochrome, FlAsH. Negative and positive controls of intermolecular FRET were also used. We compared these FRET standards with up to four FRET quantification methods: ratioing of acceptor to donor emission, donor intensity recovery upon acceptor photobleach, sensitized emission after spectral unmixing of raw images, and fluorescence lifetime imaging (FLIM). The latter was obtained with a frequency-domain setup able to provide high quality lifetime images in less than a second, and is thus very well suited for live cell studies. The FRET rates or indexes of the standards were in good agreement regardless of the method used. For the CFP-tetraCys/FlAsH pair, the rate calculated from CFP quenching was faster than that obtained by FLIM.

Impact of supplemental training programs on improving medical students' confidence in providing diabetes self-management education and support.

BACKGROUND/PURPOSE: The purpose of this study was to evaluate the effectiveness of supplemental diabetes-related training modalities and volunteer activities in increasing first-year medical students' knowledge/comfort in providing diabetes self-management education and support (DSMES) to patients. METHODS: A group of medical students developed supplemental diabetes-related training/volunteer programs. The training modalities included an optional 7-session interprofessionally taught Diabetes Enrichment Elective and a 3-hour endocrinologist-led training session intended to prepare students for involvement in an inpatient DSMES volunteer program. The volunteer program provided the students with the opportunity to provide DSMES to patients with diabetes admitted to an academic medical center. Those participating in any of the stated programs were compared to those with no such training regarding confidence in providing DSMES using an optional online survey. The results were analyzed by using Mann-Whitney U test and descriptive analyses. RESULTS: A total of 18 first-year medical students responded to the optional survey with a response rate of ~30% (10 of 33) among participants in any training/volunteer program. First-year medical students who attended any of the offered optional programs had statistically significant higher comfort level in 4 of the 6 areas assessed regarding providing DSMES compared with those with no such training (p<0.05), with medium to large effect size (r=0.48-0.59). CONCLUSION: This study suggests that the supplemental preclerkship diabetes-specific training modalities/volunteer programs can provide benefit in providing medical students with practical knowledge while improving their confidence in providing DSMES to patients with diabetes.

A student-implemented elective to improve medical student confidence in providing diabetes self-management support.

BACKGROUND: The purpose of this study was to develop a preclerkship elective and assess its effectiveness in supplementing medical students' education. METHODS: A group of medical students under the guidance of two faculty advisors developed an elective consisting of six sessions covering a variety of practical aspects of diabetes care/education taught by an interprofessional team. Following the course completion, a survey was emailed to the enrollees who attended at least one session. The results were analyzed using Wilcoxon signed-rank and descriptive analyses. RESULTS: A total of 14 medical students were enrolled (nine first year and five second year). An average of 4.4 sessions/student was attended. Thirteen students attended at least one session and were surveyed. The survey response rate was ~62% (8/13). All eight students indicated that the course was valuable and would recommend it to their colleagues. A Wilcoxon signed-rank test revealed a statistically significant increase in students' confidence in all five areas assessed following participation in the course, P<0.05 with a large effect (r>0.5). CONCLUSION: This study suggests the feasibility of developing disease state-specific preclerkship elective courses and that such courses can be beneficial in supplementing medical student education with practical knowledge.

Red fluorescent protein-aequorin fusions as improved bioluminescent Ca2+ reporters in single cells and mice.

Bioluminescence recording of Ca(2+) signals with the photoprotein aequorin does not require radiative energy input and can be measured with a low background and good temporal resolution. Shifting aequorin emission to longer wavelengths occurs naturally in the jellyfish Aequorea victoria by bioluminescence resonance energy transfer (BRET) to the green fluorescent protein (GFP). This process has been reproduced in the molecular fusions GFP-aequorin and monomeric red fluorescent protein (mRFP)-aequorin, but the latter showed limited transfer efficiency. Fusions with strong red emission would facilitate the simultaneous imaging of Ca(2+) in various cell compartments. In addition, they would also serve to monitor Ca(2+) in living organisms since red light is able to cross animal tissues with less scattering. In this study, aequorin was fused to orange and various red fluorescent proteins to identify the best acceptor in red emission bands. Tandem-dimer Tomato-aequorin (tdTA) showed the highest BRET efficiency (largest energy transfer critical distance R(0)) and percentage of counts in the red band of all the fusions studied. In addition, red fluorophore maturation of tdTA within cells was faster than that of other fusions. Light output was sufficient to image ATP-induced Ca(2+) oscillations in single HeLa cells expressing tdTA. Ca(2+) rises caused by depolarization of mouse neuronal cells in primary culture were also recorded, and changes in fine neuronal projections were spatially resolved. Finally, it was also possible to visualize the Ca(2+) activity of HeLa cells injected subcutaneously into mice, and Ca(2+) signals after depositing recombinant tdTA in muscle or the peritoneal cavity. Here we report that tdTA is the brightest red bioluminescent Ca(2+) sensor reported to date and is, therefore, a promising probe to study Ca(2+) dynamics in whole organisms or tissues expressing the transgene.

Imaging local estrogen production in single living cells with recombinant fluorescent indicators.

Estrogens are steroid hormones with many systemic effects in addition to development and maintenance of the female reproductive system, and ligands of estrogen receptors are of clinical importance because of their use as oral contraceptive, hormone replacement and antitumoral therapy. In addition, tumoral tissues have been found to express aromatase and other steroidogenic enzymes synthesizing estradiol. To aid in the understanding of these processes, we have developed assays to image the local production of estrogens in isolated living mammalian cells. We constructed biosensors based on estrogen receptor α ligand binding domain and fluorescent proteins by following two approaches. First, the ligand binding domain and a short fragment of steroid receptor coactivator-1 were appended to a circularly permuted yellow fluorescent protein to construct an excitation ratio estrogen indicator. In the second strategy, we constructed emission ratio sensors based on fluorescence resonance energy transfer, containing the ligand binding domain flanked by donor and acceptor fluorescent proteins. Estrogens altered the fluorescence signal of cells transfected with the indicators in a dose-dependent manner. We imaged local estrogen production in adrenocortical H295 cells expressing aromatase and transfected with the fluorescent sensors. In addition, paracrine detection was observed in HeLa cells harboring the indicators and co-cultured with H295 cells. This imaging approach may allow detection of physiological levels of these hormones in suitable animal models.

Wide-Field Multi-Parameter FLIM: long-term minimal invasive observation of proteins in living cells.

Time-domain Fluorescence Lifetime Imaging Microscopy (FLIM) is a remarkable tool to monitor the dynamics of fluorophore-tagged protein domains inside living cells. We propose a Wide-Field Multi-Parameter FLIM method (WFMP-FLIM) aimed to monitor continuously living cells under minimum light intensity at a given illumination energy dose. A powerful data analysis technique applied to the WFMP-FLIM data sets allows to optimize the estimation accuracy of physical parameters at very low fluorescence signal levels approaching the lower bound theoretical limit. We demonstrate the efficiency of WFMP-FLIM by presenting two independent and relevant long-term experiments in cell biology: 1) FRET analysis of simultaneously recorded donor and acceptor fluorescence in living HeLa cells and 2) tracking of mitochondrial transport combined with fluorescence lifetime analysis in neuronal processes.