Ultrafast Two-Photon Imaging of a High-Gain Voltage Indicator in Awake Behaving Mice.

Optical interrogation of voltage in deep brain locations with cellular resolution would be immensely useful for understanding how neuronal circuits process information. Here, we report ASAP3, a genetically encoded voltage indicator with 51% fluorescence modulation by physiological voltages, submillisecond activation kinetics, and full responsivity under two-photon excitation. We also introduce an ultrafast local volume excitation (ULoVE) method for kilohertz-rate two-photon sampling in vivo with increased stability and sensitivity. Combining a soma-targeted ASAP3 variant and ULoVE, we show single-trial tracking of spikes and subthreshold events for minutes in deep locations, with subcellular resolution and with repeated sampling over days. In the visual cortex, we use soma-targeted ASAP3 to illustrate cell-type-dependent subthreshold modulation by locomotion. Thus, ASAP3 and ULoVE enable high-speed optical recording of electrical activity in genetically defined neurons at deep locations during awake behavior.

Simultaneous dual-color fluorescence lifetime imaging with novel red-shifted fluorescent proteins.

We describe a red-shifted fluorescence resonance energy transfer (FRET) pair optimized for dual-color fluorescence lifetime imaging (FLIM). This pair utilizes a newly developed FRET donor, monomeric cyan-excitable red fluorescent protein (mCyRFP1), which has a large Stokes shift and a monoexponential fluorescence lifetime decay. When used together with EGFP-based biosensors, the new pair enables simultaneous imaging of the activities of two signaling molecules in single dendritic spines undergoing structural plasticity.

Fluorescent indicators for simultaneous reporting of all four cell cycle phases.

A robust method for simultaneous visualization of all four cell cycle phases in living cells is highly desirable. We developed an intensiometric reporter of the transition from S to G2 phase and engineered a far-red fluorescent protein, mMaroon1, to visualize chromatin condensation in mitosis. We combined these new reporters with the previously described Fucci system to create Fucci4, a set of four orthogonal fluorescent indicators that together resolve all cell cycle phases.

A red fluorescent protein with improved monomericity enables ratiometric voltage imaging with ASAP3.

A ratiometric genetically encoded voltage indicator (GEVI) would be desirable for tracking transmembrane voltage changes in the presence of sample motion. We performed combinatorial multi-site mutagenesis on a cyan-excitable red fluorescent protein to create the bright and monomeric mCyRFP3, which proved to be uniquely non-perturbing when fused to the GEVI ASAP3. The green/red ratio from ASAP3-mCyRFP3 (ASAP3-R3) reported voltage while correcting for motion artifacts, allowing the visualization of membrane voltage changes in contracting cardiomyocytes and throughout the cell cycle of motile cells.

Remission and Relapse of Dyslipidemia After Vertical Sleeve Gastrectomy vs Roux-en-Y Gastric Bypass in a Racially and Ethnically Diverse Population.

Importance: The comparative effectiveness of the most common operations in the long-term management of dyslipidemia is not clear. Objective: To compare 4-year outcomes associated with vertical sleeve gastrectomy (VSG) vs Roux-en-Y gastric bypass (RYGB) for remission and relapse of dyslipidemia. Design, Setting, and Participants: This retrospective comparative effectiveness study was conducted from January 1, 2009, to December 31, 2016, with follow-up until December 31, 2018. Participants included patients with dyslipidemia at the time of surgery who underwent VSG (4142 patients) or RYGB (2853 patients). Patients were part of a large integrated health care system in Southern California. Analysis was conducted from January 1, 2018, to December 31, 2021. Exposures: RYGB and VSG. Main Outcomes and Measures: Dyslipidemia remission and relapse were assessed in each year of follow-up for as long as 4 years after surgery. Results: A total of 8265 patients were included, with a mean (SD) age of 46 (11) years; 6591 (79.8%) were women, 3545 (42.9%) were Hispanic, 1468 (17.8%) were non-Hispanic Black, 2985 (36.1%) were non-Hispanic White, 267 (3.2%) were of other non-Hispanic race, and the mean (SD) body mass index (calculated as weight in kilograms divided by height in meters squared) was 44 (7) at the time of surgery. Dyslipidemia outcomes at 4 years were ascertained for 2168 patients (75.9%) undergoing RYGB and 3999 (73.9%) undergoing VSG. Remission was significantly higher for those who underwent RYGB (824 [38.0%]) compared with VSG (1120 [28.0%]) (difference in the probability of remission, 0.10; 95% CI, 0.01-0.19), with no differences in relapse (455 [21.0%] vs 960 [24.0%]). Without accounting for relapse, remission of dyslipidemia after 4 years was 58.9% (1279) for those who underwent RYGB and 51.9% (2079) for those who underwent VSG. Four-year differences between operations were most pronounced for patients 65 years or older (0.39; 95% CI, 0.27-0.51), those with cardiovascular disease (0.43; 95% CI, 0.24-0.62), or non-Hispanic Black patients (0.13; 95% CI, 0.01-0.25) and White patients (0.13; 95% CI, 0.03-0.22). Conclusions and Relevance: In this large, racially and ethnically diverse cohort of patients who underwent bariatric and metabolic surgery in clinical practices, RYGB was associated with higher rates of dyslipidemia remission after 4 years compared with VSG. However, almost one-quarter of all patients experienced relapse, suggesting that patients should be monitored closely throughout their postoperative course to maximize the benefits of these operations for treatment of dyslipidemia.

Comparative effectiveness of gastric bypass and sleeve gastrectomy on predicted 10-year risk of cardiovascular disease 5 years after surgery.

BACKGROUND: Comparative evidence is needed when deciding which bariatric operation to undergo for long-term cardiovascular risk reduction. OBJECTIVES: The Effectiveness of Gastric Bypass vs. Gastric Sleeve for Cardiovascular Disease (ENGAGE CVD) study compared the effectiveness of vertical sleeve gastrectomy (VSG) and Roux-en-Y gastric bypass (RYGB) operations for reduction of the American College of Cardiology and the American Heart Association-predicted 10-year atherosclerotic cardiovascular disease (ASCVD) risk 5 years after surgery. SETTING: Data for this study came from a large integrated healthcare system in the Southern California region of the United States. This is one of the most ethnically diverse (64% non-White) bariatric populations in the literature. METHODS: The ENGAGE CVD cohort consisted of 22,095 patients who underwent VSG or RYGB from 2009-2016. The VSG and RYGB were compared using a local instrumental variable approach to address observed and unobserved confounding, as well as to conduct heterogeneity of treatment effects for patients of different age groups, baseline-predicted 10-year CVD risk using the ASCVD risk score, and those who had type 2 diabetes (T2D) at the time of surgery. RESULTS: Patients (2771 RYGB and 6256 VVSG) were primarily women (80.6%), Hispanic or non-Hispanic Black (63.7%), and 46 ± 10 years of age, with a body mass index of 43.40 ± 6.5 kg/m2. The predicted 10-year ASCVD risk at surgery was 4.1% for VSG and 5.1% for RYGB, decreasing to 2.6% for VSG and 2.8% for RYGB 1 year postoperatively. By 5 years after surgery, patients remained with relatively low risk levels (3.0% for VSG and 3.3% for RYGB) and there were no significant differences in predicted 10-year ASCVD risk between VSG and RYGB at any time. CONCLUSION: Predicted 10-year ASCVD risk was low in this population and remained low up to 5 years for those with diabetes, Black and Hispanic patients, and older adults. Literature reporting significant differences between VSG and RYGB in 10-year ASCVD risk may be a result of residual confounding.

Minimum Threshold of Bariatric Surgical Weight Loss for Initial Diabetes Remission.

OBJECTIVE: There are few studies testing the amount of weight loss necessary to achieve initial remission of type 2 diabetes mellitus (T2DM) following bariatric surgery and no published studies with use of weight loss to predict initial T2DM remission in sleeve gastrectomy (SG) patients. RESEARCH DESIGN AND METHODS: With Cox proportional hazards models we examined the relationship between initial T2DM remission and percent total weight loss (%TWL) after bariatric surgery. Categories of %TWL were included in the model as time-varying covariates. RESULTS: Of patients (N = 5,928), 73% were female; mean age was 49.8 ± 10.3 years and BMI 43.8 ± 6.92 kg/m2, and 57% had Roux-en-Y gastric bypass (RYGB). Over an average follow-up of 5.9 years, 71% of patients experienced initial remission of T2DM (mean time to remission 1.0 year). With 0-5% TWL used as the reference group in Cox proportional hazards models, patients were more likely to remit with each 5% increase in TWL until 20% TWL (hazard ratio range 1.97-2.92). When categories >25% TWL were examined, all patients had a likelihood of initial remission similar to that of 20-25% TWL. Patients who achieved >20% TWL were more likely to achieve initial T2DM remission than patients with 0-5% TWL, even if they were using insulin at the time of surgery. CONCLUSIONS: Weight loss after bariatric surgery is strongly associated with initial T2DM remission; however, above a threshold of 20% TWL, rates of initial T2DM remission did not increase substantially. Achieving this threshold is also associated with initial remission even in patients who traditionally experience lower rates of remission, such as patients taking insulin.

Comparative Effectiveness of Gastric Bypass and Vertical Sleeve Gastrectomy for Hypertension Remission and Relapse: The ENGAGE CVD Study.

[Figure: see text].

Revision of primary sleeve gastrectomy to Roux-en-Y gastric bypass: indications and outcomes from a high-volume center.

BACKGROUND: Laparoscopic sleeve gastrectomy (SG) has become an accepted primary bariatric operation. Like other bariatric operations, inadequate weight loss and complications have been reported. OBJECTIVES: The aim of this study was to assess the indications and outcomes of revision of SG to laparoscopic Roux-en-Y gastric bypass (RYGB) at a single community hospital. SETTING: Community hospital, United States. METHODS: Retrospective review of a prospectively collected database identifying SG operations done from February 2009 to June 2014. All patients who underwent revision from SG to RYGB were studied. RESULTS: Forty-eight patients underwent revision of SG to RYGB. Mean time to revision was 26 months (range, 2-60 mo) and mean follow up after RYGB was 20 months (range, 4-48 mo). Indications for revision were reflux (n = 14), inadequate weight loss (n = 11), reflux and inadequate weight loss (n = 16), stricture (n = 4), chronic leak (n = 1), and recurrent diabetes and reflux (n = 2). Reflux symptoms resolved in 96% of patients after revision, and hiatal hernias were repaired in 50% of patients. Percentage total weight loss at 3, 6, 12, 24, and 36 months was 9.0%, 12.9%, 15.7%, 13.3%, and 6.5%, respectively. The overall rate of complication was 31%. There were no mortalities. CONCLUSIONS: Revision of SG to RYGB is a potentially effective means of treating SG complications, particularly reflux. Reflux was the most common indication for revision and was often associated with a hiatal hernia. Further studies will be necessary to evaluate the long-term maintenance of additional weight loss after revision of SG to RYGB.

A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo.

Orange-red fluorescent proteins (FPs) are widely used in biomedical research for multiplexed epifluorescence microscopy with GFP-based probes, but their different excitation requirements make multiplexing with new advanced microscopy methods difficult. Separately, orange-red FPs are useful for deep-tissue imaging in mammals owing to the relative tissue transmissibility of orange-red light, but their dependence on illumination limits their sensitivity as reporters in deep tissues. Here we describe CyOFP1, a bright, engineered, orange-red FP that is excitable by cyan light. We show that CyOFP1 enables single-excitation multiplexed imaging with GFP-based probes in single-photon and two-photon microscopy, including time-lapse imaging in light-sheet systems. CyOFP1 also serves as an efficient acceptor for resonance energy transfer from the highly catalytic blue-emitting luciferase NanoLuc. An optimized fusion of CyOFP1 and NanoLuc, called Antares, functions as a highly sensitive bioluminescent reporter in vivo, producing substantially brighter signals from deep tissues than firefly luciferase and other bioluminescent proteins.

Results from the Endologix PowerLink multicenter trial.

The Endologix PowerLink system is a one-piece, bifurcated, fully supported endoluminal graft for the treatment of abdominal aortic aneurysms (AAA). In a recently completed multicenter trial, the device has proven to be effective with a high technical success rate, low morbidity and mortality rates, and a mean decrease in AAA size over time. A 16-month mean follow-up has found low endoleak and migration rates. There have been no ruptures to date. The Endologix PowerLink device compares favorably with previously published results of other endoluminal grafts.