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In vivo imaging is facilitated by a bright, cyan-excitable orange fluorescent protein that is the basis of an improved bioluminescent protein. 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.

The far-red fluorescent protein mMaroon1 and a reporter based on stem-loop binding protein enables the generation of Fucci4, a 4-color cell cycle reporter system that can be used to distinguish all phases of the cell cycle. Also online, a paper by Laviv et al . uses mMaroon1 as a FRET acceptor for the newly developed CyRFP1. 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 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.

Two red fluorescent proteins with long Stokes shift enable simultaneous multicolor 2p imaging. CyRFP1 is well-suited for 2p structural imaging, and FRET sensors made with mCyRFP1 and mMaroon1enable multicolor 2pFLIM in brain slices. Also online, a paper by Bajar et al . reports the development of mMaroon1. 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.

ImportanceThe comparative effectiveness of the most common operations in the long-term management of dyslipidemia is not clear.ObjectiveTo 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 ParticipantsThis 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.ExposuresRYGB and VSG.Main Outcomes and MeasuresDyslipidemia remission and relapse were assessed in each year of follow-up for as long as 4 years after surgery.ResultsA 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 RelevanceIn 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.

Abstract A ratiometric genetically encoded voltage indicator (GEVI) would be desirable for tracking transmembrane voltage changes in cells that are undergoing motion. To create a high-performance ratiometric GEVI, we explored the possibility of adding a voltage-independent red fluorophore to ASAP3, a high-gain green fluorescent GEVI. We performed combinatorial multi-site mutagenesis on the cyan-excitable red fluorescent protein mCyRFP1 to enhance brightness and monomericity, creating mCyRFP3. Among red fluorescent proteins tested, mCyRFP3 proved to be the least perturbing when fused to ASAP3. We demonstrate that the red fluorescence of ASAP3-mCyRFP3 (ASAP3-R3) provides an effective reference channel to remove motion artifacts from voltage-induced changes in green fluorescence. Finally we use ASAP3-R3 to visualize membrane voltage changes throughout the cell cycle of motile cells. Competing Interest Statement The authors have declared no competing interest. Footnotes * mCyRFP2 name changed to mCyRFP3, as a different mCyRFP2 was recently published

Analyzing the spatial symmetries of three-dimensional (3D) crystal structures has led to the discovery of exotic types of quasiparticles and topologically nontrivial materials. Wieder et al. focus on the symmetry groups of 2D surfaces of 3D materials—the so-called wallpaper groups—and find that some of them allow for an additional topological class. This class hosts a single fourfold-degenerate Dirac fermion on the surface of the material and, on the basis of the authors' calculations, is expected to occur in the compound Sr 2 Pb 3 . Science , this issue p. 246 Symmetry considerations and ab initio calculations predict an unusual type of topological state. Materials whose gapless surface states are protected by crystal symmetries include mirror topological crystalline insulators and nonsymmorphic hourglass insulators. There exists only a very limited set of possible surface crystal symmetries, captured by the 17 “wallpaper groups.” Here we show that a consideration of symmetry-allowed band degeneracies in the wallpaper groups can be used to understand previously described topological crystalline insulators and to predict phenomenologically distinct examples. In particular, the two wallpaper groups with multiple glide lines, pgg and p 4 g , allow for a topological insulating phase whose surface spectrum consists of only a single, fourfold-degenerate, true Dirac fermion, representing an exception to a symmetry-enhanced fermion-doubling theorem. We theoretically predict the presence of this phase in Sr 2 Pb 3 in space group 127 ( P 4 /mbm) .

The El Niño–Southern Oscillation, which originates in the tropical Pacific, affects the rest of the world's tropics by perturbing global atmospheric circulation. Less appreciated than this influence is how the tropical Atlantic and Indian Oceans affect the Pacific. Cai et al. review what we know about these pantropical interactions, discuss possible ways of improving predictions of current climate variability, and consider how projecting future climate under different anthropogenic forcing scenarios may be improved. They argue that making progress in this field will require sustained global climate observations, climate model improvements, and theoretical advances. Science , this issue p. eaav4236 The El Niño–Southern Oscillation (ENSO), which originates in the Pacific, is the strongest and most well-known mode of tropical climate variability. Its reach is global, and it can force climate variations of the tropical Atlantic and Indian Oceans by perturbing the global atmospheric circulation. Less appreciated is how the tropical Atlantic and Indian Oceans affect the Pacific. Especially noteworthy is the multidecadal Atlantic warming that began in the late 1990s, because recent research suggests that it has influenced Indo-Pacific climate, the character of the ENSO cycle, and the hiatus in global surface warming. Discovery of these pantropical interactions provides a pathway forward for improving predictions of climate variability in the current climate and for refining projections of future climate under different anthropogenic forcing scenarios.

In an interim analysis of a trial involving 296 patients with ALK -positive non–small-cell lung cancer, lorlatinib, an anaplastic lymphoma kinase inhibitor, was superior to crizotinib in response (in 76% vs. 58%), 12-month progression-free survival (78% vs. 39%), and intracranial disease response (82% vs. 23%). Altered lipid levels were the major toxic effect associated with lorlatinib.

The ALK inhibitor crizotinib as first-line therapy was associated with a significantly better response rate, longer progression-free survival, and greater improvement in quality of life measures than standard chemotherapy in patients with ALK -positive lung cancer. Rearrangements of the anaplastic lymphoma kinase ( ALK ) gene are present in 3 to 5% of non–small-cell lung cancers (NSCLCs). 1 , 2 They define a distinct subgroup of NSCLC that typically occurs in younger patients who have never smoked or have a history of light smoking and that has adenocarcinoma histologic characteristics. 3 – 5 Crizotinib is an oral small-molecule tyrosine kinase inhibitor of ALK, MET, and ROS1 kinases. 6 In phase 1 and 2 studies, crizotinib treatment resulted in objective tumor responses in approximately 60% of patients with ALK -positive NSCLC and in progression-free survival of 7 to 10 months. 7 – 9 In . . .