De novo design of proteins housing excitonically coupled chlorophyll special pairs.

Natural photosystems couple light harvesting to charge separation using a 'special pair' of chlorophyll molecules that accepts excitation energy from the antenna and initiates an electron-transfer cascade. To investigate the photophysics of special pairs independently of the complexities of native photosynthetic proteins, and as a first step toward creating synthetic photosystems for new energy conversion technologies, we designed C2-symmetric proteins that hold two chlorophyll molecules in closely juxtaposed arrangements. X-ray crystallography confirmed that one designed protein binds two chlorophylls in the same orientation as native special pairs, whereas a second designed protein positions them in a previously unseen geometry. Spectroscopy revealed that the chlorophylls are excitonically coupled, and fluorescence lifetime imaging demonstrated energy transfer. The cryo-electron microscopy structure of a designed 24-chlorophyll octahedral nanocage with a special pair on each edge closely matched the design model. The results suggest that the de novo design of artificial photosynthetic systems is within reach of current computational methods.

Comparison of post-operative transesophageal and transthoracic echocardiogram findings following atrioventricular septal defect repair.

Success of atrioventricular septal defect repair is defined by post-operative atrioventricular valve function and presence of residual intracardiac shunting. We evaluated differences in interpretation of atrioventricular valve function and residual defects between transesophageal and transthoracic echocardiography in a contemporary cohort of infants undergoing atrioventricular septal defect repair. Among 106 patients, we identified an increase in left and right atrioventricular valve regurgitation, right atrioventricular valve inflow gradient, and increased detection rate of residual intracardiac shunting on transthoracic compared to transesophageal echocardiograms, although residual shunts identified only on transthoracic echocardiogram were not haemodynamically significant. Findings may help inform expectation of post-operative transthoracic echocardiogram findings based on intraoperative assessment.

Sulfoquinovosyl diacylglycerol is required for dimerization of the Rhodobacter sphaeroides RC-LH1 core complex.

The reaction centre-light harvesting 1 (RC-LH1) core complex is indispensable for anoxygenic photosynthesis. In the purple bacterium Rhodobacter (Rba.) sphaeroides RC-LH1 is produced both as a monomer in which 14 LH1 subunits form a C-shaped antenna around one RC, and as a dimer, where 28 LH1 subunits form an S-shaped antenna surrounding two RCs. Alongside the five RC and LH1 subunits, an additional polypeptide known as PufX provides an interface for dimerization and also prevents LH1 ring closure, introducing a channel for quinone exchange that is essential for photoheterotrophic growth. Structures of Rba. sphaeroides RC-LH1 complexes revealed several new components; protein-Y, which helps to form the quinone channel; protein-Z, of unknown function and seemingly unique to dimers; and a tightly bound sulfoquinovosyl diacylglycerol (SQDG) lipid that interacts with two PufX arginine residues. This lipid lies at the dimer interface alongside weak density for a second molecule, previously proposed to be an ornithine lipid. In this work we have generated strains of Rba.sphaeroides lacking protein-Y, protein-Z, SQDG or ornithine lipids to assess the roles of these previously unknown components in the assembly and activity of RC-LH1. We show that whilst the removal of either protein-Y, protein-Z or ornithine lipids has only subtle effects, SQDG is essential for the formation of RC-LH1 dimers but its absence has no functional effect on the monomeric complex.

Photophysics-informed two-photon voltage imaging using FRET-opsin voltage indicators.

Microbial rhodopsin-derived genetically encoded voltage indicators (GEVIs) are powerful tools for mapping bioelectrical dynamics in cell culture and in live animals. Förster resonance energy transfer (FRET)-opsin GEVIs use voltage-dependent changes in opsin absorption to modulate the fluorescence of an attached fluorophore, achieving high brightness, speed, and voltage sensitivity. However, the voltage sensitivity of most FRET-opsin GEVIs has been reported to decrease or vanish under two-photon (2P) excitation. Here we investigated the photophysics of the FRET-opsin GEVIs Voltron1 and 2. We found that the voltage sensitivity came from a photocycle intermediate, not from the opsin ground state. The voltage sensitivities of both GEVIs were nonlinear functions of illumination intensity; for Voltron1, the sensitivity reversed sign under low-intensity illumination. Using photocycle-optimized 2P illumination protocols, we demonstrate 2P voltage imaging with Voltron2 in barrel cortex of a live mouse. These results open the door to high-speed 2P voltage imaging of FRET-opsin GEVIs in vivo.

Targeting TRIP13 in favorable histology Wilms tumor with nuclear export inhibitors synergizes with doxorubicin.

Wilms tumor (WT) is the most common renal malignancy of childhood. Despite improvements in the overall survival, relapse occurs in ~15% of patients with favorable histology WT (FHWT). Half of these patients will succumb to their disease. Identifying novel targeted therapies remains challenging in part due to the lack of faithful preclinical in vitro models. Here we establish twelve patient-derived WT cell lines and demonstrate that these models faithfully recapitulate WT biology using genomic and transcriptomic techniques. We then perform loss-of-function screens to identify the nuclear export gene, XPO1, as a vulnerability. We find that the FDA approved XPO1 inhibitor, KPT-330, suppresses TRIP13 expression, which is required for survival. We further identify synergy between KPT-330 and doxorubicin, a chemotherapy used in high-risk FHWT. Taken together, we identify XPO1 inhibition with KPT-330 as a potential therapeutic option to treat FHWTs and in combination with doxorubicin, leads to durable remissions in vivo.

Generalized biomolecular modeling and design with RoseTTAFold All-Atom.

Deep-learning methods have revolutionized protein structure prediction and design but are presently limited to protein-only systems. We describe RoseTTAFold All-Atom (RFAA), which combines a residue-based representation of amino acids and DNA bases with an atomic representation of all other groups to model assemblies that contain proteins, nucleic acids, small molecules, metals, and covalent modifications, given their sequences and chemical structures. By fine-tuning on denoising tasks, we developed RFdiffusion All-Atom (RFdiffusionAA), which builds protein structures around small molecules. Starting from random distributions of amino acid residues surrounding target small molecules, we designed and experimentally validated, through crystallography and binding measurements, proteins that bind the cardiac disease therapeutic digoxigenin, the enzymatic cofactor heme, and the light-harvesting molecule bilin.

Effects of older age on visual and self-motion sensory cue integration in navigation.

Older adults demonstrate impairments in navigation that cannot be explained by general cognitive and motor declines. Previous work has shown that older adults may combine sensory cues during navigation differently than younger adults, though this work has largely been done in dark environments where sensory integration may differ from full-cue environments. Here, we test whether aging adults optimally combine cues from two sensory systems critical for navigation: vision (landmarks) and body-based self-motion cues. Participants completed a homing (triangle completion) task using immersive virtual reality to offer the ability to navigate in a well-lit environment including visibility of the ground plane. An optimal model, based on principles of maximum-likelihood estimation, predicts that precision in homing should increase with multisensory information in a manner consistent with each individual sensory cue's perceived reliability (measured by variability). We found that well-aging adults (with normal or corrected-to-normal sensory acuity and active lifestyles) were more variable and less accurate than younger adults during navigation. Both older and younger adults relied more on their visual systems than a maximum likelihood estimation model would suggest. Overall, younger adults' visual weighting matched the model's predictions whereas older adults showed sub-optimal sensory weighting. In addition, high inter-individual differences were seen in both younger and older adults. These results suggest that older adults do not optimally weight each sensory system when combined during navigation, and that older adults may benefit from interventions that help them recalibrate the combination of visual and self-motion cues for navigation.

Hypoplastic Left Heart Syndrome with Mitral Stenosis and Aortic Atresia-Echocardiographic Findings and Early Outcomes.

BACKGROUND: Mitral stenosis/aortic atresia (MS/AA) has been reported as a high-risk variant of hypoplastic left heart syndrome (HLHS), potentially related to ventriculocoronary connections (VCCs) or endocardial fibroelastosis (EFE) and myocardial hypoperfusion. We aimed to identify echocardiographic and clinical factors associated with early death or transplant in this group. METHODS: Patients with HLHS MS/AA treated at our center between 2000 and 2020 were included. Pre-stage I palliation echocardiograms were reviewed. Certain imaging factors, such as determination of VCC, EFE, and measurement of tricuspid annular plane systolic excursion were measured from retrospective review of preoperative images; others were derived from clinical reports. Groups were compared according to primary outcome of death or transplant prior to stage II palliation. RESULTS: Of 141 patients included, 39 (27.7%) experienced a primary outcome. Ventriculocoronary connections were identified in 103 (73.0%) patients and EFE in 95 (67.4%) patients. Among imaging variables, smaller ascending aorta size (median, 2.2 [interquartile range (IQR) 1.7-2.8] vs 2.6 [2.2-3.4] mm, P = .01) was associated with primary outcome. There was similar frequency of VCC (74.4% vs 72.5%, P = .83), EFE (59.0% vs 72.5%, P = .19), moderate or greater tricuspid regurgitation (5.1% vs 5.9%, P = 1.00), and similar right ventricular systolic function (indexed tricuspid annular plane systolic excursion 32.5 ± 7.3 vs 31.4 ± 7.2 mm/m2, P = .47) in the primary outcome group compared to other patients. Clinical factors associated with primary outcome included lower birth weight (mean, 2.8 ± SD 0.8 vs 3.3 ± 0.5 kg, P = .0003), gestational age <37 weeks (31.6% vs 4.9%, P < .0001), longer cardiopulmonary bypass time (median, 112 [IQR, 93-162] vs 82 [71-119] minutes, P = .001), longer intensive care unit length of stay (median, 19 [IQR, 10-30] vs 10 [7-15] days, P = .001), and extracorporeal membrane oxygenation following stage I palliation (43.6% vs 8.8%, P < .0001). Presence of VCCs and EFE was not associated with death or transplant after controlling for birth weight and era of stage I palliation. CONCLUSIONS: In one of the largest reported single-center cohorts of HLHS MS/AA, there were few pre-stage I palliation imaging characteristics associated with primary outcome. Imaging findings evaluated in this study, including the presence of VCC and/or EFE as determined using highly sensitive echocardiogram criteria, should not preclude intervention, although impact on long-term outcomes requires further evaluation.

Structured Excitation Energy Transfer: Tracking Exciton Diffusion below Sunlight Intensity.

With the increasing demand for new materials for light-harvesting applications, spatiotemporal microscopy techniques are receiving increasing attention as they allow direct observation of the nanoscale diffusion of excitons. However, the use of pulsed and tightly focused laser beams generates light intensities far above those expected under sunlight illumination, leading to photodamage and nonlinear effects that seriously limit the accuracy and applicability of these techniques, especially in biological or atomically thin materials. In this work, we present a novel spatiotemporal microscopy technique that exploits structured excitation in order to dramatically decrease the excitation intensity, up to 10,000-fold when compared with previously reported spatiotemporal photoluminescence microscopy experiments. We tested our method in two different systems, reporting the first exciton diffusion measurement at illumination conditions below sunlight, both considering average power and peak exciton densities in an organic photovoltaic sample (Y6), where we tracked the excitons for up to five recombination lifetimes. Next, nanometer-scale energy transport was directly observed for the first time in both space and time in a printed monolayer of the light-harvesting complex 2 from purple bacteria.

A longitudinal single-cell and spatial multiomic atlas of pediatric high-grade glioma.

Pediatric high-grade glioma (pHGG) is an incurable central nervous system malignancy that is a leading cause of pediatric cancer death. While pHGG shares many similarities to adult glioma, it is increasingly recognized as a molecularly distinct, yet highly heterogeneous disease. In this study, we longitudinally profiled a molecularly diverse cohort of 16 pHGG patients before and after standard therapy through single-nucleus RNA and ATAC sequencing, whole-genome sequencing, and CODEX spatial proteomics to capture the evolution of the tumor microenvironment during progression following treatment. We found that the canonical neoplastic cell phenotypes of adult glioblastoma are insufficient to capture the range of tumor cell states in a pediatric cohort and observed differential tumor-myeloid interactions between malignant cell states. We identified key transcriptional regulators of pHGG cell states and did not observe the marked proneural to mesenchymal shift characteristic of adult glioblastoma. We showed that essential neuromodulators and the interferon response are upregulated post-therapy along with an increase in non-neoplastic oligodendrocytes. Through in vitro pharmacological perturbation, we demonstrated novel malignant cell-intrinsic targets. This multiomic atlas of longitudinal pHGG captures the key features of therapy response that support distinction from its adult counterpart and suggests therapeutic strategies which are targeted to pediatric gliomas.

Premature endocycling of Drosophila follicle cells causes pleiotropic defects in oogenesis.

Endocycling cells grow and repeatedly duplicate their genome without dividing. Cells switch from mitotic cycles to endocycles in response to developmental signals during the growth of specific tissues in a wide range of organisms. The purpose of switching to endocycles, however, remains unclear in many tissues. Additionally, cells can switch to endocycles in response to conditional signals, which can have beneficial or pathological effects on tissues. However, the impact of these unscheduled endocycles on development is underexplored. Here, we use Drosophila ovarian somatic follicle cells as a model to examine the impact of unscheduled endocycles on tissue growth and function. Follicle cells normally switch to endocycles at mid-oogenesis. Inducing follicle cells to prematurely switch to endocycles resulted in the lethality of the resulting embryos. Analysis of ovaries with premature follicle cell endocycles revealed aberrant follicular epithelial structure and pleiotropic defects in oocyte growth, developmental gene amplification, and the migration of a special set of follicle cells known as border cells. Overall, these findings reveal how unscheduled endocycles can disrupt tissue growth and function to cause aberrant development.

Excitation energy transfer in proteoliposomes reconstituted with LH2 and RC-LH1 complexes from Rhodobacter sphaeroides.

Light-harvesting 2 (LH2) and reaction-centre light-harvesting 1 (RC-LH1) complexes purified from the photosynthetic bacterium Rhodobacter (Rba.) sphaeroides were reconstituted into proteoliposomes either separately, or together at three different LH2:RC-LH1 ratios, for excitation energy transfer studies. Atomic force microscopy (AFM) was used to investigate the distribution and association of the complexes within the proteoliposome membranes. Absorption and fluorescence emission spectra were similar for LH2 complexes in detergent and liposomes, indicating that reconstitution retains the structural and optical properties of the LH2 complexes. Analysis of fluorescence emission shows that when LH2 forms an extensive series of contacts with other such complexes, fluorescence is quenched by 52.6 ± 1.4%. In mixed proteoliposomes, specific excitation of carotenoids in LH2 donor complexes resulted in emission of fluorescence from acceptor RC-LH1 complexes engineered to assemble with no carotenoids. Extents of energy transfer were measured by fluorescence lifetime microscopy; the 0.72 ± 0.08 ns lifetime in LH2-only membranes decreases to 0.43 ± 0.04 ns with a ratio of 2:1 LH2 to RC-LH1, and to 0.35 ± 0.05 ns for a 1:1 ratio, corresponding to energy transfer efficiencies of 40 ± 14% and 51 ± 18%, respectively. No further improvement is seen with a 0.5:1 LH2 to RC-LH1 ratio. Thus, LH2 and RC-LH1 complexes perform their light harvesting and energy transfer roles when reconstituted into proteoliposomes, providing a way to integrate native, non-native, engineered and de novo designed light-harvesting complexes into functional photosynthetic systems.

The 50 Most Cited Publications in Adipose-Derived "Stem Cell Therapies" with Application in Orthopaedic Surgery.

Stem cell therapies have become widely popular in orthopaedic surgery, with a recent interest in adipose-derived therapeutics. Adipose-derived mesenchymal signaling cells (ADSCs) and micronized adipose tissue (MAT) are unique therapies derived from different processing methods. Characterizing the most influential studies in lipoaspirate research can help clarify controversies in definitions, identify core literature, and further collective knowledge for educational purposes. The Science Citation Index Expanded subsection of the Web of Science Core Collection was systematically searched to identify the top 50 most cited publications (based on citation/year) on orthopaedic ADSCs or MAT research. Publication and study characteristics were extracted and reported using descriptive statistics. Level of evidence was assessed for applicable studies, and Spearman correlations were calculated to assess the relationship between citation data and level of evidence. The top 50 articles were published between the years 2003 and 2020, with 78% published in the year 2010 or later. The mean number of citations was 103.1 ± 81.1. The mean citation rate was 12.4 ± 6.0 citations per year. Of the 21 studies for which level of evidence was assessed, the majority were level III (10, 47.6%). The single study design most common among the top 50 cited articles was in vitro basic science studies (17 studies, 34%). Twenty-nine articles (58%) were classified as basic science or translational. Application to treat knee osteoarthritis was the most common focus of studies (14 studies, 28%), followed by in vitro analysis of growth factor and cell signaling markers (11 studies, 22%). No correlation was found between rank, citation rate, or year of publication and level of evidence. This study provides a current landscape on the most cited articles in lipoaspirates in orthopaedic surgery. With the expansion of ADSCs and MAT in the past two decades, this study provides the first historical landmark of the literature and a launching point for future research. Studies should explicitly state their processing methodology and whether their study investigates ADSCs or MAT to avoid misinformation.

Premature endocycling of Drosophila follicle cells causes pleiotropic defects in oogenesis.

Endocycling cells grow and repeatedly duplicate their genome without dividing. Cells switch from mitotic cycles to endocycles in response to developmental signals during the growth of specific tissues in a wide range of organisms. The purpose of switching to endocycles, however, remains unclear in many tissues. Additionally, cells can switch to endocycles in response to conditional signals, which can have beneficial or pathological effects on tissues. However, the impact of these unscheduled endocycles on development is underexplored. Here, we use Drosophila ovarian somatic follicle cells as a model to examine the impact of unscheduled endocycles on tissue growth and function. Follicle cells normally switch to endocycles at mid-oogenesis. Inducing follicle cells to prematurely switch to endocycles resulted in lethality of the resulting embryos. Analysis of ovaries with premature follicle cell endocycles revealed aberrant follicular epithelial structure and pleiotropic defects in oocyte growth, developmental gene amplification, and the migration of a special set of follicle cells known as border cells. Overall, these findings reveal how unscheduled endocycles can disrupt tissue growth and function to cause aberrant development.

Contemporary transesophageal echocardiography practice patterns among paediatric cardiology centres in the United States and Canada.

OBJECTIVE: To characterise transesophageal echocardiography practice patterns among paediatric cardiac surgical centres in the United States and Canada. METHODS: A 42-question survey was sent to 80 echocardiography laboratory directors at paediatric cardiology centres with surgical programmes in the United States and Canada. Question domains included transesophageal echocardiography centre characteristics, performance and reporting, equipment use, trainee participation, and quality assurance. RESULTS: Fifty of the 80 centres (62.5%) responded to the survey. Most settings were academic (86.0%) with 42.0% of centres performing > 350 surgical cases/year. The median number of transesophageal echocardiograms performed/cardiologist/year was 50 (26, 73). Pre-operative transesophageal echocardiography was performed in most surgical cases by 91.7% of centres. Transesophageal echocardiography was always performed by most centres following Norwood, Glenn, and Fontan procedures and by < 10% of centres following coarctation repair. Many centres with a written guideline allowed transesophageal echocardiography transducer use at weights below manufacturer recommendations (50.0 and 61.1% for neonatal and paediatric transducers, respectively). Most centres (36/37, 97.3%) with categorical fellowships had rotations which included transesophageal echocardiography participation. Large surgical centres (>350 cases/year) had higher median number of transesophageal echocardiograms/cardiologist/year (75.5 [53, 86] versus 35 [20, 52], p < 0.001) and more frequently used anaesthesia for diagnostic transesophageal echocardiography ≥ 67% of time (100.0 versus 62.1%, p = 0.001). CONCLUSIONS: There is significant variability in transesophageal echocardiography practice patterns and training requirements among paediatric cardiology centres in the United States and Canada. Findings may help inform programmatic decisions regarding transesophageal echocardiography expectations, performance and reporting, equipment use, trainee involvement, and quality assurance.

The antagonistic relationship between apoptosis and polyploidy in development and cancer.

One of the important functions of regulated cell death is to prevent cells from inappropriately acquiring extra copies of their genome, a state known as polyploidy. Apoptosis is the primary cell death mechanism that prevents polyploidy, and defects in this apoptotic response can result in polyploid cells whose subsequent error-prone chromosome segregation are a major contributor to genome instability and cancer progression. Conversely, some cells actively repress apoptosis to become polyploid as part of normal development or regeneration. Thus, although apoptosis prevents polyploidy, the polyploid state can actively repress apoptosis. In this review, we discuss progress in understanding the antagonistic relationship between apoptosis and polyploidy in development and cancer. Despite recent advances, a key conclusion is that much remains unknown about the mechanisms that link apoptosis to polyploid cell cycles. We suggest that drawing parallels between the regulation of apoptosis in development and cancer could help to fill this knowledge gap and lead to more effective therapies.

Sublobar Resection, Stereotactic Body Radiation Therapy, and Percutaneous Ablation Provide Comparable Outcomes for Lung Metastasis-Directed Therapy.

BACKGROUND: Prolonged survival of patients with metastatic disease has furthered interest in metastasis-directed therapy (MDT). RESEARCH QUESTION: There is a paucity of data comparing lung MDT modalities. Do outcomes among sublobar resection (SLR), stereotactic body radiation therapy (SBRT), and percutaneous ablation (PA) for lung metastases vary in terms of local control and survival? STUDY DESIGN AND METHODS: Medical records of patients undergoing lung MDT at a single cancer center between January 2015 and December 2020 were reviewed. Overall survival, local progression, and toxicity outcomes were collected. Patient and lesion characteristics were used to generate multivariable models with propensity weighted analysis. RESULTS: Lung MDT courses (644 total: 243 SLR, 274 SBRT, 127 PA) delivered to 511 patients were included with a median follow-up of 22 months. There were 47 local progression events in 45 patients, and 159 patients died. Two-year overall survival and local progression were 80.3% and 63.3%, 83.8% and 9.6%, and 4.1% and 11.7% for SLR, SBRT, and PA, respectively. Lesion size per 1 cm was associated with worse overall survival (hazard ratio, 1.24; P = .003) and LP (hazard ratio, 1.50; P < .001). There was no difference in overall survival by modality. Relative to SLR, there was no difference in risk of local progression with PA; however, SBRT was associated with a decreased risk (hazard ratio, 0.26; P = .023). Rates of severe toxicity were low (2.1%-2.6%) and not different among groups. INTERPRETATION: This study performs a propensity weighted analysis of SLR, SBRT, and PA and shows no impact of lung MDT modality on overall survival. Given excellent local control across MDT options, a multidisciplinary approach is beneficial for patient triage and longitudinal management.

Optical constraints on two-photon voltage imaging.

Genetically encoded voltage indicators (GEVIs) are a valuable tool for studying neural circuits in vivo, but the relative merits of one-photon (1P) vs. two-photon (2P) voltage imaging are not well characterized. Here we compare the photophysical and imaging properties of commonly used GEVIs under 1P and 2P excitation. 2P excitation requires ~104-fold more illumination power per cell to produce comparable photon count rates to 1P excitation, driving a stringent tradeoff between shot noise and tissue photodamage.