Coumarin as a general switching auxiliary to prepare photochromic and spontaneously blinking fluorophores.

Single-molecule localization microscopy (SMLM) uses activatable or switchable fluorophores to create non-diffraction limited maps of molecular location in biological samples. Despite the utility of this imaging technique, the portfolio of appropriate labels for SMLM remains limited. Here, we describe a general strategy for the construction of "glitter bomb" labels by simply combining rhodamine and coumarin dyes though an amide bond. Condensation of the ortho -carboxyl group on the pendant phenyl ring of rhodamine dyes with a 7-aminocoumarin yields photochromic or spontaneously blinking fluorophores depending on the parent rhodamine structure. We apply this strategy to prepare labels useful super-resolution experiments in fixed cells using different attachment techniques. This general glitter bomb strategy should lead to improved labels for SMLM, ultimately enabling the creation of detailed molecular maps in biological samples.

A series of spontaneously blinking dyes for super-resolution microscopy.

Spontaneously blinking fluorophores permit the detection and localization of individual molecules without reducing buffers or caging groups, thus simplifying single-molecule localization microscopy (SMLM). The intrinsic blinking properties of such dyes are dictated by molecular structure and modulated by environment, which can limit utility. We report a series of tuned spontaneously blinking dyes with duty cycles that span two orders of magnitude, allowing facile SMLM in cells and dense biomolecular structures.

iATPSnFR2: A high-dynamic-range fluorescent sensor for monitoring intracellular ATP.

We developed a significantly improved genetically encoded quantitative adenosine triphosphate (ATP) sensor to provide real-time dynamics of ATP levels in subcellular compartments. iATPSnFR2 is a variant of iATPSnFR1, a previously developed sensor that has circularly permuted superfolder green fluorescent protein (GFP) inserted between the ATP-binding helices of the ε-subunit of a bacterial F0-F1 ATPase. Optimizing the linkers joining the two domains resulted in a ~fivefold to sixfold improvement in the dynamic range compared to the previous-generation sensor, with excellent discrimination against other analytes, and affinity variants varying from 4 µM to 500 µM. A chimeric version of this sensor fused to either the HaloTag protein or a suitable spectrally separated fluorescent protein provides an optional ratiometric readout allowing comparisons of ATP across cellular regions. Subcellular targeting the sensor to nerve terminals reveals previously uncharacterized single-synapse metabolic signatures, while targeting to the mitochondrial matrix allowed direct quantitative probing of oxidative phosphorylation dynamics.

A Wrist-Worn Internet of Things Sensor Node for Wearable Equivalent Daylight Illuminance Monitoring.

Light exposure is a vital regulator of physiology and behavior in humans. However, monitoring of light exposure is not included in current wearable Internet of Things (IoT) devices, and only recently have international standards defined [Formula: see text] -optic equivalent daylight illuminance (EDI) measures for how the eye responds to light. This article reports a wearable light sensor node that can be incorporated into the IoT to provide monitoring of EDI exposure in real-world settings. We present the system design, electronic performance testing, and accuracy of EDI measurements when compared to a calibrated spectral source. This includes consideration of the directional response of the sensor, and a comparison of performance when placed on different parts of the body, and a demonstration of practical use over 7 days. Our device operates for 3.5 days between charges, with a sampling period of 30 s. It has 10 channels of measurement, over the range 415-910 nm, balancing accuracy and cost considerations. Measured [Formula: see text]-opic EDI results for 13 devices show a mean absolute error of less than 0.07 log lx, and a minimum between device correlation of 0.99. These findings demonstrate that accurate light sensing is feasible, including at wrist worn locations. We provide an experimental platform for use in future investigations in real-world light exposure monitoring and IoT-based lighting control.

Publication Rates of Podium Presentations at an Annual Orthopedic Surgery Resident and Fellow Research Symposium.

Introduction Research is an important aspect of residency and fellowship programs across the country. Developing strategies to foster research productivity is worthwhile. An annual research project is one strategy that some programs implement. Methods All resident and fellow (Sports Medicine, Adult Reconstruction, Spine) presentations at an orthopedic surgery department's annual research symposium from June 2016 through June 2021 were identified. Abstract titles, title keywords, and author names were searched in PubMed and Google Scholar to identify the presence of a peer-reviewed publication. Using the total number of research symposium presentations given, the publication rate was calculated for each year, as well as collectively for 2016 to 2021. In addition to publication rate, first author percent, number of citations, Altmetric score, and journal impact factor were recorded. Current PGY-2 through PGY-5 residents completed a survey to assess the perceived value of the annual research symposium. Results Ninety-eight research symposium presentations were reviewed (69 residents, 29 fellows). Forty (58%) resident studies were published and 28 were first-author publications (70%). Thirteen (45%) fellow studies were published and seven were first-author publications (54%). Combining residents and fellows, the overall publication rate was 54% (53/98), and 66% of these (35/53) were first-author publications. There was a wide range of published manuscript journal impact factors, Altmetric scores, and number of citations. All residents surveyed reported finding value in the research symposium. Conclusion The overall publication rate of presentations at an annual orthopedic surgery department research symposium between 2016 and 2021 was 54%, consistent with publication rates reported at National Orthopedic Surgery Society meetings. All residents reported finding value in the annual research symposium. The results of this study support the academic value of implementing a required annual research project and may provide a useful gauge to inform residency and fellowship curricula at other institutions.

Decoding Suicide Decedent Profiles and Signs of Suicidal Intent Using Latent Class Analysis.

Importance: Suicide rates in the US increased by 35.6% from 2001 to 2021. Given that most individuals die on their first attempt, earlier detection and intervention are crucial. Understanding modifiable risk factors is key to effective prevention strategies. Objective: To identify distinct suicide profiles or classes, associated signs of suicidal intent, and patterns of modifiable risks for targeted prevention efforts. Design, Setting, and Participants: This cross-sectional study used data from the 2003-2020 National Violent Death Reporting System Restricted Access Database for 306 800 suicide decedents. Statistical analysis was performed from July 2022 to June 2023. Exposures: Suicide decedent profiles were determined using latent class analyses of available data on suicide circumstances, toxicology, and methods. Main Outcomes and Measures: Disclosure of recent intent, suicide note presence, and known psychotropic usage. Results: Among 306 800 suicide decedents (mean [SD] age, 46.3 [18.4] years; 239 627 males [78.1%] and 67 108 females [21.9%]), 5 profiles or classes were identified. The largest class, class 4 (97 175 [31.7%]), predominantly faced physical health challenges, followed by polysubstance problems in class 5 (58 803 [19.2%]), and crisis, alcohol-related, and intimate partner problems in class 3 (55 367 [18.0%]), mental health problems (class 2, 53 928 [17.6%]), and comorbid mental health and substance use disorders (class 1, 41 527 [13.5%]). Class 4 had the lowest rates of disclosing suicidal intent (13 952 [14.4%]) and leaving a suicide note (24 351 [25.1%]). Adjusting for covariates, compared with class 1, class 4 had the highest odds of not disclosing suicide intent (odds ratio [OR], 2.58; 95% CI, 2.51-2.66) and not leaving a suicide note (OR, 1.45; 95% CI, 1.41-1.49). Class 4 also had the lowest rates of all known psychiatric illnesses and psychotropic medications among all suicide profiles. Class 4 had more older adults (23 794 were aged 55-70 years [24.5%]; 20 100 aged ≥71 years [20.7%]), veterans (22 220 [22.9%]), widows (8633 [8.9%]), individuals with less than high school education (15 690 [16.1%]), and rural residents (23 966 [24.7%]). Conclusions and Relevance: This study identified 5 distinct suicide profiles, highlighting a need for tailored prevention strategies. Improving the detection and treatment of coexisting mental health conditions, substance and alcohol use disorders, and physical illnesses is paramount. The implementation of means restriction strategies plays a vital role in reducing suicide risks across most of the profiles, reinforcing the need for a multifaceted approach to suicide prevention.

Intraosseous Versus Intravenous Vancomycin in Tourniquetless Primary Total Knee Arthroplasty: A Randomized Trial.

BACKGROUND: Intraosseous (IO) administration of vancomycin at the time of total knee arthroplasty (TKA) has been shown to be safer and more effective than intravenous (IV) administration at preventing early periprosthetic joint infection. Previous studies have relied on tourniquet inflation to enhance local tissue concentrations and mitigate systemic release. METHODS: A single-blinded, randomized clinical trial was performed on 20 patients (10 IV, 10 IO) undergoing primary TKA. The control (IV) group received weight-dosed vancomycin approximately 1 hour prior to the incision and weight-dosed cefazolin immediately prior to the incision. The interventional (IO) group received weight-dosed cefazolin immediately prior to the incision and 500 mg of vancomycin delivered via the IO technique at the time of the incision. Systemic samples for vancomycin levels were taken prior to the incision and at closure. During the procedure, tissue samples were taken from the distal femur, proximal tibia, and suprapatellar synovium. There were no differences in patient demographics or changes in serum creatinine from preoperative to postoperatively between groups. RESULTS: Significant differences in systemic vancomycin levels (ug/mL) were found at the start of the case (IV = 27.9 ± 4.9 versus IO = 0 ± 0, P = .0004) and at the end of the case (IV = 19.6 ± 2.6 versus IO = 7.8 ± 1.0, P = .001). No significant differences were seen in the average vancomycin concentration in the distal femur (IV = 61.0 ± 16.0 versus IO = 66.2 ± 12.3, P = .80), proximal tibia (IV = 52.8 ± 13.5 versus IO = 57.1 ± 17.0, P = .84), or suprapatellar synovial tissue (IV = 10.7 ± 5.3 versus IO = 9.0 ± 3.3, P = .80). There were no complications associated with vancomycin administration in either group. CONCLUSIONS: This study demonstrates the utility of IO vancomycin in tourniquetless TKA with similar local tissue and significantly lower systemic concentrations than IV administration. LEVEL OF EVIDENCE: Level 1 therapeutic randomized trial.

Recommendations for measuring and standardizing light for laboratory mammals to improve welfare and reproducibility in animal research.

Light enables vision and exerts widespread effects on physiology and behavior, including regulating circadian rhythms, sleep, hormone synthesis, affective state, and cognitive processes. Appropriate lighting in animal facilities may support welfare and ensure that animals enter experiments in an appropriate physiological and behavioral state. Furthermore, proper consideration of light during experimentation is important both when it is explicitly employed as an independent variable and as a general feature of the environment. This Consensus View discusses metrics to use for the quantification of light appropriate for nonhuman mammals and their application to improve animal welfare and the quality of animal research. It provides methods for measuring these metrics, practical guidance for their implementation in husbandry and experimentation, and quantitative guidance on appropriate light exposure for laboratory mammals. The guidance provided has the potential to improve data quality and contribute to reduction and refinement, helping to ensure more ethical animal use.

An engineered biosensor enables dynamic aspartate measurements in living cells.

Intracellular levels of the amino acid aspartate are responsive to changes in metabolism in mammalian cells and can correspondingly alter cell function, highlighting the need for robust tools to measure aspartate abundance. However, comprehensive understanding of aspartate metabolism has been limited by the throughput, cost, and static nature of the mass spectrometry (MS)-based measurements that are typically employed to measure aspartate levels. To address these issues, we have developed a green fluorescent protein (GFP)-based sensor of aspartate (jAspSnFR3), where the fluorescence intensity corresponds to aspartate concentration. As a purified protein, the sensor has a 20-fold increase in fluorescence upon aspartate saturation, with dose-dependent fluorescence changes covering a physiologically relevant aspartate concentration range and no significant off target binding. Expressed in mammalian cell lines, sensor intensity correlated with aspartate levels measured by MS and could resolve temporal changes in intracellular aspartate from genetic, pharmacological, and nutritional manipulations. These data demonstrate the utility of jAspSnFR3 and highlight the opportunities it provides for temporally resolved and high-throughput applications of variables that affect aspartate levels.

A ratiometric ER calcium sensor for quantitative comparisons across cell types and subcellular regions.

The endoplasmic reticulum (ER) is an important regulator of Ca2+ in cells and dysregulation of ER calcium homeostasis can lead to numerous pathologies. Understanding how various pharmacological and genetic perturbations of ER Ca2+ homeostasis impacts cellular physiology would likely be facilitated by more quantitative measurements of ER Ca2+ levels that allow easier comparisons across conditions. Here, we developed a ratiometric version of our original ER-GCaMP probe that allows for more quantitative comparisons of the concentration of Ca2+ in the ER across cell types and sub-cellular compartments. Using this approach we show that the resting concentration of ER Ca2+ in primary dissociated neurons is substantially lower than that in measured in embryonic fibroblasts.

Patient Perceptions of Same-Day Discharge Versus Overnight Stay After Total Joint Arthroplasty: Results of a Survey.

Background: In the COVID-19 era, there has been increasing interest in same-day discharge (SDD) after total joint arthroplasty (TJA). However, patient perception of SDD is not well reported. Purpose: We sought to understand patients' perceptions and preferences of postoperative care by surveying patients who have completed both an overnight stay (ONS) and an SDD after TJA. Methods: We emailed survey links to 67 patients who previously underwent either 2 total hip arthroplasties (THAs) or 2 total knee arthroplasties (TKAs). Results: Fifty-two patients (78%) responded to the survey. Thirty-four (65%) patients underwent staged, bilateral TKAs, and 18 (35%) patients underwent staged, bilateral THAs. Overall, 63% of patients preferred their SDD, 12% had no preference, and 25% preferred their ONS, with no difference in preference between TKA and THA groups. Those who preferred their SDD reported being more comfortable at home. Those who preferred their ONS felt their pain and concerns were better addressed. No differences were found in comfort, sleep quality, appetite, burden on family, return to function, feelings of being discharged too soon, overall experience, 30-day emergency department (ED) visits, or readmissions within 30 days between patients' SDD and ONS. There was a small statistically significant difference between patients' perception of safety between SDD and ONS. Conclusion: Our survey found that most patients reported a preference for SDD after TJA over ONS. Although there was a small difference in patient perception of safety, there were no differences in return to the ED or readmissions after SDD and ONS.

Linguistic Disparities in Diabetes Care Quality in California Community Health Centers Before and During the COVID-19 Pandemic.

BACKGROUND: Disparities in diabetes care quality may have increased for patients with limited English language proficiency (LEP) compared to non-LEP patients during the COVID-19 pandemic. Changes in diabetes care quality for adult LEP and non-LEP patients of community health centers (CHCs) were examined from 2019 to 2020. METHODS: Adults with Type 2 diabetes (n = 15 965) of 88 CHC sites in California and with 1+ visit/year in 2019 and 2020 from OCHIN electronic health record data were included. Multivariable regression models estimated the association of LEP status and changes in diabetes care quality from 2019 to 2020, controlling for patient sociodemographic and clinical characteristics. Interaction terms (LEP × 2020) were used to estimate differential over time changes in (1) blood pressure screening, (2) blood pressure control (<140/90 mm Hg), and (3) hemoglobin A1c control (HbA1c <8%) for LEP versus non-LEP patients. RESULTS: LEP and non-LEP patients with diabetes had comparable blood pressure screening and control in 2019 and in 2020. LEP patients were less likely than non-LEP patients to have their HbA1c under control in 2019 (OR = 0.85, 95% CI = 0.77, 0.96, P = .006) and 2020 (OR = 0.83, 95% CI = 0.75, 0.92, P = .001). There were no differential changes in HbA1c control over time for LEP and non-LEP patients. DISCUSSION: Although LEP patients were less likely than non-LEP patients to have their HbA1c under control, CHCs maintained quality of care equally for LEP and non-LEP patients with diabetes during the early pandemic period.

Automated Scanning Probe Tip State Classification without Machine Learning.

The manual identification and in situ correction of the state of the scanning probe tip is one of the most time-consuming and tedious processes in atomic-resolution scanning probe microscopy. This is due to the random nature of the probe tip on the atomic level, and the requirement for a human operator to compare the probe quality via manual inspection of the topographical images after any change in the probe. Previous attempts to automate the classification of the scanning probe state have focused on the use of machine learning techniques, but the training of these models relies on large, labeled data sets for each surface being studied. These data sets are extremely time-consuming to create and are not always available, especially when considering a new substrate or adsorbate system. In this paper, we show that the problem of tip classification from a topographical image can be solved by using only a single image of the surface along with a small amount of prior knowledge of the appearance of the system in question with a method utilizing template matching (TM). We find that by using these TM methods, comparable accuracy and precision can be achieved to values obtained with the use of machine learning. We demonstrate the efficacy of this technique by training a machine learning-based classifier and comparing the classifications with the TM classifier for two prototypical silicon-based surfaces. We also apply the TM classifier to a number of other systems where supervised machine learning-based training was not possible due to the nature of the training data sets. Finally, the applicability of the TM method to surfaces used in the literature, which have been classified using machine learning-based methods, is considered.

Precise determination of molecular adsorption geometries by room temperature non-contact atomic force microscopy.

High resolution force measurements of molecules on surfaces, in non-contact atomic force microscopy, are often only performed at cryogenic temperatures, due to needing a highly stable system, and a passivated probe tip (typically via CO-functionalisation). Here we show a reliable protocol for acquiring three-dimensional force map data over both single organic molecules and assembled islands of molecules, at room temperature. Isolated cobalt phthalocyanine and islands of C60 are characterised with submolecular resolution, on a passivated silicon substrate (B:Si(111)-[Formula: see text]). Geometries of cobalt phthalocyanine are determined to a ~ 10 pm accuracy. For the C60, the protocol is sufficiently robust that areas spanning 10 nm × 10 nm are mapped, despite the difficulties of room temperature operation. These results provide a proof-of-concept for gathering high-resolution three-dimensional force maps of networks of complex, non-planar molecules on surfaces, in conditions more analogous to real-world application.

Psychometric properties of the Distress Tolerance Scale in a clinical sample.

The factor structure, reliability, and concurrent validity of the Distress Tolerance Scale were evaluated in a large outpatient sample (N = 775). Prior research demonstrates mixed findings regarding the most appropriate factor structure, finding evidence for the presence of four subfactors as well as a potential second-order (hierarchical) General Distress Tolerance factor. Competing factor structures were compared using confirmatory factor analyses. A second-order hierarchical model with correlated residuals fit the data well, though results suggested poor factor discrimination. A bifactor hierarchical model also demonstrated acceptable fit. However, all subfactors except for Regulation demonstrated small or nonsignificant loadings and/or variances. The model was respecified with all items loading onto a General Distress Tolerance factor and three items loading onto the Regulation factor, which also demonstrated acceptable fit. In support of its concurrent validity, General Distress Tolerance was more strongly associated with neuroticism and a measure of difficulties with emotion regulation than with symptoms of anxiety and depression. The present study extends the literature by demonstrating support for a hierarchical bifactor structure and the favorable psychometric properties of the Distress Tolerance Scale in a large clinical sample. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

White matter and literacy: A dynamic system in flux.

Cross-sectional studies have linked differences in white matter tissue properties to reading skills. However, past studies have reported a range of, sometimes conflicting, results. Some studies suggest that white matter properties act as individual-level traits predictive of reading skill, whereas others suggest that reading skill and white matter develop as a function of an individual's educational experience. In the present study, we tested two hypotheses: a) that diffusion properties of the white matter reflect stable brain characteristics that relate to stable individual differences in reading ability or b) that white matter is a dynamic system, linked with learning over time. To answer these questions, we examined the relationship between white matter and reading in a five-year longitudinal dataset and a series of large-scale, single-observation, cross-sectional datasets (N = 14,249 total participants). We find that gains in reading skill correspond to longitudinal changes in the white matter. However, in the cross-sectional datasets, we find no evidence for the hypothesis that individual differences in white matter predict reading skill. These findings highlight the link between dynamic processes in the white matter and learning.

Oral Carbon Monoxide Enhances Autophagy Modulation in Prostate, Pancreatic, and Lung Cancers.

Modulation of autophagy, specifically its inhibition, stands to transform the capacity to effectively treat a broad range of cancers. However, the clinical efficacy of autophagy inhibitors has been inconsistent. To delineate clinical and epidemiological features associated with autophagy inhibition and a positive oncological clinical response, a retrospective analysis of patients is conducted treated with hydroxychloroquine, a known autophagy inhibitor. A direct correlation between smoking status and inhibition of autophagy with hydroxychloroquine is identified. Recognizing that smoking is associated with elevated circulating levels of carbon monoxide (CO), it is hypothesized that supplemental CO can amplify autophagy inhibition. A novel, gas-entrapping material containing CO in a pre-clinical model is applied and demonstrated that CO can dramatically increase the cytotoxicity of autophagy inhibitors and significantly inhibit the growth of tumors when used in combination. These data support the notion that safe, therapeutic levels of CO can markedly enhance the efficacy of autophagy inhibitors, opening a promising new frontier in the quest to improve cancer therapies.