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.

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 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.

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.

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).

Alcohol Use and Mental Health Symptoms in Nurses during the Early Months of COVID-19.

OBJECTIVE: The current study examined associations of symptoms of posttraumatic stress disorder [PTSD], depression, and generalized anxiety disorder [GAD] with alcohol consumption and drinking to cope in a sample of 310 nurses during the first six months of the COVID-19 pandemic. METHOD: Using a cross-sectional design, nurses completed online surveys. RESULTS: Over 50% of the sample reported alcohol misuse and 12.2% reported drinking to cope. Further, 38.7% reported elevated symptoms of PTSD, 29.7% moderate-to-high symptoms of depression, and 56.8% elevated symptoms of GAD symptoms. Hierarchical regression analyses were conducted to examine how mental health symptoms were associated with alcohol outcomes, controlling for age, gender pronouns, education, and race. No significant predictors emerged for alcohol consumption. Significant associations of symptoms of PTSD and depression were found for drinking to cope, such that higher levels of mental health symptoms were associated with greater endorsement of drinking to cope. CONCLUSION: Results are discussed in light of increasing prevention and support services for nurses.

Optimized Red-Absorbing Dyes for Imaging and Sensing.

Rhodamine dyes are excellent scaffolds for developing a broad range of fluorescent probes. A key property of rhodamines is their equilibrium between a colorless lactone and fluorescent zwitterion. Tuning the lactone-zwitterion equilibrium constant (KL-Z) can optimize dye properties for specific biological applications. Here, we use known and novel organic chemistry to prepare a comprehensive collection of rhodamine dyes to elucidate the structure-activity relationships that govern KL-Z. We discovered that the auxochrome substituent strongly affects the lactone-zwitterion equilibrium, providing a roadmap for the rational design of improved rhodamine dyes. Electron-donating auxochromes, such as julolidine, work in tandem with fluorinated pendant phenyl rings to yield bright, red-shifted fluorophores for live-cell single-particle tracking (SPT) and multicolor imaging. The N-aryl auxochrome combined with fluorination yields red-shifted Förster resonance energy transfer (FRET) quencher dyes useful for creating a new semisynthetic indicator to sense cAMP using fluorescence lifetime imaging microscopy (FLIM). Together, this work expands the synthetic methods available for rhodamine synthesis, generates new reagents for advanced fluorescence imaging experiments, and describes structure-activity relationships that will guide the design of future probes.

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 based measurements that are typically employed to measure aspartate levels. To address these issues, we have developed a 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 mass spectrometry 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.

Direct and Interactive Effects of Personality and Experiencing Changes in Relationships on Symptoms of Internalizing Psychopathology During the COVID-19 Pandemic.

Purpose: The present study examined the prospective direct and interactive effects of personality (neuroticism, extraversion) and experiencing changes in friendships during the pandemic on symptoms of stress, anxiety, and depression. Methods: A sample of patients (N = 77) at an outpatient treatment clinic who had received a diagnostic assessment in the 6 months prior to the COVID-19 lockdown was re-contacted during the pandemic (May-June 2020) and completed a survey assessing stressors and symptoms of internalizing psychopathology. Results: Neuroticism had main effects on anxiety, whereas experiencing changes in friendships had main effects on stress and depression. Extraversion did not have main effects on stress, depression, or anxiety. The relationship between experiencing changes in friendships and stress and anxiety was moderated by extraversion, such that the strength of the relationship between changes in friendships and stress and anxiety waned as the level of extraversion increased. Neuroticism was not a moderator of the association between changes in friendships and emotional disorder symptoms. Conclusion: These results suggest that higher levels of extraversion may protect against symptoms of stress reactivity and anxiety that are associated with COVID-related changes in friendships, while neuroticism may have limited prospective associations with symptoms during the pandemic. Supplementary Information: The online version of this article contains supplementary material available 10.1007/s10608-023-10364-x.

Developing Transdiagnostic Internalizing Disorder Prognostic Indices for Outpatient Cognitive Behavioral Therapy.

A growing literature is devoted to understanding and predicting heterogeneity in response to cognitive behavioral therapy (CBT), including using supervised machine learning to develop prognostic models that could be used to inform treatment planning. The current study developed CBT prognostic models using data from a broad dimensionally oriented pretreatment assessment (324 predictors) of 1,210 outpatients with internalizing psychopathology. Super learning was implemented to develop prognostic indices for three outcomes assessed at 12-month follow-up: principal diagnosis improvement (attained by 65.8% of patients), principal diagnosis remission (56.8%), and transdiagnostic full remission (14.3%). The models for principal diagnosis remission and transdiagnostic remission performed best (AUROCs = 0.71-0.73). Calibration was modest for all three models. Three-quarters (77.3%) of patients in the top tertile of the predicted probability distribution achieved principal diagnosis remission, compared to 35.0% in the bottom tertile. One-third (35.3%) of patients in the top two deciles of predicted probabilities for transdiagnostic complete remission achieved this outcome, compared to 2.7% in the bottom tertile. Key predictors included principal diagnosis severity, social anxiety diagnosis/severity, hopelessness, temperament, and global impairment. While additional work is needed to improve performance, integration of CBT prognostic models ultimately could lead to more effective and efficient treatment of patients with internalizing psychopathology.

Longitudinal associations of chronic health stress and COVID-19-related anxiety among outpatients with anxiety and mood disorders.

BACKGROUND: The 2019 coronavirus (COVID-19) pandemic led to elevated levels of psychological distress on a global scale. Given that individuals with pre-existing physical conditions are at risk for worse COVID-19 outcomes, those dealing with the stress of physical health problems (including knowing someone with health problems) may experience more severe distress during the pandemic. METHODS: Patients with emotional disorders who completed a diagnostic assessment in the 6 months prior to COVID-19 were surveyed in May-June 2020 on their emotional reactions to COVID-19 (N = 77). RESULTS: Multiple linear regression was used to test the hypothesis that chronic stress due to having and knowing others with physical health problems would predict COVID-related worries and behaviours, holding pre-COVID levels of depression, anxiety and worry about health constant. Chronic stress surrounding the health of others was significantly associated with experiencing more severe COVID-related worry and behaviours. In comparison, chronic stress due to one's own health problems had weak and non-significant associations with COVID-related worries and behaviours. CONCLUSIONS: Results indicate that outpatients who report stress about surrounding loved one's health are at risk for experiencing more severe distress during a health pandemic and thus, may benefit from targeted outreach, assessment and intervention.

A Sulfur-Controlled Approach to the Synthesis of Linerixibat.

Establishing the two stereocenters in the benzothiazepine ring of linerixibat (GSK2330672) has been a long-standing problem at GSK. Our solution rests on an episulfonium-controlled Ritter reaction followed by a sulfoxide-directed reduction. A rationale for both steps is based on a mixture of literature precedent and computational experiments. Transition state modeling suggests the sulfoxide-directed reduction proceeds through electronic repulsion between the lone pair of electrons on sulfur and the incoming borohydride anion.

Understanding the role of COVID-19-related workplace stress and institutional betrayal on mental health in nurses: Some heroes wear scrubs.

This study examined the association of three specific COVID-19-related workplace stressors (percentage of nursing work with COVID-positive [COVID+] patients, number of COVID-19-related patient deaths witnessed, and living separately from family for safety) and their associations with posttraumatic stress symptoms (PTSS) and symptoms of major depressive disorder (MDD) and generalized anxiety disorder (GAD) among 391 nurses (93.6% White, 93.4% utilize she/her pronouns). Cross-sectional data were collected via an online survey. Institutional betrayal (i.e., the perception that an institution failed to protect a member who depends on and trusts it) was examined as a moderator of these associations. Although institutional betrayal was not a significant moderator in the three individual models, it held small-to-medium-sized positive main effects with PTSS and symptoms of GAD and MDD in both the individual and combined models. In the individual models, the percentage of nursing work with COVID+ patients was significantly positively associated with all three mental health conditions, f2 = .019-.195, whereas it only showed a significant effect with PTSS in the combined model, f2 = .138. Living separately from family was significantly positively associated with PTSS and MDD symptoms in both the individual, f2 = .037 and .015, respectively, and combined models, f2 = .025 and .013, respectively. Number of patient deaths held a significant positive association with PTSS alone, f2 = .022, in the individual model only. The findings are discussed in light of ways in which health care settings can better support and prioritize mental health among nursing staff.

2,7-Diaminobenzopyrylium Dyes Are Live-Cell Mitochondrial Stains.

Small-molecule fluorescent stains enable the imaging of cellular structures without the need for genetic manipulation. Here, we introduce 2,7-diaminobenzopyrylium (DAB) dyes as live-cell mitochondrial stains excited with violet light. This amalgam of the coumarin and rhodamine fluorophore structures yields dyes with high photostability and tunable spectral properties.

Thermal and Catalytic Decomposition of 2-Hydroxyethylhydrazine and 2-Hydroxyethylhydrazinium Nitrate Ionic Liquid.

To develop chemical kinetics models for the combustion of ionic liquid-based monopropellants, identification of the elementary steps in the thermal and catalytic decomposition of components such as 2-hydroxyethylhydrazinium nitrate (HEHN) is needed but is currently not well understood. The first decomposition step in protic ionic liquids such as HEHN is typically the proton transfer from the cation to the anion, resulting in the formation of 2-hydroxyethylhydrazine (HEH) and HNO3. In the first part of this investigation, the high-temperature thermal decomposition of HEH is probed with flash pyrolysis (<1400 K) and vacuum ultraviolet (10.45 eV) photoionization time-of-flight mass spectrometry (VUV-PI-TOFMS). Next, the investigation into the thermal and catalytic decomposition of HEHN includes two mass spectrometric techniques: (1) tunable VUV-PI-TOFMS (7.4-15 eV) and (2) ambient ionization mass spectrometry utilizing both plasma and laser ionization techniques whereby HEHN is introduced onto a heated inert or iridium catalytic surface and the products are probed. The products can be identified by their masses, their ionization energies, and their collision-induced fragmentation patterns. Formation of product species indicates that catalytic surface recombination is an important reaction process in the decomposition mechanism of HEHN. The products and their possible elementary reaction mechanisms are discussed.

Facets of Emotion Regulation and Emotional Disorder Symptom Dimensions: Differential Associations and Incremental Validity in a Large Clinical Sample.

A growing literature suggests robust associations between dimensions of emotion regulation and emotional disorder psychopathology. However, limited research has investigated associations of emotion regulation dimensions across several emotional disorders (transdiagnostic associations), or the incremental validity of emotion regulation versus the higher-order construct of neuroticism. The current study used exploratory structural equation modeling and a large clinical sample (N = 1,138) to: (a) develop a multidimensional emotion regulation measurement model, (b) evaluate the differential associations between latent emotion regulation dimensions and five latent emotional disorder symptom dimensions (social anxiety, depression, agoraphobia/panic, obsessions/compulsions, generalized worry), and (c) determine the incremental contribution of emotion regulation in predicting symptom dimensions beyond neuroticism. The best-fitting measurement model of emotion regulation included four dimensions: Problematic Responses, Poor Recognition/Clarity, Negative Thinking, and Emotional Inhibition/Suppression. Although many zero-order associations between the four latent emotion regulation dimensions and five latent symptom dimensions were significant, few associations remained significant in a structural regression model that included neuroticism. Specifically, Negative Thinking and Problematic Responses incrementally predicted depression symptoms, while Emotional Inhibition/Suppression predicted both social anxiety and depression symptoms. Associations between neuroticism and the emotional disorder dimensions were similar regardless of whether the emotion regulation dimensions were held constant. These results suggest that self-reported emotion regulation dimensions are associated with the severity and expression of a range of emotional disorder symptoms, but that some emotion regulation dimensions have limited incremental validity after accounting for general emotional reactivity. Studies of emotion regulation should assess neuroticism as a key covariate.

A General Method to Improve Fluorophores Using Deuterated Auxochromes.

Fluorescence microscopy relies on dyes that absorb and then emit photons. In addition to fluorescence, fluorophores can undergo photochemical processes that decrease quantum yield or result in spectral shifts and irreversible photobleaching. Chemical strategies that suppress these undesirable pathways-thereby increasing the brightness and photostability of fluorophores-are crucial for advancing the frontier of bioimaging. Here, we describe a general method to improve small-molecule fluorophores by incorporating deuterium into the alkylamino auxochromes of rhodamines and other dyes. This strategy increases fluorescence quantum yield, inhibits photochemically induced spectral shifts, and slows irreparable photobleaching, yielding next-generation labels with improved performance in cellular imaging experiments.

Visual attention toward emotional stimuli: Anxiety symptoms correspond to distinct gaze patterns.

Decades of research have established a link between emotional disorders and attentional biases for emotional stimuli, but the relationship between symptom severity and visual attention is still not fully understood. Depression has been associated with increased attention towards dysphoric stimuli and decreased attention on positive stimuli ("negativity bias"), and some studies have also shown this trend in anxiety disorders. We examined eye fixation variables in 47 participants with emotional disorders completing an emotion recognition task. Results showed that depression severity was not associated with increased fixations on dysphoric stimuli, however, higher levels of generalized anxiety predicted increased fixations in the mouth region of sad and happy faces. Higher levels of social interaction anxiety predicted reduced fixations in the eye region of happy faces. While we did not replicate the negativity bias that has been shown in prior studies, our sample was highly comorbid, indicating the need to consider comorbidity, disorder severity, and the task itself when conducting research on visual attention in clinical samples. Additionally, more attention should be paid to the mouth region of emotional faces, as it may provide more specific information regarding the visual processing of emotions.

Developing and Validating Clinical Questionnaires.

Coinciding with the development and revision of conceptual models of psychopathology, there has been a proliferation in the number of self-report clinical questionnaires and studies evaluating their psychometric properties. Unfortunately, many clinical measures are constructed and evaluated using suboptimal methods. This review provides current guidelines for the conceptualization, development, and psychometric validation of clinical questionnaires using latent variable methods. A two-stage exploratory-confirmatory framework is provided. The exploratory stage includes item selection and revision, initial structural evaluation, and preliminary tests of concurrent validity (e.g., convergent and discriminant). The confirmatory stage involves replicating factor structure using a more restrictive model, identifying areas of model strain, conducting additional tests of concurrent and predictive validity, and evaluating measurement invariance. Recommendations are provided for (a) item generation, (b) how to use different types of exploratory and confirmatory factor models to determine structure, and (c) evaluating reliability and validity using a latent variable measurement model approach.