Fast and sensitive GCaMP calcium indicators for imaging neural populations.

Calcium imaging with protein-based indicators1,2 is widely used to follow neural activity in intact nervous systems, but current protein sensors report neural activity at timescales much slower than electrical signalling and are limited by trade-offs between sensitivity and kinetics. Here we used large-scale screening and structure-guided mutagenesis to develop and optimize several fast and sensitive GCaMP-type indicators3-8. The resulting 'jGCaMP8' sensors, based on the calcium-binding protein calmodulin and a fragment of endothelial nitric oxide synthase, have ultra-fast kinetics (half-rise times of 2 ms) and the highest sensitivity for neural activity reported for a protein-based calcium sensor. jGCaMP8 sensors will allow tracking of large populations of neurons on timescales relevant to neural computation.

Glutamate indicators with improved activation kinetics and localization for imaging synaptic transmission.

The fluorescent glutamate indicator iGluSnFR enables imaging of neurotransmission with genetic and molecular specificity. However, existing iGluSnFR variants exhibit low in vivo signal-to-noise ratios, saturating activation kinetics and exclusion from postsynaptic densities. Using a multiassay screen in bacteria, soluble protein and cultured neurons, we generated variants with improved signal-to-noise ratios and kinetics. We developed surface display constructs that improve iGluSnFR's nanoscopic localization to postsynapses. The resulting indicator iGluSnFR3 exhibits rapid nonsaturating activation kinetics and reports synaptic glutamate release with decreased saturation and increased specificity versus extrasynaptic signals in cultured neurons. Simultaneous imaging and electrophysiology at individual boutons in mouse visual cortex showed that iGluSnFR3 transients report single action potentials with high specificity. In vibrissal sensory cortex layer 4, we used iGluSnFR3 to characterize distinct patterns of touch-evoked feedforward input from thalamocortical boutons and both feedforward and recurrent input onto L4 cortical neuron dendritic spines.

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.

Eosinophilic Esophagitis: Etiology and Therapy.

Eosinophilic esophagitis (EoE) is a relatively recently identified but now frequently encountered antigen/immune-mediated disease which places significant burden on patients and the healthcare system. With its growing prevalence and recognition by healthcare providers in multiple disciplines, substantial progress has been made regarding the diagnostic criteria, clinical evaluation, tools for disease assessment, and immune pathways related to pathogenesis. Current treatment goals focus on the amelioration of inflammation and prevention of remodeling consequences using proton pump inhibitors, swallowed topical steroids, elimination diets, and esophageal dilation. Ongoing research holds promise for more efficacious and targeted therapies as well as a personalized approach to the care of patients with EoE.

A general method to optimize and functionalize red-shifted rhodamine dyes.

Expanding the palette of fluorescent dyes is vital to push the frontier of biological imaging. Although rhodamine dyes remain the premier type of small-molecule fluorophore owing to their bioavailability and brightness, variants excited with far-red or near-infrared light suffer from poor performance due to their propensity to adopt a lipophilic, nonfluorescent form. We report a framework for rationalizing rhodamine behavior in biological environments and a general chemical modification for rhodamines that optimizes long-wavelength variants and enables facile functionalization with different chemical groups. This strategy yields red-shifted 'Janelia Fluor' (JF) dyes useful for biological imaging experiments in cells and in vivo.

jYCaMP: an optimized calcium indicator for two-photon imaging at fiber laser wavelengths.

Femtosecond lasers at fixed wavelengths above 1,000 nm are powerful, stable and inexpensive, making them promising sources for two-photon microscopy. Biosensors optimized for these wavelengths are needed for both next-generation microscopes and affordable turn-key systems. Here we report jYCaMP1, a yellow variant of the calcium indicator jGCaMP7 that outperforms its parent in mice and flies at excitation wavelengths above 1,000 nm and enables improved two-color calcium imaging with red fluorescent protein-based indicators.

Comparison of pharmacokinetics, pharmacodynamics, safety, and immunogenicity of teriparatide biosimilar with EU- and US-approved teriparatide reference products in healthy men and postmenopausal women.

Biosimilar teriparatide (INTG-8) was tested in a healthy population of males and postmenopausal females to assess pharmacokinetic bioequivalence to originator teriparatide comparator products. Primary pharmacokinetic comparison confirmed bioequivalence. Pharmacodynamics, safety, and tolerability were comparable to the originator products. INTG-8 was therefore confirmed to be biosimilar to originator products. INTRODUCTION: The purpose of this present study was to demonstrate pharmacokinetic (PK) equivalence of a biosimilar teriparatide (INTG8) to EU- and US-approved teriparatide reference products in healthy men and postmenopausal women. Secondary objectives included comparison of the pharmacodynamics (PD), safety, and tolerability. METHODS: One hundred and five subjects randomly (1:1:1) received single subcutaneous 20 µg injection of teriparatide biosimilar, EU- and US-teriparatide on 3 consecutive days in this assessor-blind, three-period, single-dose, crossover study. Maximum serum concentration (Cmax), area under the curve (AUC) from time zero to t (AUC0-t), and AUC from time zero extrapolated to infinity (AUC0-∞) were primary PK parameters, analyzed by non-compartmental methods. The secondary PD endpoints were maximum observed effect (Emax), area under the effect curve (AUE) from time zero to the last measurable concentration (AUE0-t), and time to maximum observed effect (Tmax) for total serum calcium levels. Safety, tolerability, and immunogenicity were also evaluated. This study was registered with ctri.nic.in/ (CTRI/2020/10/028627) on 26 October 2020. RESULTS: Baseline demographics were similar across the three-treatment sequence groups. The 90% confidence intervals (CI) for the geometric mean ratios (test:reference) of Cmax, AUC0-t, and AUC0-∞ were within the predefined bioequivalence criterion of 80.00% to 125.00%, which demonstrated PK equivalence of teriparatide biosimilar to EU- and US-teriparatide for all primary endpoints. The PD comparability was demonstrated by similar serum calcium levels. Study treatments were generally well tolerated and showed no meaningful differences in safety or immunogenicity profiles. There were no deaths, or serious AEs were reported during this study. CONCLUSION: The study demonstrated PK bioequivalence of teriparatide biosimilar to the EU- and US-teriparatide reference products with comparable PD, safety, and immunogenicity profiles.

Ubiquilin-2 differentially regulates polyglutamine disease proteins.

Divergent protein context helps explain why polyglutamine expansion diseases differ clinically and pathologically. This heterogeneity may also extend to how polyglutamine disease proteins are handled by cellular pathways of proteostasis. Studies suggest, for example, that the ubiquitin-proteasome shuttle protein Ubiquilin-2 (UBQLN2) selectively interacts with specific polyglutamine disease proteins. Here we employ cellular models, primary neurons and mouse models to investigate the potential differential regulation by UBQLN2 of two polyglutamine disease proteins, huntingtin (HTT) and ataxin-3 (ATXN3). In cells, overexpressed UBQLN2 selectively lowered levels of full-length pathogenic HTT but not of HTT exon 1 fragment or full-length ATXN3. Consistent with these results, UBQLN2 specifically reduced accumulation of aggregated mutant HTT but not mutant ATXN3 in mouse models of Huntington's disease (HD) and spinocerebellar ataxia type 3 (SCA3), respectively. Normally a cytoplasmic protein, UBQLN2 translocated to the nuclei of neurons in HD mice but not in SCA3 mice. Remarkably, instead of reducing the accumulation of nuclear mutant ATXN3, UBQLN2 induced an accumulation of cytoplasmic ATXN3 aggregates in neurons of SCA3 mice. Together these results reveal a selective action of UBQLN2 toward polyglutamine disease proteins, indicating that polyglutamine expansion alone is insufficient to promote UBQLN2-mediated clearance of this class of disease proteins. Additional factors, including nuclear translocation of UBQLN2, may facilitate its action to clear intranuclear, aggregated disease proteins like HTT.

High-performance calcium sensors for imaging activity in neuronal populations and microcompartments.

Calcium imaging with genetically encoded calcium indicators (GECIs) is routinely used to measure neural activity in intact nervous systems. GECIs are frequently used in one of two different modes: to track activity in large populations of neuronal cell bodies, or to follow dynamics in subcellular compartments such as axons, dendrites and individual synaptic compartments. Despite major advances, calcium imaging is still limited by the biophysical properties of existing GECIs, including affinity, signal-to-noise ratio, rise and decay kinetics and dynamic range. Using structure-guided mutagenesis and neuron-based screening, we optimized the green fluorescent protein-based GECI GCaMP6 for different modes of in vivo imaging. The resulting jGCaMP7 sensors provide improved detection of individual spikes (jGCaMP7s,f), imaging in neurites and neuropil (jGCaMP7b), and may allow tracking larger populations of neurons using two-photon (jGCaMP7s,f) or wide-field (jGCaMP7c) imaging.

Clinical comparison of V122I genotypic variant of transthyretin amyloid cardiomyopathy with wild-type and other hereditary variants: a systematic review.

V122I genotype variant (pV142I) is the most common hereditary transthyretin amyloidosis (hATTR) in the USA, with 3-3.5% of African-Americans being the carriers of this mutation. We aimed to compare baseline clinical features, cardiac parameters, and mortality in V122I-ATTR with the wild-type ATTR and other hATTR subtypes. We systematically searched PubMed/Medline and Google Scholar databases to identify relevant studies from inception to 10th September, 2020 reporting phenotypic, echocardiographic, and/or laboratory parameters in patients with hereditary and wild types of cardiac amyloidoses. A total of 2843 patients from 7 individual studies with 67-100% males and an overall follow-up duration of 51.6 ± 30.4 months were identified. The mean age of diagnosis among wild-type ATTR patients was 77 years, followed by 71.2 and 65 years in V122I and T60A group patients, respectively. V122I patients were mostly black, had a poor quality of life, and highest mortality risk compared with other subtypes. Merely, the presence of V122I mutation was identified as an independent predictor of mortality. V30M subtype correlated with the least severe cardiac disease and a median survival duration comparable with T60A subtype. V122I ATTR is an aggressive disease, prevalent in African-Americans, and is associated with a greater morbidity and mortality, which is partly attributed to its misdiagnosis and/or late diagnosis. Current advances in non-invasive studies to diagnose hATTR coupled with concurrent drug therapies have improved quality of life and provide a survival benefit to these patients.

Efficacy of remote physiological monitoring-guided care for chronic heart failure: an updated meta-analysis.

Previous studies have reported contradictory findings on the utility of remote physiological monitoring (RPM)-guided management of patients with chronic heart failure (HF). Multiple databases were searched for studies that evaluated the clinical efficacy of RPM-guided management versus standard of care (SOC) for HF patients. The primary outcome was HF-related hospitalization (HFH). The secondary outcomes were all-cause mortality, cardiovascular-related (CV) mortality, and emergency department (ED) visits. Pooled relative risk (RR) and corresponding 95% confidence intervals (CIs) were calculated and combined using a random-effects model. A total of 16 randomized controlled trials, including 8679 HF patients (4574 managed with RPM-guided therapy vs. 4105 managed with SOC), were included in the final analysis. The average follow-up period was 15.2 months. There was no significant difference in HFH rate between the two groups (RR: 0.94; 95% CI: 0.84-1.07; P = 0.36). Similarly, there were no significant differences in CV mortality (RR 0.86, 95% CI 0.73-1.02, P = 0.08) or in ED visits (RR 0.80, 95% CI 0.59-1.08, P = 0.14). However, RPM-guided therapy was associated with a borderline statistically significant reduction in all-cause mortality (RR: 0.88; 95% CI: 0.78-1.00; P = 0.05). Subgroup analysis based on the strategy of RPM showed that both hemodynamic and arrhythmia telemonitoring-guided management can reduce the risk of HFH (RR: 0.79; 95% CI: 0.64-0.97; P = 0.02) and (RR: 0.79; 95% CI: 0.67-0.94; P = 0.008) respectively. Our study demonstrated that RPM-guided diuretic therapy of HF patients did not reduce the risk of HFH but can improve survival. Hemodynamic and arrhythmia telemonitoring-guided management could reduce the risk of HF-related hospitalizations.

Neuroenhancement of surgeons during robotic suturing.

BACKGROUND: The initial phases of robotic surgical skills acquisition are associated with poor technical performance, such as low knot-tensile strength (KTS). Transcranial direct-current stimulation (tDCS) can improve force and accuracy in motor tasks but research in surgery is limited to open and laparoscopic tasks in students. More recently, robotic surgery has gained traction and is now the most common approach for certain procedures (e.g. prostatectomy). Early-phase robotic suturing performance is dependent on prefrontal cortex (PFC) activation, and this study aimed to determine whether performance can be improved with prefrontal tDCS. METHODS: Fifteen surgical residents were randomized to either active then sham tDCS or sham then active tDCS, in two counterbalanced sessions in a double-blind crossover study. Within each session, participants performed a robotic suturing task repeated in three blocks: pre-, intra- and post-tDCS. During the intra-tDCS block, participants were randomized to either active tDCS (2 mA for 15 min) to the PFC or sham tDCS. Primary outcome measures of technical quality included KTS and error scores. RESULTS: Significantly faster completion times were observed longitudinally, regardless of active (p < 0.001) or sham stimulation (p < 0.001). KTS was greater following active compared to sham stimulation (median: active = 44.35 N vs. sham = 27.12 N, p < 0.001). A significant reduction in error scores from "pre-" to "post-" (p = 0.029) were only observed in the active group. CONCLUSION: tDCS could reduce error and enhance KTS during robotic suturing and warrants further exploration as an adjunct to robotic surgical training.

Case report of superficial femoral artery and popliteal artery aneurysm repair using brachial vein.

BACKGROUND: Several veins have been well-recognized as acceptable conduits for infrainguinal bypass surgery when the ipsilateral greater saphenous vein is unavailable. However, there is a paucity of literature describing the brachial vein as an adequate alternative. In the absence of other viable autogenous conduits, we describe the use of a brachial vein as a successful alternative for lower extremity revascularization. METHODS: A 70-year-old man presented with a chief complaint of right calf pain. Duplex ultrasound imaging of his right lower extremity revealed right-sided 2.5 cm acutely thrombosed superficial femoral artery and popliteal artery aneurysms. The patient underwent a suction thrombectomy with tissue plasminogen activator using the Power Pulse feature and Solent catheter from the AngioJet® (Boston-Scientific, Marlborough, MA) system. To repair the thrombosed aneurysms, an open bypass was planned. Due to lack of viable alternative traditionally used venous conduits, a bypass was created using the patient's brachial vein. RESULTS: A bypass was created from the superficial femoral artery to the P2 segment of the popliteal artery using a non-reversed brachial vein with ligation of the side branches of the superficial femoral artery and popliteal artery aneurysm from within the sac lumen. Completion angiogram revealed runoff through the anterior tibial artery only. Follow-up imaging at three months demonstrated a patent brachial bypass. CONCLUSION: Brachial veins can be safely used as viable venous conduits for lower extremity bypass surgery and should therefore be considered as an alternative when more commonly used veins are unsuitable or unavailable. However, more research is needed to determine the potential opportunities and challenges this alternative may present.

Corruption and disasters in the built environment: a literature review.

This paper presents the findings of a review of academic literature concerning the degree to which corruption worsens disasters triggered by natural hazards in the built environment. The research employed a 'systematic literature review' methodology to analyse leading academic databases, resulting in a detailed analysis of 59 peer-reviewed, published papers. It found that while much of the literature focuses on earthquakes (relating to building and infrastructure collapse), the quality of governance, and the drivers of corruption, there is presently very limited scholarship on the general scope, reach, and scale with respect to how disasters are worsened by corruption. It is notable that the Sendai Framework for Disaster Risk Reduction 2015-2030 and a number of other high-level reports fail to mention corruption. The paper argues that this serious gap in understanding and expressing how corruption increases vulnerability in the built environment within disaster studies perversely supports the furtherance of corruption in worsening disasters.

Exploring COVID-19 vaccine hesitancy at a rural historically black college and university.

OBJECTIVES: Minorities have been disproportionately affected by the coronavirus disease 2019 (COVID-19) yet have the lowest COVID-19 vaccine rate. Vaccine hesitancy has been reported at higher rates in African Americans (AAs) and young adults. This study aimed to assess COVID-19 vaccine hesitancy, determine the rationale for receiving or declining the COVID-19 vaccine, and propose strategies to address confidence in faculty, staff, and students at a rural historically black college and university (HBCU). METHODS: A study was conducted using an electronic survey administered to a convenient sample of 210 faculty, students, and staff at the University of Maryland Eastern Shore, an HBCU in a rural community. RESULTS: Most participants were 18 to 24 years old (69%), college students (73.89%), AA (70%), and identified as a woman (70%). Notably, 87% of participants were nonhesitant (received one dose or intended to be vaccinated). Approximately 54% had already received at least one dose of a COVID-19 vaccine. Only 13% of participants were hesitant and did not plan to receive the COVID-19 vaccine. The most common rationale for vaccine hesitancy was mistrust of the health care system or government toward AAs. CONCLUSION: The results show that vaccine hesitancy was low in the predominantly young-adult AA population at a rural HBCU. However, opportunities exist for pharmacists and other accessible health care professionals to contribute to efforts aimed at decreasing vaccine hesitancy and improving vaccine confidence.

Fat grafting rescues radiation-induced joint contracture.

The aim of this study was to explore the therapeutic effects of fat grafting on radiation-induced hind limb contracture. Radiation therapy (RT) is used to palliate and/or cure a range of malignancies but causes inevitable and progressive fibrosis of surrounding soft tissue. Pathological fibrosis may lead to painful contractures which limit movement and negatively impact quality of life. Fat grafting is able to reduce and/or reverse radiation-induced soft tissue fibrosis. We explored whether fat grafting could improve extensibility in irradiated and contracted hind limbs of mice. Right hind limbs of female 60-day-old CD-1 nude mice were irradiated. Chronic skin fibrosis and limb contracture developed. After 4 weeks, irradiated hind limbs were then injected with (a) fat enriched with stromal vascular cells (SVCs), (b) fat only, (c) saline, or (d) nothing (n = 10/group). Limb extension was measured at baseline and every 2 weeks for 12 weeks. Hind limb skin then underwent histological analysis and biomechanical strength testing. Irradiation significantly reduced limb extension but was progressively rescued by fat grafting. Fat grafting also reduced skin stiffness and reversed the radiation-induced histological changes in the skin. The greatest benefits were found in mice injected with fat enriched with SVCs. Hind limb radiation induces contracture in our mouse model which can be improved with fat grafting. Enriching fat with SVCs enhances these beneficial effects. These results underscore an attractive approach to address challenging soft tissue fibrosis in patients following RT.

Anxiety and depression affect performance on the symbol digit modalities test over time in MS and other immune disorders.

BACKGROUND: Longitudinal studies assessing depression and anxiety effects on cognition in multiple sclerosis (MS) are limited. OBJECTIVE: We tested whether within-person fluctuations in symptoms of depression or anxiety over time affect cognition in persons with MS, inflammatory bowel disease (IBD), rheumatoid arthritis (RA), and a lifetime history of depression/anxiety disorders (DEP/ANX) but without an immune-mediated inflammatory diseases (IMID). METHODS: We followed participants (MS: 255, IBD: 247, RA: 154, and DEP/ANX: 306) for 3 years. Annually, they completed the hospital anxiety and depression scale (HADS) and cognitive tests including the symbol digit modalities test (SDMT). We evaluated associations of elevated symptoms (scores ⩾ 11) of anxiety (HADS-A) and depression (HADS-D) with SDMT z-scores using multivariable linear models-estimating between-person and within-person effects. RESULTS: Participants with MS performed worse on the SDMT than participants in the DEP/ANX cohort (ß = -0.68; 95% CI: -0.88, -0.48). Participants with elevated HADS-A scores performed worse on the SDMT than those without elevated scores (ß = -0.43; 95% CI: -0.65, -0.21), particularly those with RA. Time-varying within-person elevations in depressive symptoms were associated with worse SDMT performance (ß = -0.12; 95% CI: -0.21, -0.021). CONCLUSIONS: Across persons, elevated symptoms of anxiety adversely affected information processing. Elevated symptoms of depression within-persons over time were associated with declines in information processing speed.

Endoxifen: A new, protein kinase C inhibitor to treat acute and mixed mania associated with bipolar I disorder.

OBJECTIVES: Endoxifen is a protein kinase C inhibitor. The objective of the present phase III study was to demonstrate the safety and efficacy of endoxifen in treating bipolar I disorder (BPD I) patients. METHODS: A multicenter, double-blind, active-controlled study was conducted using a daily dose of 8 mg endoxifen compared to 1000 mg divalproex, the current standard treatment, in patients with BPD I acute manic episodes with/without mixed features. The primary endpoint of our study was the mean change in total Young Mania Rating Scale (YMRS) score at day 21. RESULTS: Endoxifen (n = 116) significantly (p < 0.0001) reduced total YMRS score (from 33.1 to 17.8. A significant (p < 0.001) improvement in Montgomery-Åsberg Depression Rating Scale (MADRS) score was observed for endoxifen (4.8 to 2.5). Early time to remission of the disease was observed with endoxifen compared to divalproex. None of the patients required rescue medication and there was no drug-associated withdrawals. Changes in Clinical Global Impressions-Bipolar Disorder and Clinical Global Impression-Severity of Illness scores showed that treatment with endoxifen was well-tolerated. CONCLUSIONS: Endoxifen at a low daily dose of 8 mg was as efficacious and safe in patients with BPD I acute manic episodes with/without mixed features.

Malignant Transformation of Nonhealing Ulcer-Basal Cell Carcinoma.

We discuss the rare case of a 72-year-old female with a history of a nonhealing lower extremity ulcer that was biopsied, revealing malignant transformation to basal cell carcinoma (BCC). Although BCC is the most common malignancy worldwide, malignant transformation of nonhealing wounds is more often associated with squamous cell carcinoma. Current literature estimates the rate of BCC arising from venous stasis ulcer to occur between 1.5 and 15%. When diagnosed early, BCC can have cure rates of up to 95%. However, metastatic BCC has a median survival of roughly 8 months. We believe it is important to raise awareness of this rare, but often curable, clinical diagnosis to improve long-term outcomes.

Mutant UBQLN2 promotes toxicity by modulating intrinsic self-assembly.

UBQLN2 is one of a family of proteins implicated in ubiquitin-dependent protein quality control and integrally tied to human neurodegenerative disease. Whereas wild-type UBQLN2 accumulates in intraneuronal deposits in several common age-related neurodegenerative diseases, mutations in the gene encoding this protein result in X-linked amyotrophic lateral sclerosis/frontotemporal dementia associated with TDP43 accumulation. Using in vitro protein analysis, longitudinal fluorescence imaging and cellular, neuronal, and transgenic mouse models, we establish that UBQLN2 is intrinsically prone to self-assemble into higher-order complexes, including liquid-like droplets and amyloid aggregates. UBQLN2 self-assembly and solubility are reciprocally modulated by the protein's ubiquitin-like and ubiquitin-associated domains. Moreover, a pathogenic UBQLN2 missense mutation impairs droplet dynamics and favors amyloid-like aggregation associated with neurotoxicity. These data emphasize the critical link between UBQLN2's role in ubiquitin-dependent pathways and its propensity to self-assemble and aggregate in neurodegenerative diseases.