Engineering luminopsins with improved coupling efficiencies.

Significance: Luminopsins (LMOs) are bioluminescent-optogenetic tools with a luciferase fused to an opsin that allow bimodal control of neurons by providing both optogenetic and chemogenetic access. Determining which design features contribute to the efficacy of LMOs will be beneficial for further improving LMOs for use in research. Aim: We investigated the relative impact of luciferase brightness, opsin sensitivity, pairing of emission and absorption wavelength, and arrangement of moieties on the function of LMOs. Approach: We quantified efficacy of LMOs through whole cell patch clamp recordings in HEK293 cells by determining coupling efficiency, the percentage of maximum LED induced photocurrent achieved with bioluminescent activation of an opsin. We confirmed key results by multielectrode array recordings in primary neurons. Results: Luciferase brightness and opsin sensitivity had the most impact on the efficacy of LMOs, and N-terminal fusions of luciferases to opsins performed better than C-terminal and multi-terminal fusions. Precise paring of luciferase emission and opsin absorption spectra appeared to be less critical. Conclusions: Whole cell patch clamp recordings allowed us to quantify the impact of different characteristics of LMOs on their function. Our results suggest that coupling brighter bioluminescent sources to more sensitive opsins will improve LMO function. As bioluminescent activation of opsins is most likely based on Förster resonance energy transfer, the most effective strategy for improving LMOs further will be molecular evolution of luciferase-fluorescent protein-opsin fusions.

Engineering luminopsins with improved coupling efficiencies.

Significance: Luminopsins (LMOs) are bioluminescent-optogenetic tools with a luciferase fused to an opsin that allow bimodal control of neurons by providing both optogenetic and chemogenetic access. Determining which design features contribute to the efficacy of LMOs will be beneficial for further improving LMOs for use in research. Aim: We investigated the relative impact of luciferase brightness, opsin sensitivity, pairing of emission and absorption wavelength, and arrangement of moieties on the function of LMOs. Approach: We quantified efficacy of LMOs through whole cell patch clamp recordings in HEK293 cells by determining coupling efficiency, the percentage of maximum LED induced photocurrent achieved with bioluminescent activation of an opsin. We confirmed key results by multielectrode array (MEAs) recordings in primary neurons. Results: Luciferase brightness and opsin sensitivity had the most impact on the efficacy of LMOs, and N-terminal fusions of luciferases to opsins performed better than C-terminal and multi-terminal fusions. Precise paring of luciferase emission and opsin absorption spectra appeared to be less critical. Conclusions: Whole cell patch clamp recordings allowed us to quantify the impact of different characteristics of LMOs on their function. Our results suggest that coupling brighter bioluminescent sources to more sensitive opsins will improve LMO function. As bioluminescent activation of opsins is most likely based on Förster resonance energy transfer (FRET), the most effective strategy for improving LMOs further will be molecular evolution of luciferase-fluorescent protein-opsin fusions.

Climate change alters shellfish reef communities: A temperate mesocosm experiment.

Climate change is expected to cause significant changes to rocky shore diversity. This study used outdoor mesocosms to test the predictions that warming and ocean acidification will alter the responses of native Trichomya hirsuta and introduced Mytilus galloprovincialis mussels, and their associated communities of infauna. Experiments consisted of orthogonal combinations of temperature (ambient 22 °C or elevated 25 °C), pCO2 (ambient 400 µatm or elevated 1000 µatm), mussel species (T. hirsuta or M. galloprovincialis), and mussel configuration (native, introduced, or both), with n = 3 replicates. Elevated pCO2 reduced the growth of T. hirsuta but not that of M. galloprovincialis, and warming and pCO2 influenced the infauna that colonised both species of mussels. There was a reduction in infaunal molluscs and an increase in polychaetes; there was, however, no effect on crustaceans. Results from this study suggest that climate-driven changes from one mussel species to another can significantly influence infaunal communities.

Alienation and beauty in medical photography.

Though photography was initially touted to overtake medical illustration as a more objective medium, today photographs are underused in medical texts. The concern with aesthetics and the relationship between the body and the patient combined to shape the future of medical photography, and in some ways medicine itself. Closely examining two cases - Duchenne's 'Mécanisme de la Physionomie Humaine' (1856), and Grant's 'An Atlas of Anatomy' (1962) - I consider the role of alienation and beauty in medical photography and the evocative questions each raised in medical history. This is adapted from a talk given at RCPE.

Relationship of anxiety with joint pain and its management: A population survey.

OBJECTIVES: The aims of the present study was to examine the associations between the severity of pain and anxiety in a community population reporting joint pain, and to investigate the management of joint pain in the presence of comorbid anxiety. METHODS: A population survey was carried out of people aged ≥45 years, registered with eight general practices in Cheshire, Shropshire and Staffordshire, UK. Respondents were asked to report pain intensity in their hands, hips, knees and feet (on a numerical rating scale), anxiety symptoms (Generalized Anxiety Disorder seven-item [GAD-7] scale) and guideline-recommended treatments used to manage pain. Clinical anxiety was defined by a GAD-7 score of 10 or more. RESULTS: A total of 11,222 respondents with joint pain were included in the analysis, with 1,802 (16.1%) reporting clinical anxiety. Respondents reporting more severe pain were more likely to report clinical anxiety (severe versus mild pain, odds ratio [OR] 5.36, 95% confidence interval [CI] 4.56 to 6.31). The number of pain sites was also positively associated with clinical anxiety (four versus one site; OR 3.64, 95% CI 3.09 to 4.30). Those with clinical anxiety were less likely to undertake general fitness exercises (OR 0.60, 95% CI 0.52 to 0.70), but more likely to diet (OR 1.43, 95% CI 1.21 to 1.69), use walking aids (OR 1.53, 95% CI 1.32 to 1.77) and assistive devices (OR 1.24, 95% CI 1.04 to 1.49), and more likely to use opioids (OR 1.34, 95% CI 1.18 to1.52). CONCLUSIONS: Anxiety is common among patients presenting to primary care with joint pain. Patients with anxiety are likely to manage their joint pain differently to those without. Case-finding to identify and treat anxiety would be appropriate in this population, with caution about opioid prescribing and consideration of exercise as an intervention.

Chest pain and shortness of breath in cardiovascular disease: a prospective cohort study in UK primary care.

OBJECTIVE: To determine characteristics associated with monthly chest pain and shortness of breath (SoB) during activity in cardiovascular disease (CVD) and trajectories of these symptoms over 10 months. STUDY DESIGN AND SETTING: Baseline questionnaire was sent to patients aged ≥40 years from 10 UK general practices. Responders were sent monthly questionnaires for 10 months. For patients with CVD (ischaemic heart disease and heart failure), the association of sociodemographic characteristics, pain elsewhere and anxiety and depression with monthly reports of chest pain and SoB during activity were determined using multilevel, multinomial logistic regression. Common symptom trajectories were determined using dual trajectory latent class growth analysis. RESULTS: 661 patients with CVD completed at least 5 monthly questionnaires. Multiple other pain sites (relative risk ratio: 4.03; 95% CI 1.64 to 9.91) and anxiety or depression (relative risk ratio: 3.31; 95% CI 1.89 to 5.79) were associated with reporting weekly chest pain. Anxiety or depression (relative risk ratio: 4.10; 95% CI 2.72 to 6.17), obesity (relative risk ratio: 2.53; 95% CI 1.49 to 4.30), older age (80+: relative risk ratio: 2.51; 95% CI 1.19 to 5.26), increasing number of pain sites (4+: relative risk ratio: 4.64; 95% CI 2.35 to 9.18) and female gender (relative risk ratio: 1.81; 95% CI 1.20 to 2.75) were associated with reporting weekly SoB. Eight symptom trajectories were identified, with SoB symptoms more common than chest pain. CONCLUSIONS: Potentially modifiable characteristics are associated with the experience of chest pain and SoB. Identified symptom trajectories may facilitate tailored care to improve outcomes in patients with CVD.

Does metformin protect against osteoarthritis? An electronic health record cohort study.

BACKGROUND: Obesity is a major risk factor for osteoarthritis (OA) whilst there is some evidence that diabetes also increases risk. Metformin is a common oral treatment for those with diabetes. OBJECTIVE: The aim is to investigate whether metformin reduces the risk of OA. METHODS: This was a cohort study set within the Consultations in Primary Care Archive, with 3217 patients with type 2 diabetes. Patients at 13 general practices with recorded type 2 diabetes in the baseline period (2002-2003) and no prior record of OA were identified. Exposure was a prescription for metformin. Outcome was an OA record during follow up. Cox proportional hazard models with Gamma frailty term were fitted: adjusted for age, gender, deprivation, and comorbidity. RESULTS: There was no association between prescribed metformin treatment at baseline and OA (adjusted HR: 1.02, 95% CI: 0.91, 1.15). A similar non- significant association was found when allowing exposure status of prescription of metformin to vary over time.

Retrospective Chart Analysis of Concussion Discharge Instructions in the Emergency Department.

BACKGROUND: Recognition and diagnosis of concussion is increasing, but current research shows these patients are discharged from the emergency department (ED) with a wide variability of recommendations and instructions. OBJECTIVE: To assess the adequacy of documentation of discharge instructions given to patients discharged from the ED with concussions. METHODS: This was a quality-improvement study conducted at a University-based Level I trauma center. A chart review was performed on all patients discharged with closed head injury or concussion over a 1-year period. Chi-squared measures of association and Fisher's exact test were used to compare the proportion of patients receiving discharge instructions (printed or documented in the chart as discussed by the physician). Multivariable logistic regression was used to assess the relationship between whether the concussion was sport-related in relation to our primary outcomes. RESULTS: There were 1855 charts that met inclusion criteria. The physician documented discussion of concussion discharge instructions in 41% (95% confidence interval [CI] 39.2-43.7) and printed instructions were given in 71% (95% CI 69.1-73.2). Physicians documented discussion of instructions more often for sport-related vs. non-sport-related concussion (58% vs. 39%, p = 0.008) with an odds ratio (OR) of 2.1 (95% CI 1.6-2.8). Discharge instructions were given more often for sport-related injuries than those without sport-related injuries (85% vs. 69%, p = 0.047), with an OR of 2.2 (95% CI 1.6-3.1). Children were more likely to have had physician-documented discussion of instructions (56%, 95% CI 52.3-59.1 vs. 31%, 95% CI 28.0-33.6), printed discharge instructions (86%, 95% CI 83.2-88.1 vs. 61%, 95% CI 57.6-63.4), and return-to-play precautions given (11.2%, 95% CI 9.2-13.6 vs. 4.5%, 95% CI 3.4-5.9) compared with adults. CONCLUSIONS: Documentation of discharge instructions given to ED patients with concussions was inadequate, overall.

Deciphering the molecular mechanism responsible for GCaMP6m's Ca2+-dependent change in fluorescence.

The goal of this work is to determine how GCaMP6m's fluorescence is altered in response to Ca2+-binding. Our detailed spectroscopic study reveals the simplest explanation for how GCaMP6m changes fluorescence in response to Ca2+ is with a four-state model, in which a Ca2+-dependent change of the chromophore protonation state, due to a shift in pKa, is the predominant factor. The pKa shift is quantitatively explained by a change in electrostatic potential around the chromophore due to the conformational changes that occur in the protein when calmodulin binds Ca2+ and interacts with the M13 peptide. The absolute pKa values for the Ca2+-free and Ca2+-saturated states of GCaMP6m are critical to its high signal-to-noise ratio. This mechanism has important implications for further improvements to GCaMP6m and potentially for other similarly designed biosensors.

Emergency department management of patients with rib fracture based on a clinical practice guideline.

BACKGROUND: Clinical practice guidelines (CPGs) have the ability to increase efficiency and standardize care. A CPG based on forced vital capacity (FVC) for rib fractures was developed as a tool for triage of these patients. The objectives of this study were to assess the efficacy and compliance of physicians with this rib fracture CPG. METHODS: Patients >18 that were discharged from an urban level 2 trauma center emergency department (ED) between the dates of January 1, 2014, to December 31, 2016, were eligible for the study. Demographics, mechanism, outcomes and FVC were abstracted by review of the electronic medical record. Compliance with the CPG was examined, and comparisons were made between patients successfully discharged and patients who returned. RESULTS: 455 patients met were identified during the study period. 233 were eligible after exclusions. 64% of the cohort was male with median age of 53 years. Falls were the most common mechanism (59.6%). The median number of rib fractures was 2 and median FVC 2500 mL. 28 (12.0%) of the 233 returned to the ED after discharge. The groups were well matched with no significant differences. The most common reason for return was pain (95%). Adjusted analysis showed that increasing age (adjusted OR (AOR) 0.968) and FVC (AOR 0.999) were independent predictors. Adherence with the CPG was good for hemothorax/pneumothorax and bilateral fractures (96%), but lagged with the number of fractures (74%). CONCLUSIONS: This study confirms that the rib fracture CPG is safe and an FVC of 1500 mL is a safe criterion for discharging patients with rib fractures. Interestingly, it appears that older age is protective. More work needs to be done on effective pain control to decrease return to ED visits using this CPG. LEVEL OF EVIDENCE: IV. TYPE OF STUDY: Therapeutic.

Patterns of routine primary care for osteoarthritis in the UK: a cross-sectional electronic health records study.

OBJECTIVE: To determine common patterns of recorded primary care for osteoarthritis (OA), and patient and provider characteristics associated with the quality of recorded care. DESIGN: An observational study nested within a cluster-randomised controlled trial. SETTING: Eight UK general practices who were part of the Management of Osteoarthritis in Consultations study. PARTICIPANTS: Patients recorded as consulting within the eight general practices for clinical OA. PRIMARY OUTCOMES: Achievement of seven quality indicators of care (pain/function assessment, information provision, exercise/weight advice, analgesics, physiotherapy), recorded through an electronic template or routinely recorded in the electronic healthcare records, was identified for patients aged ≥45 years consulting over a 6-month period with clinical OA. Latent class analysis was used to cluster patients based on care received. Clusters were compared on patient and clinician-level characteristics. RESULTS: 1724 patients (median by practice 183) consulted with clinical OA. Common patterns of recorded quality care were: cluster 1 (38%, High) received most quality indicators of care; cluster 2 (11%, Moderate) had pain and function assessment, and received or were considered for other indicators; cluster 3 (17%, Low) had pain and function assessment, and received or were considered for paracetamol or topical non-steroidal anti-inflammatory drugs; cluster 4 (35%, None) had no recorded quality indicators. Patients with higher levels of recorded care consulted a clinician who saw more patients with OA, consulted multiple times and had less morbidity. Those in the High cluster were more likely to have recorded diagnosed OA and have knee/hip OA. CONCLUSIONS: Patterns of recorded care for OA fell into four natural clusters. Appropriate delivery of core interventions and relatively safe pharmacological options for OA are still not consistently recorded as provided in primary care. Further research to understand clinical recording behaviours and determine potential barriers to quality care alongside effective training for clinicians is needed. TRIAL REGISTRATION NUMBER: ISRCTN06984617; Results.

Two-photon directed evolution of green fluorescent proteins.

Directed evolution has been used extensively to improve the properties of a variety of fluorescent proteins (FPs). Evolutionary strategies, however, have not yet been used to improve the two-photon absorption (2PA) properties of a fluorescent protein, properties that are important for two-photon imaging in living tissues, including the brain. Here we demonstrate a technique for quantitatively screening the two-photon excited fluorescence (2PEF) efficiency and 2PA cross section of tens of thousands of mutant FPs expressed in E. coli colonies. We use this procedure to move EGFP through three rounds of two-photon directed evolution leading to new variants showing up to a 50% enhancement in peak 2PA cross section and brightness within the near-IR tissue transparency wavelength range.

Multiphoton photochemistry of red fluorescent proteins in solution and live cells.

Genetically encoded fluorescent proteins (FPs), and biosensors based on them, provide new insights into how living cells and tissues function. Ultimately, the goal of the bioimaging community is to use these probes deep in tissues and even in entire organisms, and this will require two-photon laser scanning microscopy (TPLSM), with its greater tissue penetration, lower autofluorescence background, and minimum photodamage in the out-of-focus volume. However, the extremely high instantaneous light intensities of femtosecond pulses in the focal volume dramatically increase the probability of further stepwise resonant photon absorption, leading to highly excited, ionizable and reactive states, often resulting in fast bleaching of fluorescent proteins in TPLSM. Here, we show that the femtosecond multiphoton excitation of red FPs (DsRed2 and mFruits), both in solution and live cells, results in a chain of consecutive, partially reversible reactions, with individual rates driven by a high-order (3-5 photon) absorption. The first step of this process corresponds to a three- (DsRed2) or four-photon (mFruits) induced fast isomerization of the chromophore, yielding intermediate fluorescent forms, which then subsequently transform into nonfluorescent products. Our experimental data and model calculations are consistent with a mechanism in which ultrafast electron transfer from the chromophore to a neighboring positively charged amino acid residue triggers the first step of multiphoton chromophore transformations in DsRed2 and mFruits, consisting of decarboxylation of a nearby deprotonated glutamic acid residue.

A fluorescent, genetically-encoded voltage probe capable of resolving action potentials.

There is a pressing need in neuroscience for genetically-encoded, fluorescent voltage probes that can be targeted to specific neurons and circuits to allow study of neural activity using fluorescent imaging. We created 90 constructs in which the voltage sensing portion (S1-S4) of Ciona intestinalis voltage sensitive phosphatase (CiVSP) was fused to circularly permuted eGFP. This led to ElectricPk, a probe that is an order of magnitude faster (taus ~1-2 ms) than any currently published fluorescent protein-based voltage probe. ElectricPk can follow the rise and fall of neuronal action potentials with a modest decrease in fluorescence intensity (~0.7% ΔF/F). The probe has a nearly linear fluorescence/membrane potential response to both hyperpolarizing and depolarizing steps. This is the first probe based on CiVSP that captures the rapid movements of the voltage sensor, suggesting that voltage probes designed with circularly permuted fluorescent proteins may have some advantages.