Endoplasmic Reticulum Calcium Mediates Drosophila Wing Development.

Background: The temporal dynamics of morphogen presentation impacts transcriptional responses and tissue patterning. However, the mechanisms controlling morphogen release are far from clear. We found that inwardly rectifying potassium (Irk) channels regulate endogenous transient increases in intracellular calcium and bone morphogenetic protein (BMP/Dpp) release for Drosophila wing development. Inhibition of Irk channels reduces BMP/Dpp signaling, and ultimately disrupts wing morphology. Ion channels impact development of several tissues and organisms in which BMP signaling is essential. In neurons and pancreatic beta cells, Irk channels modulate membrane potential to affect intracellular Ca++ to control secretion of neurotransmitters and insulin. Based on Irk activity in neurons, we hypothesized that electrical activity controls endoplasmic reticulum (ER) Ca++ release into the cytoplasm to regulate the release of BMP. Materials and Methods: To test this hypothesis, we reduced expression of four proteins that control ER calcium, Stromal interaction molecule 1 (Stim), Calcium release-activated calcium channel protein 1 (Orai), SarcoEndoplasmic Reticulum Calcium ATPase (SERCA), small conductance calcium-activated potassium channel (SK), and Bestrophin 2 (Best2) using RNAi and documented wing phenotypes. We use live imaging to study calcium and Dpp release within pupal wings and larval wing discs. Additionally, we employed immunohistochemistry to characterize Small Mothers Against Decapentaplegic (SMAD) phosphorylation downstream of the BMP/Dpp pathway following RNAi knockdown. Results: We found that reduced Stim and SERCA function decreases amplitude and frequency of endogenous calcium transients in the wing disc and reduced BMP/Dpp release. Conclusion: Our results suggest control of ER calcium homeostasis is required for BMP/Dpp release, and Drosophila wing development.

Inpatient long-term video-electroencephalographic monitoring event capture audiovisual diagnostic quality.

OBJECTIVES: Long-term video-electroencephalographic monitoring (LTVEM) represents the gold-standard method to evaluate whether events represent electrographic seizures, but limited work has evaluated the quality of inpatient event capture. We evaluated the frequency of audiovisual factors impairing the ideal electroclinical correlation of seizure-like episodes during LTVEM. METHODS: We retrospectively reviewed consecutive inpatient LTVEM studies (11/2019-12/2019) from three academic epilepsy centers. We evaluated all pushbutton events for audiovisual characteristics such as whether the event was narrated, whether the patient was blocked on camera, and what diagnostic challenges impaired the electroencephalographer's ability to understand either the reason the event button was pushed or clinical semiology ("electroclinical correlation"). We determined the percent of events and studies with each outcome. RESULTS: There were 154 studies with 520 pushbutton events. The pushbutton was most commonly activated by patients (41%), followed by nurses (31%) or family (17%). Twenty-nine percent of events represented electrographic seizures, and 78% occurred in the Epilepsy Monitoring Unit. The reason for the push was not stated in 45% of events, and inadequate narration impaired electroclinical correlation in 19% of events. At least one relevant part of the patient's body was blocked during 12% of events, but this impaired electroclinical correlation in only 1% of events. There was at least one factor impairing electroclinical correlation in 21% of events, most commonly due to incomplete narration (N = 99), lights off (N = 15), or blankets covering the patient (N = 15). At least one factor impaired electroclinical correlation for any event in 36% of studies. CONCLUSION: Audiovisual factors impairing the electroencephalographer's ability to render an electroclinical correlation were common, particularly related to inadequate narration from bedside observers to explain the reason for pushing the button or event semiology. Future efforts to develop targeted countermeasures should address narration challenges and improve inpatient seizure monitoring quality metrics.

Mechanisms Underlying Influence of Bioelectricity in Development.

To execute the intricate process of development, cells coordinate across tissues and organs to determine where each cell divides and differentiates. This coordination requires complex communication between cells. Growing evidence suggests that bioelectrical signals controlled via ion channels contribute to cell communication during development. Ion channels collectively regulate the transmembrane potential of cells, and their function plays a conserved role in the development of organisms from flies to humans. Spontaneous calcium oscillations can be found in nearly every cell type and tissue, and disruption of these oscillations leads to defects in development. However, the mechanism by which bioelectricity regulates development is still unclear. Ion channels play essential roles in the processes of cell death, proliferation, migration, and in each of the major canonical developmental signaling pathways. Previous reviews focus on evidence for one potential mechanism by which bioelectricity affects morphogenesis, but there is evidence that supports multiple different mechanisms which are not mutually exclusive. Evidence supports bioelectricity contributing to development through multiple different mechanisms. Here, we review evidence for the importance of bioelectricity in morphogenesis and provide a comprehensive review of the evidence for several potential mechanisms by which ion channels may act in developmental processes.

Identifying the activities of physiotherapy practitioners through primary and secondary models of care provided in New South Wales emergency departments.

BACKGROUND: Following the introduction of the emergency department (ED) primary contact physiotherapy role, emergency physiotherapy models of care have evolved and are increasingly being adopted in the Australian ED. This has occurred due to growing ED patient demand and a need for greater workforce flexibility. Since introduction, there here has been limited evaluation of the scope of work physiotherapists are providing in Australian ED. OBJECTIVES: To identify the activities of ED physiotherapists provided through different models of care in NSW. METHODS: Prospective observation study in 19 participating EDs conducted over 6 months between September 2014 and April 2015. RESULTS: The study identified different models of care across participating hospitals where physiotherapists worked independently or in conjunction with a team through a referral service. The individual's scope of work was determined by organisational policy, culture, individual competence, knowledge and skills, and varied significantly between sites. CONCLUSIONS: These findings could guide both ED work flow and the development of multidisciplinary workforce structures to improve the utilisation of the physiotherapy service in EDs. This will allow for better service levels in hospitals, better access for patients and better use of resources.

Imaging Dpp Release from a Drosophila Wing Disc.

The transforming Growth Factor-beta (TGF-ß) superfamily is essential for early embryonic patterning and development of adult structures in multicellular organisms. The TGF-ß superfamily includes TGF-ß, bone morphogenetic protein (BMPs), Activins, Growth and Differentiation Factors, and Nodals. It has long been known that the amount of ligand exposed to cells is important for its effects. It was thought that long-range concentration gradients set up embryonic pattern. However, recently it has become clear that the timing of exposure to these ligands is also important for their downstream transcriptional consequences. A TGF-ß superfamily ligand cannot have a developmental consequence until it is released from the cell in which it was produced. Until recently, it was difficult to determine when these ligands were released from cells. Here we show how to measure the release of a Drosophila BMP called Decapentaplegic (Dpp) from the cells of the wing primordium or wing disc. This method could be modified for other systems or signaling ligands.

Green Methodologies for Copper(I)-Catalyzed Azide-Alkyne Cycloadditions: A Comparative Study.

Successful copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reactions may be achieved by several methods. In this paper, four synthetic protocols were performed for direct comparison of time required for the synthesis, yield, and purity of the 1H-1,2,3-triazole products. The methods with Cu(I) catalysts were conventional, microwave heating, solvent-free, and a method using glycerol solvent. The compounds synthesized in this paper were known non-fluorinated triazoles and new fluorinated triazoles. The results lead to the conclusion that the microwave method should be strongly considered for CuAAC syntheses.

Ion Channel Contributions to Wing Development in Drosophila melanogaster.

During morphogenesis, cells communicate with each other to shape tissues and organs. Several lines of recent evidence indicate that ion channels play a key role in cellular signaling and tissue morphogenesis. However, little is known about the scope of specific ion-channel types that impinge upon developmental pathways. The Drosophila melanogaster wing is an excellent model in which to address this problem as wing vein patterning is acutely sensitive to changes in developmental pathways. We conducted a screen of 180 ion channels expressed in the wing using loss-of-function mutant and RNAi lines. Here we identify 44 candidates that significantly impacted development of the Drosophila melanogaster wing. Calcium, sodium, potassium, chloride, and ligand-gated cation channels were all identified in our screen, suggesting that a wide variety of ion channel types are important for development. Ion channels belonging to the pickpocket family, the ionotropic receptor family, and the bestrophin family were highly represented among the candidates of our screen. Seven new ion channels with human orthologs that have been implicated in human channelopathies were also identified. Many of the human orthologs of the channels identified in our screen are targets of common general anesthetics, anti-seizure and anti-hypertension drugs, as well as alcohol and nicotine. Our results confirm the importance of ion channels in morphogenesis and identify a number of ion channels that will provide the basis for future studies to understand the role of ion channels in development.

Ion Channels in Bone Morphogenetic Protein Signaling.

How a single fertilized egg develops into a complex multicellular organism is one of the great mysteries of life. Developmental biology textbooks describe cascades of ligands, receptors, kinases, and transcription factors that designate proliferation, migration, and ultimately fate of cells organized into a multicellular organism. Recently, it has become apparent that ion channels are integral to the process of developmental signaling. Ion channels provide bioelectric signals that must intersect with the known developmental signaling pathways. We review some evidence that bioelectric signaling contributes to bone morphogenetic protein signaling.

Plasma cell output from germinal centers is regulated by signals from Tfh and stromal cells.

Germinal centers (GCs) are the sites where B cells undergo affinity maturation. The regulation of cellular output from the GC is not well understood. Here, we show that from the earliest stages of the GC response, plasmablasts emerge at the GC-T zone interface (GTI). We define two main factors that regulate this process: Tfh-derived IL-21, which supports production of plasmablasts from the GC, and TNFSF13 (APRIL), which is produced by a population of podoplanin+ CD157high fibroblastic reticular cells located in the GTI that are also rich in message for IL-6 and chemokines CXCL12, CCL19, and CCL21. Plasmablasts in the GTI express the APRIL receptor TNFRSF13B (TACI), and blocking TACI interactions specifically reduces the numbers of plasmablasts appearing in the GTI. Plasma cells generated in the GTI may provide an early source of affinity-matured antibodies that may neutralize pathogens or provide feedback regulating GC B cell selection.

A Thorough QTc Study Confirms Early Pharmacokinetics/QTc Modeling: A Supratherapeutic Dose of Omarigliptin, a Once-Weekly DPP-4 Inhibitor, Does Not Prolong the QTc Interval.

Omarigliptin is a dipeptidyl peptidase-4 inhibitor being developed as a once-weekly treatment for type 2 diabetes. This double-blind, double-dummy, randomized, 3-period balanced crossover study definitively evaluated the effects of a supratherapeutic omarigliptin dose on QTc interval. Population-specific correction of QT interval (QTcP) was used for the primary analysis. Healthy subjects (n = 60) were enrolled and received treatments separated by a ≥4-week washout: (1) single-dose 25 mg omarigliptin (day 1), single-dose 175 mg omarigliptin (day 2); (2) placebo (day 1) followed by single-dose 400 mg moxifloxacin (day 2); (3) placebo (days 1 and 2). Day 2 QTcP intervals were analyzed. The primary hypothesis was supported if the 90%CIs for the least-squares mean differences between omarigliptin 175 mg and placebo in QTcP interval change from baseline were all < 10 milliseconds at every postdose point on day 2. The upper bounds of the 90%CIs for the differences (omarigliptin-placebo) in QTcP change from baseline for omarigliptin 175 mg were < 10 milliseconds at all postdose times on day 2. In conclusion, a supratherapeutic dose of omarigliptin does not prolong the QTcP interval to a clinically meaningful degree relative to placebo, confirming the results of the earlier concentration-QTc analysis.

Efavirenz modulation of sleep spindles and sleep spectral profile.

Non-nucleoside reverse transcriptase inhibitors are important antiretroviral agents for the treatment of human immunodeficiency virus. Some non-nucleoside reverse transcriptase inhibitors, in particular efavirenz, have prominent effects on sleep, cognition and psychiatric variables that limit their tolerability. To avoid confounds due to drug-drug and drug-disease interactions, we assessed the effects of efavirenz in healthy volunteers on sleep, cognition and psychological endpoints during the first week of treatment. Forty healthy male subjects were randomized to receive placebo or efavirenz 600 mg nightly for 7 days after completion of a 3-day placebo run-in period. Treatment with efavirenz was associated with reduced time to sleep onset in the Maintenance of Wakefulness Test, an increase in non-rapid eye movement sleep, a large exposure-related decrease in sigma band spectral density and sleep spindle density during non-rapid eye movement sleep, and reduced performance on an attention switching task. Because efavirenz has been shown to have serotonin 2A receptor partial-agonist properties, we reasoned that antagonism of serotonin 2A receptor signalling in the thalamic reticular nucleus, which generates sleep spindles and promotes attention, may be responsible. Consistent with predictions, treatment of healthy volunteers with a single dose of a serotonin 2A receptor antagonist was found to significantly suppress sigma band spectral density in an exposure-related manner and modulated the overall spectral profile in a manner highly similar to that observed with efavirenz, consistent with the notion that efavirenz exhibits serotonin 2A receptor partial-agonist pharmacology in humans.

Germinal center dysregulation by histone methyltransferase EZH2 promotes lymphomagenesis.

Protection against deadly pathogens requires the production of high-affinity antibodies by B cells, which are generated in germinal centers (GCs). Alteration of the GC developmental program is common in many B cell malignancies. Identification of regulators of the GC response is crucial to develop targeted therapies for GC B cell dysfunctions, including lymphomas. The histone H3 lysine 27 methyltransferase enhancer of zeste homolog 2 (EZH2) is highly expressed in GC B cells and is often constitutively activated in GC-derived non-Hodgkin lymphomas (NHLs). The function of EZH2 in GC B cells remains largely unknown. Herein, we show that Ezh2 inactivation in mouse GC B cells caused profound impairment of GC responses, memory B cell formation, and humoral immunity. EZH2 protected GC B cells against activation-induced cytidine deaminase (AID) mutagenesis, facilitated cell cycle progression, and silenced plasma cell determinant and tumor suppressor B-lymphocyte-induced maturation protein 1 (BLIMP1). EZH2 inhibition in NHL cells induced BLIMP1, which impaired tumor growth. In conclusion, EZH2 sustains AID function and prevents terminal differentiation of GC B cells, which allows antibody diversification and affinity maturation. Dysregulation of the GC reaction by constitutively active EZH2 facilitates lymphomagenesis and identifies EZH2 as a possible therapeutic target in NHL and other GC-derived B cell diseases.

Germinal center B cells govern their own fate via antibody feedback.

Affinity maturation of B cells in germinal centers (GCs) is a process of evolution, involving random mutation of immunoglobulin genes followed by natural selection by T cells. Only B cells that have acquired antigen are able to interact with T cells. Antigen acquisition is dependent on the interaction of B cells with immune complexes inside GCs. It is not clear how efficient selection of B cells is maintained while their affinity matures. Here we show that the B cells' own secreted products, antibodies, regulate GC selection by limiting antigen access. By manipulating the GC response with monoclonal antibodies of defined affinities, we show that antibodies in GCs are in affinity-dependent equilibrium with antibodies produced outside and that restriction of antigen access influences B cell selection, seen as variations in apoptosis, plasma cell output, T cell interaction, and antibody affinity. Feedback through antibodies produced by GC-derived plasma cells can explain how GCs maintain an adequate directional selection pressure over a large range of affinities throughout the course of an immune response, accelerating the emergence of B cells of highest affinities. Furthermore, this mechanism may explain how spatially separated GCs communicate and how the GC reaction terminates.

Self-determination and compliance in directly observed therapy of tuberculosis treatment in the Kingdom of Lesotho.

Public health officials have created directly observed therapy, short course (DOTS) to treat tuberculosis (TB) and to guarantee that drugs are taken in the right combination and for the appropriate duration. The World Health Organization (WHO) uses supervisors (nurses, nursing assistants, and community health workers [CHWs], as well as community members and relatives), to watch the patient take his or her TB medication. This article explores the observation component of DOTS, and how DOTS supervisors enhance patients' ability to comply with TB treatment by providing enablers, education, and supportive relationships. It also explores how supervisors can achieve the balance between patients' right to self-determination and a community's need for social accountability. Social work can contribute to the efficacy of TB treatment by helping public health officials understand the importance of balancing patients' rights and society's needs in determining the allocation of program resources. The results of this study are based on data gathered from 71 researcher-administered questionnaires and 25 semi-structured interviews in seven of the Kingdom of Lesotho's 19 health service areas. It was found that by providing enablers, education, and supportive relationships, DOTS supervisors ameliorate some of the logistical problems that prevent compliance. Noncompliance for patients is often an institutional/managerial problem that denies patients access to services and resources and should not be blamed on them.

Open-label, pilot protocol of patients with rheumatoid arthritis who switch to infliximab after an incomplete response to etanercept: the opposite study.

OBJECTIVE: To incorporate a new trial design to examine clinical response, cytokine expression and joint imaging in patients with rheumatoid arthritis (RA) switching from etanercept to infliximab treatment. METHODS: A randomised, open-label, clinical trial of 28 patients with an inadequate response to etanercept was conducted. Eligible patients received background methotrexate and were randomised 1:1 to discontinue etanercept and receive infliximab 3 mg/kg at weeks 0, 2, 6, 14 and 22, or to continue etanercept 25 mg twice weekly. Data were analysed for clinical response, serum biomarker levels, radiographic progression, MRI and adverse events. RESULTS: At week 16, 62% of infliximab-treated patients achieved American College of Rheumatology 20% criteria for improvement in RA (ACR20) responses compared with 29% of etanercept-treated patients. A 30.8% decrease from baseline in Disease Activity Score 28 was observed in patients receiving infliximab, compared with a 16.0% decrease in patients receiving etanercept. ACR20 and American College of Rheumatology 50% criteria for improvement in RA responses correlated at least minimally with intracellular adhesion molecule-1 and interleukin 8 in patients receiving infliximab. 38% of patients who were switched to infliximab showed reductions in Health Assessment Questionnaire scores (>0.4), compared with 0% of patients receiving etanercept. MRI analyses were inconclusive. Both drugs were well tolerated; 54% of infliximab-treated patients and 50% of etanercept-treated patients reported adverse events. CONCLUSIONS: In this exploratory, open-label trial (with single-blind evaluator), patients were randomised to continue with etanercept or switch to infliximab. The small sample size of this hypothesis-generating study was underpowered to show statistical differences between groups. There was a numerical trend favouring patients who switched to infliximab, therefore warranting further study with a more rigorous design.

Effects of irradiation on nerve graft vasculature.

Increasing doses of therapeutic irradiation are known to impair nerve regeneration after grafting. One possible factor is the effect of irradiation on the endoneurial vasculature. This study investigates the effects of postoperative irradiation on the size, number, and cross-sectional area of endoneurial vessels in the rat posterior tibial nerve graft model. Sixty-five Sprague-Dawley rats underwent 1.5-cm interposition grafts to the tibial nerve. Postoperatively, they were assigned to one of five groups. The animals in Group 1 were unirradiated controls. Groups 2 to 5 received postoperative irradiation in the amounts of 46, 66, 86, and 106 Gy, respectively. One hundred and twenty days after grafting, sections of the proximal, grafted, and distal nerve were harvested and analyzed with digital morphometry. Statistical analysis of the average vessel area, number of vessels, and total vascular area was performed. The grafted segments of Groups 4 and 5 and the distal segments of all irradiated groups showed a statistically significant decrease in the number of vessels, compared to controls. The average size of the vessel was smaller in the proximal segment of the irradiated groups, compared to controls. There was no difference in size in either the grafted or distal segments of the irradiated groups, compared to controls. The observed changes in the endoneurial vasculature resulted from both the action of regeneration and the effects of irradiation. The irradiation effects appear to be dose-dependent.

A subjective rating scale for evaluating the appearance outcome of autologous breast reconstruction.

BACKGROUND: Subjective evaluations of the appearance outcome of autologous breast reconstruction are usually performed by surgeons and not by patients. Such surgeon-based evaluations are rarely reproducible and show little interobserver agreement. Among existing patient-based subjective scales, none has been tested for reliability, and no study to date has evaluated the reliability when both surgeons and patients use the same scale. METHODS: The authors developed a new instrument for assessing the appearance of autologous breast reconstruction. The instrument's use by four plastic surgeons and 36 transverse rectus musculocutaneous flap patients was assessed for test-retest reliability, internal consistency, surgeon-patient and surgeon-surgeon interobserver agreement, and interitem correlation. RESULTS: The instrument demonstrated high overall internal consistency when used by patients (Cronbach alpha = 0.92). Test-retest reliability on each aesthetic subitem in the scale was higher among patients than among surgeons (weighted kappa range, 0.57 to 0.88 versus 0.25 to 0.66). Interrater agreement was poor among both patients and surgeons (weighted kappa, 0.0 to 0.39). Poor correlation was found between surgeons' evaluations of aesthetic subitems and patients' overall appearance and overall satisfaction scores. CONCLUSIONS: The instrument both demonstrates better internal consistency and is more reliable when used by patients to evaluate their own reconstructions. By contrast, the instrument's use by surgeons is not as internally consistent and reproducible. The poor interobserver agreement among surgeons and the weak correlation between surgeon and patient evaluations suggest that patient input regarding item-specific criteria should be included in evaluations of breast reconstructions.

Functional recovery and histomorphometric assessment following tibial nerve injury in the mouse.

Longitudinal studies have established that functional recovery following sciatic nerve injury can be evaluated in the mouse. Injury to the tibial nerve offers several advantages to sciatic nerve injury, including improved lower extremity sensation and end-organ reinnervation. Functional recovery following tibial nerve crush injury was studied in 55 C3H mice randomized into five groups harvested for histomorphometric evaluation from either normal nerves or 2, 3, 4, or 6 weeks postoperatively. Walking tracks were obtained preoperatively, and at regular intervals postoperatively, and foot print lengths measured. Significant normalization of print length occurred 14 days postoperatively, and complete recovery was noted 28 days postoperatively. Significant histomorphologic evidence of neuroregeneration was detected between 2 and 4 weeks postoperatively. Injury to the tibial nerve is a viable alternative to the sciatic nerve for studying neural regeneration in mice, and the print length factor can be used to monitor functional recovery in this model.

Evaluation of sample preparation techniques for mass measurements of PCR products using ESI-FT-ICR mass spectrometry.

Elimination of PCR buffer components and alkali metal cations (i.e., Na+, K+) is of critical importance to allow for accurate mass measurements of PCR products for genotyping and sequencing applications. Ethanol precipitation followed by microdialysis has been repeatedly shown to efficiently desalt PCR products for analysis by mass spectrometry and is considered the gold standard. Alternative cleanup techniques that are compatible with automation are explored here with the intent of expanding the bottleneck that exists between the production of PCR products and analysis by electrospray ionization mass spectrometry (ESI-MS). Numerous combinations of approaches were evaluated that included PCR purification kits and alcohol precipitations. The data shown here support alternative approaches to an ethanol precipitation followed by microdialysis that have comparable desalting efficiency and can be utilized for cleanup of PCR products generated from single reactions.