High-yield recombinant adeno-associated viral vector production by multivariate optimization of bioprocess and transfection conditions.

Recombinant adeno-associated viral vectors (rAAVs) are one of the most used vehicles for gene therapy, with five rAAV therapeutics commercially approved by the FDA. To improve product yield, we optimized the suspension production process of rAAV8 vectors carrying a proprietary transgene using a commercially available transfection reagent, FectoVIR-AAV. Using a miniaturized automated 250 mL scale bioreactor system, we generated models of vector genome (vg) titer, capsid (cp) titer, and Vg:Cp percentage from two multivariate design of experiment studies, one centered around bioreactor operating parameters, and another based on the transfection conditions. Using the optimized process returned from these models, the vector genome titer from the bioreactor was improved to beyond 1 × 1012 vg/mL. Five critical parameters were identified that had large effects on the pre-purification vector quantity-the transfection pH, production pH, complexation time, viable cell density at transfection, and transfection reagent to DNA ratio. The optimized process was further assessed for its performance extending to six AAV serotypes, namely AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9 carrying a transgene encoding for green fluorescent protein (GFP). Five of the six serotypes returned higher vector genome titers than the control condition. These data suggest that the choice of transfection reagent is a major factor in improving vector yield. The multivariate design of experiment approach is a powerful way to optimize production processes, and the optimized process from one AAV vector can to some extent be generalized to other serotypes and transgenes to accelerate development timelines of new programs.

Prognosis and oncogenomic profiling of patients with tropomyosin receptor kinase fusion cancer in the 100,000 genomes project.

INTRODUCTION: Neurotrophic tyrosine receptor kinase (NTRK) gene fusions are oncogenic drivers in various tumor types. Limited data exist on the overall survival (OS) of patients with tumors with NTRK gene fusions and on the co-occurrence of NTRK fusions with other oncogenic drivers. MATERIALS AND METHODS: This retrospective study included patients enrolled in the Genomics England 100,000 Genomes Project who had linked clinical data from UK databases. Patients who had undergone tumor whole genome sequencing between March 2016 and July 2019 were included. Patients with and without NTRK fusions were matched. OS was analyzed along with oncogenic alterations in ALK, BRAF, EGFR, ERBB2, KRAS, and ROS1, and tumor mutation burden (TMB) and microsatellite instability (MSI). RESULTS: Of 15,223 patients analyzed, 38 (0.25%) had NTRK gene fusions in 11 tumor types, the most common were breast cancer, colorectal cancer (CRC), and sarcoma. Median OS was not reached in both the NTRK gene fusion-positive and -negative groups (hazard ratio 1.47, 95% CI 0.39-5.57, P = 0.572). A KRAS mutation was identified in two (5%) patients with NTRK gene fusions, and both had hepatobiliary cancer. High TMB and MSI were both more common in patients with NTRK gene fusions, due to the CRC subset. While there was a higher risk of death in patients with NTRK gene fusions compared to those without, the difference was not statistically significant. CONCLUSION: This study supports the hypothesis that NTRK gene fusions are primary oncogenic drivers and the co-occurrence of NTRK gene fusions with other oncogenic alterations is rare.

Rationale and design of ON-TRK: a novel prospective non-interventional study in patients with TRK fusion cancer treated with larotrectinib.

BACKGROUND: Tropomyosin receptor kinase (TRK) fusion proteins resulting from neurotrophic tyrosine receptor kinase (NTRK) gene fusions are rare primary oncogenic drivers in a wide array of tumors. Larotrectinib is a first-in-class, highly selective, central nervous system-active TRK inhibitor approved by the US Food and Drug Administration (FDA), European Medicines Agency (EMA), and over 40 countries for the treatment of TRK fusion solid tumors in adult and pediatric patients. Due to the rarity of TRK fusion cancer, larotrectinib was granted accelerated approval based on a relatively small number of patients enrolled in three early phase trials. ON-TRK aims to evaluate the safety profile of larotrectinib in a broader population and over extended time periods. METHODS: ON-TRK is a prospective, non-interventional, open-label, multicenter, multi-cohort, post-approval study in adult and pediatric patients with locally advanced or metastatic TRK fusion cancer treated with larotrectinib that will describe the safety and effectiveness of larotrectinib in real-world practice conditions. Adult patients will be grouped by tumor type and followed for at least 2 years. Patients < 18 years old will be enrolled under a 'pediatric' cohort regardless of tumor type and will be followed for 5 years to evaluate the risk of potential long-term adverse effects of larotrectinib on their growth and development. The effectiveness of larotrectinib in the overall study population as well as in patient subgroups will also be evaluated. Procedures avoided in patients with infantile fibrosarcoma (e.g., amputation) and the number of patients who were able to undergo surgery with a curative intent (excluding amputation) because of the use of larotrectinib will be described. Larotrectinib treatment patterns in real-world practice, including dosing and duration of treatment, will be described. DISCUSSION: The FDA Accelerated Approval Program allows for earlier approval of and patient access to drugs that treat serious conditions and fill an unmet medical need. This study is designed to fulfill post-approval requirements set by the FDA as well as post-marketing requirements set forth by local regulatory bodies and is part of the risk management plan for the EMA. STUDY REGISTRATION: This study is registered at ClinicalTrials.gov ( NCT04142437 ). PROTOCOL VERSION: v2.5, 25 March 2021.

Real-world patient characteristics associated with survival of 2 years or more after radium-223 treatment for metastatic castration-resistant prostate cancer (EPIX study).

BACKGROUND: The real-world EPIX study was conducted to gather information about the characteristics of patients with metastatic castration-resistant prostate cancer (mCRPC) who survived ≥2 years after treatment with the alpha-emitter radium-223. METHODS: This retrospective study of electronic health records in the US Flatiron database (NCT04516161) included patients with mCRPC treated with radium-223 between January 2013 and June 2019. Median overall survival (OS) and prostate-specific antigen (PSA) response (≥50% reduction) from start of radium-223 treatment were the primary and secondary endpoints, respectively. Patient characteristics were compared between those who survived ≥2 years versus <2 years, including a subgroup who survived <6 months. RESULTS: In the 1180 patients identified, median OS was 12.9 months (95% CI: 12.1-13.7), and 13% of patients with data at 6 months had a PSA response. The survival groups included 775 patients (65.7%) who survived <2 years (including 264 (22.4%) who survived <6 months) and 185 patients (15.7%) who survived ≥2 years; 220 patients (18.6%) had incomplete follow-up data and were censored. On multivariate analysis, age >75 years, Eastern Cooperative Oncology Group performance status (ECOG PS) 2-4, visceral metastases, prior symptomatic skeletal events (SSEs), and prior chemotherapy were independently prognostic of reduced OS. For patients with survival ≥2 years versus <2 years, median age was 71 versus 75 years, 4% versus 14% had ECOG PS 2-4, 4% versus 10% had visceral metastases, 38% versus 44% had prior SSEs, and 16% versus 32% had prior chemotherapy. CONCLUSIONS: In this study of men with mCRPC treated in real-world clinical practice, median OS was consistent with that seen in the phase 3 ALSYMPCA trial. Patients who survived ≥2 years after the start of radium-223 were younger and had better ECOG PS, lower disease burden, and less use of prior chemotherapy than those who survived <2 years.

Hyper-IgE Syndrome due to an Elusive Novel Intronic Homozygous Variant in DOCK8.

Rare, biallelic loss-of-function mutations in DOCK8 result in a combined immune deficiency characterized by severe and recurrent cutaneous infections, eczema, allergies, and susceptibility to malignancy, as well as impaired humoral and cellular immunity and hyper-IgE. The advent of next-generation sequencing technologies has enabled the rapid molecular diagnosis of rare monogenic diseases, including inborn errors of immunity. These advances have resulted in the implementation of gene-guided treatments, such as hematopoietic stem cell transplant for DOCK8 deficiency. However, putative disease-causing variants revealed by next-generation sequencing need rigorous validation to demonstrate pathogenicity. Here, we report the eventual diagnosis of DOCK8 deficiency in a consanguineous family due to a novel homozygous intronic deletion variant that caused aberrant exon splicing and subsequent loss of expression of DOCK8 protein. Remarkably, the causative variant was not initially detected by clinical whole-genome sequencing but was subsequently identified and validated by combining advanced genomic analysis, RNA-seq, and flow cytometry. This case highlights the need to adopt multipronged confirmatory approaches to definitively solve complex genetic cases that result from variants outside protein-coding exons and conventional splice sites.

Emergency Front-of-Neck Airway Rescue Via the Cricothyroid Membrane: A High-Resolution Computed Tomography Study of Airway Anatomy in Adults.

BACKGROUND: Emergency front-of-neck airway rescue is recommended in a can't intubate, can't oxygenate clinical scenario. Cannula cricothyroidotomy has been reported as having a high failure rate. Our primary aim was to estimate the angle of the trachea in relation to the horizontal axis in a simulated emergency front-of-neck airway rescue position. Our secondary aims were to estimate the optimal cannula angle of approach and evaluate the anatomical relationship of the cricothyroid membrane (CTM) to adjacent structures. We also assessed whether the CTM lies above or below the neck midpoint, a point equidistant from the suprasternal notch (SSN), and the chin surface landmarks. All measurements were compared between the male and female subjects. METHODS: Subjects having elective computed tomography of their thorax were consented to have extension of the computed tomography to include their neck. A preliminary radiation dose and risk assessment deemed the additional radiation to be of very low risk (level IIa). Subjects were positioned supinely on the computed tomography table. Standard neck extension was achieved by placing a pillow under the scapulae and a rolled towel under the neck to simulate emergency front-of-neck airway rescue positioning. RESULTS: Fifty-two subjects were included in this study: 31 men and 21 women. The mean angle of the trachea in relation to the horizontal axis was 25.5° (95% confidence interval [CI], 21.8-29.1) in men and 14.0° (95% CI, 11.5-16.5) in women. The mean minimum angles required for hypothetical cannula cricothyroidotomy for men and women were 55.2° (95% CI, 51.8-58.7) and 50.5° (95% CI, 45.4-55.6), respectively. The CTM was located lower in the neck in men compared to women. The CTM was located below the neck midpoint in 30 of 30 (100%) male subjects and 11 of 20 (55%) female subjects (P < .001). CONCLUSIONS: The trachea angulates posteriorly in a simulated emergency front-of-neck airway rescue position in supine subjects and to a greater degree in men compared to women (P < .001). The minimum angle required for hypothetical cannula cricothyroidotomy was >45° in the majority (75%) of subjects studied. A steeper cannula angle of approach may be more reliable and warrants further clinical study. If airway anatomy is indistinct and performing a vertical scalpel cricothyroidotomy, consideration should be given to performing this incision lower in the neck in men compared to women.

Larotrectinib versus Prior Therapies in Tropomyosin Receptor Kinase Fusion Cancer: An Intra-Patient Comparative Analysis.

Randomized controlled basket trials investigating drugs targeting a rare molecular alteration are challenging. Using patients as their own control overcomes some of these challenges. Growth modulation index (GMI) is the ratio of progression-free survival (PFS) on the current therapy to time to progression (TTP) on the last prior line of therapy; GMI ≥ 1.33 is considered a threshold of meaningful clinical activity. In a retrospective, exploratory analysis among patients with advanced tropomyosin receptor kinase (TRK) fusion cancer treated with the selective TRK inhibitor larotrectinib who received ≥1 prior line of therapy for locally advanced/metastatic disease, we determined the proportion of patients with GMI ≥ 1.33; patients who had not progressed by data cut-off were censored for PFS. Among 72 eligible patients, median GMI was 2.68 (range 0.01-48.75). Forty-seven patients (65%) had GMI ≥ 1.33; 13/25 patients (52%) with GMI < 1.33 had not yet progressed on larotrectinib. Kaplan-Meier estimates showed a median GMI of 6.46. The probability of attaining GMI ≥ 1.33 was 0.75 (95% confidence interval (CI), 0.65-0.85). Median TTP on previous treatment was 3.0 months (95% CI, 2.6-4.4). Median PFS on larotrectinib was not estimable ((NE); 95% CI, NE; hazard ratio, 0.220 (95% CI, 0.146-0.332)). This analysis suggests larotrectinib improves PFS for patients with TRK fusion cancer compared with prior therapy.

Disease Characteristics and Completion of Treatment in Patients With Metastatic Castration-Resistant Prostate Cancer Treated With Radium-223 in an International Early Access Program.

BACKGROUND: Radium-223 is approved by the US Food and Drug Administration and European Medicines Agency for the treatment of metastatic castration-resistant prostate cancer (mCRPC). There are currently no markers for selecting patients most likely to complete radium-223 treatment. PATIENTS AND METHODS: In this phase IIIb, international, single-arm study, patients received radium-223, 55 kBq/kg, every 4 weeks for ≤6 cycles. Primary end points were safety and overall survival. In post hoc analyses patients were grouped according to number of radium-223 injections received (1-4 or 5-6). Associations between baseline covariates and number of injections were investigated. RESULTS: Of 696 eligible patients, 473 (68%) had received 5 to 6 radium-223 injections and 223 (32%) 1 to 4 injections. Patients with less pain (moderate-severe vs. none-mild, odds ratio [OR], 0.41; P < .0001), lower Eastern Cooperative Oncology Group performance status (≥2 vs. 0-1, OR, 0.51; P = .0074), lower prostate-specific antigen level (>141 µg/L vs. ≤141 µg/L, OR, 0.40; P < .0001), and higher hemoglobin level (<10 g/dL vs. ≥10 g/dL, OR, 0.50; P = .0206) were more likely to receive 5 to 6 than 1 to 4 injections. Median overall survival was not reached and was 6.3 months (95% confidence interval, 5.4-7.4) in patients who had received 5 to 6 and 1 to 4 radium-223 injections, respectively. Adverse events were less common in patients who received 5 to 6 than 1 to 4 injections; anemia was reported in 87 (18%) and 64 (29%) patients, respectively. CONCLUSION: Patients with less advanced mCRPC are more likely to receive 5 to 6 radium-223 injections and to achieve better overall survival. Consideration of baseline and disease characteristics is recommended before initiation of radium-223 treatment.

Radium-223 in asymptomatic patients with castration-resistant prostate cancer and bone metastases treated in an international early access program.

BACKGROUND: Radium-223, a targeted alpha therapy, is used to treat symptomatic patients with castration-resistant prostate cancer (CRPC) and bone metastases. Data for radium-223 in asymptomatic CRPC patients with bone metastases are lacking. METHODS: This was a prospective, single-arm phase 3b study. Patients with metastatic CRPC (malignant lymphadenopathy not exceeding 6 cm was allowed, visceral disease was excluded) received radium-223, 55 kBq/kg intravenously, every 4 weeks for up to 6 cycles. Co-primary endpoints were safety and overall survival. Post hoc analyses were performed according to baseline asymptomatic or symptomatic disease status. Asymptomatic status was defined as no pain and no opioid use at baseline. RESULTS: Seven hundred eight patients received ≥1 radium-223 injection: 548 (77%) were symptomatic to various degrees, and 135 (19%) were asymptomatic. Asymptomatic patients had more favorable baseline disease characteristics than symptomatic. A lower proportion of asymptomatic versus symptomatic patients had received prior abiraterone (25% vs 35%) and prior docetaxel (52% vs 62%). A higher proportion of asymptomatic (71%) versus symptomatic (55%) patients completed radium-223 treatment. Overall survival (hazard ratio [HR] 0.486), time to disease progression (HR 0.722) and time to first symptomatic skeletal event (HR 0.328) were better in asymptomatic than symptomatic patients. Alkaline phosphatase (ALP) response rates were similar (46% vs 47%), and ALP normalization (44% vs 25%) and prostate-specific antigen response rates (21% vs 13%) were higher in asymptomatic than symptomatic patients. A lower proportion of asymptomatic patients reported treatment-emergent adverse events (TEAEs, 61% vs 79%), grade 3-4 TEAEs (29% vs 40%) and drug-related TEAEs (28% vs 44%). There were two treatment-related deaths, both in patients with baseline symptomatic disease. CONCLUSIONS: Using radium-223 earlier in the disease course, when patients are asymptomatic or minimally symptomatic, may enable patients to complete treatment and optimize treatment outcome compared to symptomatic patients, and therefore may allow sequencing with other life-prolonging therapies. TRIAL REGISTRATION: The study was registered with ClinicalTrials.gov , number NCT01618370 on June 13, 2012 and the European Union Clinical Trials Register, EudraCT number 2012-000075-16 on April 4, 2012.

Measuring the success of Medical Emergency Teams: potentially preventable deaths versus total cardiac arrest deaths. A single centre observational study.

BACKGROUND: Rapid response systems have been implemented with the aim of preventing patient deterioration, in-hospital cardiac arrests (IHCA) and related deaths. Not all 'unexpected deaths' are preventable, thus compromising the use of unexpected deaths as an outcome measure. AIMS: To assess temporal trends in potentially preventable deaths as a subset of total unexpected death rates over a 4-year period. METHODS: A single centre, cohort study of all unexpected deaths between 1 January 2010 and 31 December 2013. Unexpected deaths were identified from the rapid response systems database and patients' case histories were reviewed to reclassify the deaths into one of three categories: potentially preventable: if earlier MET activation may have prevented death; missed not for resuscitation opportunity; and not preventable. Total bed days were obtained from the hospital's patient administration system. RESULTS: The rate of potentially preventable deaths decreased from 5.3 to 0.7 per 100 000 bed days (incident rate ratio (IRR) 0.53 (95% CI 0.31-0.90), P = 0.02). The rate of total unexpected deaths was unchanged (IRR 0.96 (0.80-1.16), P = 0.70), as were the rates of non-preventable deaths (IRR 1.06 (0.78-1.42), P = 0.72) and missed NFR deaths (IRR 1.1 (0.83-1.42), P = 0.56). CONCLUSION: The rate of potentially preventable deaths has decreased by 47% per year over a 4-year period without any change in the overall rate of unexpected deaths. Distinguishing between potentially preventable deaths in contrast to total unexpected deaths enables more targeted evaluation of rapid response systems.

DHA mediates the protective effect of fish consumption on new episodes of depression among women.

In a longitudinal cohort study of young Australian adults, we reported that for women higher baseline levels of fish consumption were associated with reduced incidence of new depressive episodes during the 5-year follow-up. Fish are high in both n-3 fatty acids and tyrosine. In this study, we seek to determine whether n-3 fatty acids or tyrosine explain the observed association. During 2004-2006, a FFQ (nine fish items) was used to estimate weekly fish consumption among 546 women aged 26-36 years. A fasting blood sample was taken and high-throughput NMR spectroscopy was used to measure 233 metabolites, including serum n-3 fatty acids and tyrosine. During 2009-2011, new episodes of depression since baseline were identified using the lifetime version of the Composite International Diagnostic Interview. Relative risks were calculated using log-binomial regression and indirect effects estimated using the STATA binary_mediation command. Potential mediators were added to separate models, and mediation was quantified as the proportion of the total effect due to the mediator. The n-3 DHA mediated 25·3 % of the association between fish consumption and depression when fish consumption was analysed as a continuous variable and 16·6 % when dichotomised (reference group: <2 serves/week). Tyrosine did not mediate the association (<0·1 %). Components in fish other than n-3 fatty acids and tyrosine might be beneficial for women's mental health.

Intensive care patient and family satisfaction.

PURPOSE: The purpose of this paper is to measure family member satisfaction with the care provided in an Australian private intensive care unit (ICU) at two time points separated by two years. The study was part of a quality improvement process for ICU, and was designed with reference to the revised Australian Commission on Safety and Quality in Health care. DESIGN/METHODOLOGY/APPROACH: This prospective study involved family members of patients admitted in ICU in February 2011 and February 2013. All patients admitted to during the study month were eligible. Questionnaire addressed staff competence, treatment of family, communication, environment and overall satisfaction, using a Likert scale. There was one free text question. The first survey was done by handing the survey package to the next of kin at the time of discharge while the second involved mailing a survey package within a week of discharge from ICU. Quantitative analysis was based on ten Likert items and qualitative analysis based on the free text question. FINDINGS: The response rate was 53 percent (54/102) in 2013 (mailed) compared to 44 percent (44/100) in 2011 (hand delivered). The results from second (2013) survey showed statistically significant improvement in satisfaction associated with nursing and medical competency. Other areas with improvement were the relative's waiting room and visiting hours. The area lacking improvement was ease of finding ICU the hospital. It confirmed that families were satisfied with the care provided and highlighted areas for improvement. The results indicated high satisfaction overall, especially with the hospital staff competency and the overall care quality their relative received. Though most responses also indicated satisfaction with communication and support services, these areas did not perform as well. ORIGINALITY/VALUE: This study provided a simple and effective mechanism to monitor consumer satisfaction with ICU.

Control of type 1 error in a hybrid complete two-period vaccine efficacy trial.

A complete two-period experimental design has been defined as one in which subjects are randomized to treatment, observed for the occurrence of an event of interest, re-randomized, and observed again for the event in a second period. A 4-year vaccine efficacy trial was planned to compare a high-dose vaccine with a standard dose vaccine. Subjects would be randomized each year, and subjects who had participated in a previous year would be allowed to re-enroll in a subsequent year and would be re-randomized. A question of interest is whether positive correlation between observations on subjects who re-enrolled would inflate the variance of test statistics. The effect of re-enrollment and correlation on type 1 error in a 4-year trial is investigated by simulation. As conducted, the trial met its power requirements after two years. Subjects therefore included some who participated for a single year and others who participated in both years. Those who participated in both years constituted a complete two-period design. An algebraic expression for the variance of the treatment difference in a complete two-period design is derived. It is shown that under a 'no difference' null, correlation does not result in variance inflation in this design. When there is a treatment difference, there is variance inflation but it is small. In the vaccine efficacy trial, the effect of correlation on the statistical inference was negligible.

The rise and fall of food minerals.

Climax fertility developed in early times with higher plants and animals developing to use this high standard of mineral nutrition. This climax fertility is built on carbon fixed from the air by plants. This carbon provides the energy to soil micro-organisms. Both those dependant on soil organic matter and those that draw energy and minerals directly from the plant. These micro-organisms increase plant mineral content and provide other life compounds. Agricultural activity has broken this climax fertility leading to a fall in food chain minerals.

Successful resuscitation from cardiopulmonary arrest due to profound hypothermia using noninvasive techniques.

Profound hypothermia is defined as a core body temperature of 20.0 °C or less. Successful resuscitation from this degree of hypothermia is rare. We present a case of successful resuscitation in a 2-year-old boy found in cardiac arrest due to profound hypothermia. Invasive techniques such as cavity lavage, extracorporeal membrane oxygenation, and cardiopulmonary bypass were not used.

Hal: an automated pipeline for phylogenetic analyses of genomic data.

The rapid increase in genomic and genome-scale data is resulting in unprecedented levels of discrete sequence data available for phylogenetic analyses. Major analytical impasses exist, however, prior to analyzing these data with existing phylogenetic software. Obstacles include the management of large data sets without standardized naming conventions, identification and filtering of orthologous clusters of proteins or genes, and the assembly of alignments of orthologous sequence data into individual and concatenated super alignments. Here we report the production of an automated pipeline, Hal that produces multiple alignments and trees from genomic data. These alignments can be produced by a choice of four alignment programs and analyzed by a variety of phylogenetic programs. In short, the Hal pipeline connects the programs BLASTP, MCL, user specified alignment programs, GBlocks, ProtTest and user specified phylogenetic programs to produce species trees. The script is available at sourceforge (http://sourceforge.net/projects/bio-hal/). The results from an example analysis of Kingdom Fungi are briefly discussed.

The effects of immunological castration against GnRH with recombinant OL protein (Ovalbumin-LHRH-7) on carcass and meat quality characteristics, histological appearance of testes and pituitary gland in Kivircik male lambs.

The aims of the study were to investigate the effects of immunization against GnRH using OL protein (Ovalbumin-LHRH-7) on feedlot performance, carcass, meat quality and some reproductive traits in Kivircik ram lambs. Ram lambs in the immunization (I, n=7) group were immunized against GnRH using OL protein and boosted 2 weeks later. Control (C, n=7) group was not treated. The animals were kept at pasture for 6 weeks after the first immunization, subjected to a 70 day fattening program, and then slaughtered. Growth performance, various carcass and meat quality characteristics were not affected from the immunization. GnRH immunization induced GnRH antibody production, suppressed testosterone production and testicular growth (P<0.01). Testicular structure was negatively affected from the immunization, but not pituitary. These results suggest that immunization against GnRH with OL could be an alternative castration technique in ram lambs without negatively affecting carcass and meat quality characteristics.

Inhibition of sodium-calcium exchange by KB-R7943: Dodecylamine and sphingosine in transfected Chinese hamster ovary cells.

Inhibitors of Na(+)/Ca(2+) exchange (NCX) such as KB-R7943 and SEA0400 are thought to act by promoting an inactive state of the exchanger induced by elevated cytosolic Na(+) concentrations (Na(+)-dependent inactivation). This conclusion is based mainly on experiments in excised patches from frog oocytes expressing NCX and has not been fully tested in intact cells. Here we characterize the inhibitory effects of KB-R7943 and other amphiphilic cations on NCX activity in transfected Chinese hamster ovary (CHO) cells expressing the cardiac isoform of the Na(+)/Ca(2+) exchanger (NCX1.1). Cytosolic Na(+) concentrations were varied using the channel-forming ionophore gramicidin. KB-R7943 (10microM) inhibited NCX activity at high (140mM) but not at low (10mM) cytosolic Na(+) concentrations. Cells expressing NCX mutants that were either more (F223E) or less (K229Q) susceptible than the wild-type to Na(+)-dependent inactivation were either hypersensitive or resistant, respectively, to KB-R7943 inhibition. We recently reported that Na-dependent inactivation in transfected CHO cells could be partially but transiently reversed by increasing the external Ca(2+) concentration (Chernysh et al., 2008). The inhibitory effects of KB-R7943 were likewise partially reversed by elevated Ca(2+). Other amphiphilic cations such as dodecylamine (20microM) and sphingosine (10microM) closely mimicked the effects of KB-R7943 on NCX activity. Our results support the hypothesis that KB-R7943 inhibits NCX activity in intact cells by promoting Na(+)-dependent inactivation and suggest that this is a general mode of inhibition by amphiphilic cations.

Calcineurin activation by slow calcium release from intracellular stores suppresses protein kinase C regulation of L-type calcium channels in L6 cells.

L-type Ca(2+) channel activity was assayed in L6 cells as the rate of nifedipine-sensitive Ba(2+) influx in a depolarizing medium. In the absence of extracellular Ca(2+), activation of protein kinase C (PKC) with phorbol-12-myristate-13-acetate (PMA) or thymeleatoxin (TMX) inhibited Ba(2+) influx by 38%. Thapsigargin (Tg), a selective inhibitor of the Ca(2+)-ATPase in the sarcoplasmic reticulum, evoked a rise in the cytosolic Ca(2+) concentration ([Ca(2+)](i)) in a Ca(2+)-free medium from 30 to approximately 80 nM. This [Ca(2+)](i) increase declined slowly, giving rise to a modest elevation of [Ca(2+)](i) that persisted for >5 min. The inhibitory effects of PMA and TMX on channel activity were abolished when tested in Tg-treated cells in a Ca(2+)-free medium. However, when the Ca(2+) ionophore, ionomycin, was applied with Tg, PMA and TMX retained their inhibitory effect on L-type Ca(2+) channel activity, suggesting that a lower amplitude and prolonged release of Ca(2+) stores is necessary for abrogating PKC-mediated inhibition of LCC. Cyclosporin A (5 microM) and ascomycin (5 microM), inhibitors of the Ca(2+)/calmodulin-dependent protein phosphatase, calcineurin, fully restored the inhibitory effect of PMA and TMX on channel activity. Addition of 1mM CaCl(2) to the Tg-treated cells increased [Ca(2+)](i) to approximately 165 nM and also restored the inhibitory effects of PMA and TMX. These results indicate that a small, relatively prolonged [Ca(2+)](i) increase elicited by passive depletion of internal Ca(2+) stores led to activation of calcineurin, giving rise to an increase in protein phosphatase activity that counteracted the inhibitory effects of PKC on channel activity. A larger increase in [Ca(2+)](i) via store-dependent Ca(2+) entry enhanced the activity of PKC sufficiently to overcome the protein phosphatase activity of calcineurin. This study is the first to demonstrate that the regulation of L-type Ca(2+) channels in a myocyte model involves a balance between the differential Ca(2+) sensitivities and opposing actions of PKC and calcineurin.