Differential distribution of ivacaftor and its metabolites in plasma and human airway epithelia.

Ivacaftor is the first clinically approved monotherapy potentiator to treat CFTR channel dysfunction in people with cystic fibrosis. Ivacaftor (Iva) is a critical component for all current modulator therapies, including highly effective modulator therapies. Clinical studies show that CF patients on ivacaftor-containing therapies present various clinical responses, off-target effects, and adverse reactions, which could be related to metabolites of the compound. In this study, we reported the concentrations of Iva and two of its major metabolites (M1-Iva and M6-Iva) in capillary plasma and estimated M1-Iva and M6-Iva metabolic activity via the metabolite parent ratio in capillary plasma over 12 h. We also used the ratio of capillary plasma versus human nasal epithelial cell concentrations to evaluate entry into epithelial cells in vivo. M6-Iva was rarely detected by LC-MS/MS in epithelial cells from participants taking ivacaftor, although it was detected in plasma. To further explore this discrepancy, we performed in vitro studies, which showed that M1-Iva, but not M6-Iva, readily crossed 16HBE cell membranes. Our studies also suggest that metabolism of these compounds is unlikely to occur in airway epithelia despite evidence of expression of metabolism enzymes. Overall, our data provide evidence that there are differences between capillary and cellular concentrations of these compounds that may inform future studies of clinical response and off-target effects.

Pharmacokinetic variability of CFTR modulators from standard and alternative regimens.

Elexacaftor, tezacaftor, ivacaftor (ETI) is a CFTR modulator combination approved for use in ∼90 % of people with cystic fibrosis (pwCF) over 2 years old. While most pwCF tolerate this therapy well, some are intolerant to standard dosing, and others show little response. Clinical providers may adjust ETI dosing to combat these issues, but these adjustments are not well guided by pharmacokinetic evidence. Our post-approval study aimed to describe pharmacokinetic variability of ETI plasma concentrations in 15 participants who were administered a standard or reduced dose. ETI were quantified by LC-MS/MS in plasma samples taken prior to the morning dose. Results showed non-significant differences for each compound regardless of dosing regimen and after dose equivalence normalization. The majority of participants in both dosing groups had concentrations expected to elicit clinical response to ETI therapy. These findings indicate that dose reduction may be a viable strategy to maintain clinical benefit while managing intolerance.

Cognitive modeling, ecological psychology, and musical improvisation.

Understanding novelty and improvisation in music requires gathering insight from a variety of disciplines. One fruitful path for synthesizing these insights is via modeling. As such, my aim in this paper is to start building a bridge between traditional cognitive models and contemporary embodied and ecological approaches to cognitive science. To achieve this task, I offer a perspective on a model that would combine elements of ecological psychology (especially affordances) and the Learning Intelligent Decision Agent (LIDA) cognitive architecture. Jeff Pressing's cognitive model of musical improvisation will also be a central link between these elements. While some overlap between these three areas already exists, there are several points of tension between them, notably concerning the nature of perception and the function of artificial general intelligence modeling. I thus aim to alleviate the most worrisome concerns here, introduce several future research questions, and conclude with several points on how my account is part of a general theory, rather than merely a redescription of existent work.

A phase I/II study of preoperative letrozole, everolimus, and carotuximab in stage 2 and 3 hormone receptor-positive and Her2-negative breast cancer.

PURPOSE: In nonmetastatic hormone receptor-positive and Her2-negative breast cancer, preoperative endocrine therapies can yield outcomes similar with chemotherapy. We evaluated the tolerability and preliminary antitumor activity of preoperative letrozole, everolimus, and carotuximab, a monoclonal antibody targeting endoglin, in nonmetastatic breast cancer. METHODS: Eligible patients had newly diagnosed, stage 2 or 3, hormone receptor-positive and Her2/neu-negative breast cancer. Patients received escalating doses of everolimus; the dose of letrozole and carotuximab were fixed at 2.5 mg PO daily and 15 mg/kg intravenously every 2 weeks, respectively. The primary objective was to determine the safety and tolerability of the combination. Secondary objectives included pharmacokinetic and pharmacodynamic studies and assessments of antitumor activity. RESULTS: Fifteen patients enrolled. The recommended phase 2 dose of everolimus in combination with letrozole and carotuximab was 10 mg PO daily. The most frequent adverse events were headache (67%), fatigue (47%), facial flushing and swelling (47%), gingival hemorrhage (40%), epistaxis (33%), nausea and vomiting (27%). Headache constituted a dose-limiting toxicity. At least two signs of mucocutaneous telangiectasia developed in 92% of patients. Carotuximab accumulated in the extravascular space and accelerated the biodistribution and clearance of everolimus. All patients had residual disease. Gene expression analyses were consistent with downregulation of genes involved in proliferation and DNA repair. Among 6 patients with luminal B breast cancer, 5 converted to luminal A after one cycle of therapy. CONCLUSION: Letrozole, everolimus, and carotuximab were tolerated in combination at their single-agent doses. Pharmacokinetic studies revealed an interaction between everolimus and carotuximab. TRIAL REGISTRATION: This trial is registered with ClinicalTrials.gov (Identifier: NCT02520063), first posted on August 11, 2015, and is active, not recruiting.

Quantitation of cystic fibrosis triple combination therapy, elexacaftor/tezacaftor/ivacaftor, in human plasma and cellular lysate.

The triple combination modulator therapy (ETI, elexacaftor (ELX), tezacaftor (TEZ), and ivacaftor (IVA)) is a recent advancement for the care of patients with cystic fibrosis. To aid in the development of clinical pharmacokinetics studies of this treatment, we developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for quantifying the component compounds in human plasma and cell lysate. This assay was optimized for small volumes (10 µL), uses stably labeled isotopes of the ETI compounds as internal standards, and employs a simple methanol protein precipitation method. Chromatography was performed on an ACE Excel C18, 2.1 × 50 mm, reversed phase analytical column, using a step or bump isocratic method, with mobile phases consisting of 0.1% formic acid in water for A, and 0.1% formic acid in acetonitrile for B. Analyte and internal standard detection was conducted with ESI positive ionization tandem mass spectrometry. The precursor/product transitions (m/z) monitored were 598.0/422.0 for ELX, 521.0/449.0 for TEZ, 393.0/172.0 for IVA, 601.0/422.0 for IS-ELX, 525.0/453.0 for IS-TEZ, and 399.0/178.0 for IS-IVA, respectively. The assay has a dynamic range of 10 to 10,000 ng/mL, with a mean coefficient of determination (r2, mean ± SD) of 0.9970 ± 0.0027 (ELX), 0.9989 ± 0.0004 (TEZ), 0.9981 ± 0.0003 (IVA), regardless of specimen matrix. The mean precision values for all calibration standards ranged from 0.0 to 10.8% (ELX), 0.0 to 6.7% (TEZ), and 0.2 to 5.6% (IVA), while the accuracy for calibration standards was within the range of -5.7 to 3.5% (ELX), -3.2 to 6.0% (TEZ), and -3.8 to 5.2% (IVA). Validation results demonstrated high accuracy (≤7.3, ≤9.8, ≤10.6% deviation) and high precision (≤11.5, ≤6.3, ≤11.0% CV) for the respective ETI quality control samples. This method provides a fully validated assay for ETI quantitation for use in clinical research.

Plasma and cellular ivacaftor concentrations in patients with cystic fibrosis.

Access to cystic fibrosis transmembrane conductance regulator (CFTR) modulators has been gradually increasing for people with cystic fibrosis, the first of which was ivacaftor, a CFTR potentiator that is part of all clinically available modulator treatments. In this study, we hypothesized that the steady-state concentrations in blood and tissue are highly variable in patients taking ivacaftor in a real-world context, which may have an impact on the treatment approach. We collected nasal epithelial cells to estimate target site concentrations and blood samples to estimate pharmacokinetic parameters at a steady state. We found that patients on ivacaftor monotherapy have variable concentrations well above the maximal effective concentration and may maintain concentrations necessary for the clinical benefit even if dosing is reduced. We also are the first to provide detailed target site concentration data over time, which shows that tissue concentrations do not fluctuate significantly and do not correlate with plasma concentrations. These findings show that some patients may have higher-than-expected concentrations and may benefit from tailored dosing to balance clinical response with side effects or adherence needs.

Systemic Administration of a Brain Permeable Cdk5 Inhibitor Alters Neurobehavior.

Cyclin-dependent kinase 5 (Cdk5) is a crucial regulator of neuronal signal transduction. Cdk5 activity is implicated in various neuropsychiatric and neurodegenerative conditions such as stress, anxiety, depression, addiction, Alzheimer's disease, and Parkinson's disease. While constitutive Cdk5 knockout is perinatally lethal, conditional knockout mice display resilience to stress-induction, enhanced cognition, neuroprotection from stroke and head trauma, and ameliorated neurodegeneration. Thus, Cdk5 represents a prime target for treatment in a spectrum of neurological and neuropsychiatric conditions. While intracranial infusions or treatment of acutely dissected brain tissue with compounds that inhibit Cdk5 have allowed the study of kinase function and corroborated conditional knockout findings, potent brain-penetrant systemically deliverable Cdk5 inhibitors are extremely limited, and no Cdk5 inhibitor has been approved to treat any neuropsychiatric or degenerative diseases to date. Here, we screened aminopyrazole-based analogs as potential Cdk5 inhibitors and identified a novel analog, 25-106, as a uniquely brain-penetrant anti-Cdk5 drug. We characterize the pharmacokinetic and dynamic responses of 25-106 in mice and functionally validate the effects of Cdk5 inhibition on open field and tail-suspension behaviors. Altogether, 25-106 represents a promising preclinical Cdk5 inhibitor that can be systemically administered with significant potential as a neurological/neuropsychiatric therapeutic.

Embodied Intelligence: Smooth Coping in the Learning Intelligent Decision Agent Cognitive Architecture.

Much of our everyday, embodied action comes in the form of smooth coping. Smooth coping is skillful action that has become habituated and ingrained, generally placing less stress on cognitive load than considered and deliberative thought and action. When performed with skill and expertise, walking, driving, skiing, musical performances, and short-order cooking are all examples of the phenomenon. Smooth coping is characterized by its rapidity and relative lack of reflection, both being hallmarks of automatization. Deliberative and reflective actions provide the contrast case. In Dreyfus' classic view, smooth coping is "mindless" absorption into action, being in the flow, and any reflective thought will only interrupt this flow. Building on the pragmatist account of Dewey, others, such as Sutton, Montero, and Gallagher, insist on the intelligent flexibility built into smooth coping, suggesting that it is not equivalent to automatization. We seek to answer two complementary challenges in this article. First, how might we model smooth coping in autonomous agents (natural or artificial) at fine granularity? Second, we use this model of smooth coping to show how we might implement smooth coping in artificial intelligent agents. We develop a conceptual model of smooth coping in LIDA (Learning Intelligent Decision Agent). LIDA is an embodied cognitive architecture implementing the global workspace theory of consciousness, among other psychological theories. LIDA's implementation of consciousness enables us to account for the phenomenology of smooth coping, something that few cognitive architectures would be able to do. Through the fine granular analysis of LIDA, we argue that smooth coping is a sequence of automatized actions intermittently interspersed with consciously mediated action selection, supplemented by dorsal stream processes. In other words, non-conscious, automatized actions (whether learned or innate) often require occasional bursts of conscious cognition to achieve the skillful and flexible adjustments of smooth coping. In addition, never-conscious dorsal stream information and associated sensorimotor processes provide further online adjustments during smooth coping. To achieve smooth coping in LIDA we introduce a new module to the LIDA cognitive architecture the Automatized Action Selection sub-module. Our complex model of smooth coping borrows notions of "embodied intelligence" from enactivism and augments these by allowing representations and more detailed mechanisms of conscious control. We explore several extended examples of smooth coping, starting from basic activities like walking and scaling up to more complex tasks like driving and short-order cooking.

Routine use of low-dose glucarpidase following high-dose methotrexate in adult patients with CNS lymphoma: an open-label, multi-center phase I study.

BACKGROUND: High-dose methotrexate (HD-MTX) has broad use in the treatment of central nervous system (CNS) malignancies but confers significant toxicity without inpatient hydration and monitoring. Glucarpidase is a bacterial recombinant enzyme dosed at 50 units (u)/kg, resulting in rapid systemic MTX clearance. The aim of this study was to demonstrate feasibility of low-dose glucarpidase to facilitate MTX clearance in patients with CNS lymphoma (CNSL). METHODS: Eight CNSL patients received HD-MTX 3 or 6 g/m2 and glucarpidase 2000 or 1000u 24 h later. Treatments repeated every 2 weeks up to 8 cycles. RESULTS: Fifty-five treatments were administered. Glucarpidase 2000u yielded > 95% reduction in plasma MTX within 15 min following 33/34 doses (97.1%) and glucarpidase 1000u yielded > 95% reduction following 15/20 doses (75%). Anti-glucarpidase antibodies developed in 4 patients and were associated with MTX rebound. In CSF, glucarpidase was not detected and MTX levels remained cytotoxic after 1 (3299.5 nmol/L, n = 8) and 6 h (1254.7 nmol/L, n = 7). Treatment was safe and well-tolerated. Radiographic responses in 6 of 8 patients (75%) were as expected following MTX-based therapy. CONCLUSIONS: This study demonstrates feasibility of planned-use low-dose glucarpidase for MTX clearance and supports the hypothesis that glucarpidase does not impact MTX efficacy in the CNS. CLINICAL TRIAL REGISTRATION: NCT03684980 (Registration date 26/09/2018).

Ivacaftor-elexacaftor-tezacaftor and tacrolimus combination in cystic fibrosis.

The CFTR modulator combination elexacaftor/tezacaftor/ivacaftor (ETI) is a genetic mutation-targeted treatment in cystic fibrosis that results in profound improvements in clinical outcomes. Each of the compounds are substrates of CYP3A4/5, the cytochrome P450 enzyme family for which tacrolimus is also a substrate. The use of these compounds in an individual with a solid organ transplant has not been previously studied and there is potential for a drug interaction. In this report, we describe a pediatric liver transplant recipient with clinical decline related to cystic fibrosis who improved substantially with ETI, without significant impact on the systemic exposure of either ETI or tacrolimus.

Between Ecological Psychology and Enactivism: Is There Resonance?

Ecological psychologists and enactivists agree that the best explanation for a large share of cognition is non-representational in kind. In both ecological psychology and enactivist philosophy, then, the task is to offer an explanans that does not rely on representations. Different theorists within these camps have contrasting notions of what the best kind of non-representational explanation will look like, yet they agree on one central point: instead of focusing solely on factors interior to an agent, an important aspect of cognition is found in the link or coupling between an agent and the external world. This link is fluid, dynamic, and active in a variety of ways, and we do not need to add any internal extra something in the perception-action-cognition process. At the same time, even devout defenders of ecological psychology and enactivism recognize that plenty happens inside an agent during cognition. In particular, no one denies that the brain plays an important role. What, then, is the role of the brain if it's not in the game of representing the environment? One possible option is to describe the brain as a resonant organ instead of a representational organ. In this paper we consider the history of resonance in more detail. Particular focus will be placed on two different sets of approaches that have developed the concept of resonance: a representational reading of resonance and a non-representational, dynamic account of resonance. We then apply these accounts to a case study on music performance, specifically in the context of standard tonal jazz. From this application, we propose that a non-representational resonance account consistent with both enactivism and ecological psychology is a viable way of explaining jazz performance. We conclude with future considerations on research regarding the brain as a resonant organ.

Genital Shedding of Human Immunodeficiency Virus Type-1 (HIV) When Antiretroviral Therapy Suppresses HIV Replication in the Plasma.

BACKGROUND: During antiretroviral treatment (ART) with plasma HIV RNA below the limit of quantification, HIV RNA can be detected in genital or rectal secretions, termed discordant shedding (DS). We hypothesized that proliferating cells produce virions without HIV replication. METHODS: ART-naive Peruvians initiating ART were observed for DS over 2 years. HIV env and pol genomes were amplified from DS. Antiretrovirals and cytokines/chemokines concentrations were compared at DS and control time points. RESULTS: Eighty-two participants had ART suppression. DS was detected in 24/82 (29%) participants: 13/253 (5%) cervicovaginal lavages, 20/322 (6%) seminal plasmas, and 6/85 (7%) rectal secretions. HIV RNA in DS specimens was near the limit of quantification and not reproducible. HIV DNA was detected in 6/13 (46%) DS cervicovaginal lavages at low levels. Following DNase treatment, 5/39 DS specimens yielded HIV sequences, all without increased genetic distances. Women with and without DS had similar plasma antiretroviral levels and DS in 1 woman was associated with inflammation. CONCLUSIONS: HIV RNA and DNA sequences and therapeutic antiretroviral plasma levels did not support HIV replication as the cause of DS from the genital tract. Rather, our findings infer that HIV RNA is shed due to proliferation of infected cells with virion production.

Variable cellular ivacaftor concentrations in people with cystic fibrosis on modulator therapy.

The development of CFTR modulators has transformed the care of patients with cystic fibrosis (CF). Although the clinical efficacy of modulators depends on their concentrations in target tissues, the pharmacokinetic properties of these drugs in epithelia are not utilized to guide patient care. We developed assays to quantitate ivacaftor in cells and plasma from patients on modulator therapy, and our analyses revealed that cellular ivacaftor concentrations differ from plasma concentrations measured concurrently, with evidence of in vivo accumulation of ivacaftor in the cells of patients. While the nature of this study is exploratory and limited by a small number of patients, these findings suggest that techniques to measure modulator concentrations in vivo will be essential to interpreting their clinical impact, particularly given the evidence that ivacaftor concentrations influence the activity and stability of restored CFTR protein.

Transdermal Delivery of Enfuvirtide in a Porcine Model Using a Low-Frequency, Low-Power Ultrasound Transducer Patch.

Ultrasound-mediated transdermal delivery is a promising parenteral administration method for large-molecule or unstable medications. This study evaluated skin health and systemic delivery when administering enfuvirtide, an injectable anti-retroviral medication, over a 1-mo period in a porcine model using a low-frequency cymbal transducer. Three groups received twice-daily treatments: (i) enfuvirtide injection control (n = 12); (ii) saline ultrasound control (n = 6); and (iii) enfuvirtide ultrasound treatment (n = 13). Ultrasound parameters were as follows: 30-min exposure, 90 mW/cm², 24-26 kHz and 15% duty cycle. No statistical difference in trans-epidermal water loss, a measure of skin health and function, was seen between ultrasound-treated and control skin sites for either saline (p = 0.50) or enfuvirtide (p = 0.29) groups. Average trough plasma concentrations of enfuvirtide were 0.6 ± 0.2 and 2.8 ± 0.8 µg/mL for ultrasound and injection, respectively. Tolerability and efficacy results indicate that chronic, low-frequency ultrasound exposure can be a practical means for transdermal delivery of medications such as enfuvirtide.

HIV RNA persists in rectal tissue despite rapid plasma virologic suppression with dolutegravir-based therapy.

OBJECTIVES: Despite plasma virologic suppression with antiretroviral therapy (ART), HIV persists in gut tissue. The objectives of this study were to compare plasma and rectal tissue HIV RNA dynamics and to assess relationships with dolutegravir (DTG) plasma and tissue concentrations. DESIGN: A longitudinal cohort study of HIV-infected treatment-naïve individuals initiating DTG-based ART was conducted over 12 weeks with plasma and rectal tissue sampling (Clinicaltrials.gov:NCT02924389). METHODS: HIV RNA and DTG concentrations were quantified in plasma and rectal tissue samples collected pre-ART (baseline) and post-ART at weeks 2, 6, and 12 using Abbott Real-Time HIV-1 assays and high-performance liquid chromatography tandem mass spectroscopy, respectively. Relationships between rectal tissue RNA and DTG concentrations were modeled using binary logistic regression, controlling for repeated measures. RESULTS: Twelve participants were enrolled: six (50.0%) women, nine (75.0%) black, median age 42.0 years (Q1 31.2, Q3 52.0). All attained plasma virologic suppression by week 6. 11 of 12 (91.7%) had detectable rectal tissue HIV RNA at baseline, and only three of 11 (27.3%) achieved rectal tissue virologic suppression at any time-point. Compared with rectal tissue nonsuppressors, three of three (100.0%) of rectal tissue suppressors were women, had higher BMI, 35.9 kg/m (range 24.9-38.5) versus 20.6 (17.7-29.9), P = 0.05, and lower baseline log plasma HIV RNA: 3.7 copies/ml (range 3.6-4.4) versus 4.7 (3.8-5.4), P = 0.02. No significant relationships between rectal tissue RNA suppression and DTG concentrations were seen. CONCLUSION: Rectal tissue HIV RNA persisted in most participants and was not predicted by DTG concentrations. Impact of host factors, particularly sex, on tissue HIV viral dynamics warrants further exploration.

Monotypic low-level HIV viremias during antiretroviral therapy are associated with disproportionate production of X4 virions and systemic immune activation.

OBJECTIVE: During effective antiretroviral therapy (ART), low-level plasma viremias (LLV) (HIV RNA >30-1000 copies/ml) can be detected intermittently. We hypothesized that systemic inflammation is associated with LLV either as the cause or result of the production of virions from clonally expanded cells. METHODS: Prospective cohort study of HIV-infected ART-naive Peruvians enrolled prior to ART and followed for 2 years. Plasma HIV RNA and peripheral blood mononuclear cell (PBMC) HIV DNA concentrations were quantified pre-ART from individuals whose plasma HIV RNA was ART-suppressed. Inflammatory biomarker concentrations were measured pre and during ART. Single-genome amplification (SGA) derived HIV env and pol genotypes from pre-ART and LLV specimens. Antiretroviral levels during ART assessed adherence. Statistical associations and phylogenetic relationships were examined. RESULTS: Among 82 participants with median plasma HIV RNA less than 30 copies/ml, LLV were detected in 33 of 82 (40%), with a LLV median HIV RNA of 73 copies/ml. Participants with vs. without LLV had significantly higher pre-ART plasma HIV RNA (P < 0.001) and PBMC HIV DNA (P < 0.007); but, during ART, their antiretroviral drug levels were similar. LLV env sequences were monotypic in 17 of 28 (61%) and diverse in 11 of 28 (39%) participants. Those with the monotypic vs. diverse LLV pattern had elevated hsCRP and sCD163 (P = 0.004) and LLV with more X4 variants (P = 0.02). CONCLUSION: In individuals with monotypic LLV sequences, higher levels of pre-ART HIV DNA and RNA, systemic inflammation and X4 viruses suggest an interaction between inflammation and the production of virions from proliferating infected cells, and that naïve T cells may be a source of LLV.

G protein-coupled estrogen receptor regulates embryonic heart rate in zebrafish.

Estrogens act by binding to estrogen receptors alpha and beta (ERα, ERß), ligand-dependent transcription factors that play crucial roles in sex differentiation, tumor growth and cardiovascular physiology. Estrogens also activate the G protein-coupled estrogen receptor (GPER), however the function of GPER in vivo is less well understood. Here we find that GPER is required for normal heart rate in zebrafish embryos. Acute exposure to estrogens increased heart rate in wildtype and in ERα and ERß mutant embryos but not in GPER mutants. GPER mutant embryos exhibited reduced basal heart rate, while heart rate was normal in ERα and ERß mutants. We detected gper transcript in discrete regions of the brain and pituitary but not in the heart, suggesting that GPER acts centrally to regulate heart rate. In the pituitary, we observed gper expression in cells that regulate levels of thyroid hormone triiodothyronine (T3), a hormone known to increase heart rate. Compared to wild type, GPER mutants had reduced levels of T3 and estrogens, suggesting pituitary abnormalities. Exposure to exogenous T3, but not estradiol, rescued the reduced heart rate phenotype in gper mutant embryos, demonstrating that T3 acts downstream of GPER to regulate heart rate. Using genetic and mass spectrometry approaches, we find that GPER regulates maternal estrogen levels, which are required for normal embryonic heart rate. Our results demonstrate that estradiol plays a previously unappreciated role in the acute modulation of heart rate during zebrafish embryonic development and suggest that GPER regulates embryonic heart rate by altering maternal estrogen levels and embryonic T3 levels.

The impact of growth promoters on muscle growth and the potential consequences for meat quality.

To meet the demands of increased global meat consumption, animal production systems will have to become more efficient, or at least maintain the current efficiency utilizing feed ingredients that are not also used for human consumption. Use of growth promoters is a potential option for increasing production animal feed efficiency and increased muscle growth. The objective of this manuscript is to describe the mechanisms by which the growth promoters, beta-adrenergic agonists and growth hormone, mediate their effects, with specific consideration of the aspects which have implications for meat quality.

Dose-dependent effects of vitamin D on transdifferentiation of skeletal muscle cells to adipose cells.

Fat infiltration within muscle is one of a number of features of vitamin D deficiency, which leads to a decline in muscle functionality. The origin of this fat is unclear, but one possibility is that it forms from myogenic precursor cells present in the muscle, which transdifferentiate into mature adipocytes. The current study examined the effect of the active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), on the capacity of the C2C12 muscle cell line to differentiate towards the myogenic and adipogenic lineages. Cells were cultured in myogenic or adipogenic differentiation media containing increasing concentrations (0, 10⁻¹³, 10⁻¹¹, 10⁻9, 10⁻7 or 10⁻5  M) of 1,25(OH)2D3 for up to 6 days and markers of muscle and fat development measured. Mature myofibres were formed in both adipogenic and myogenic media, but fat droplets were only observed in adipogenic media. Relative to controls, low physiological concentrations (10⁻¹³ and 10⁻¹¹  M) of 1,25(OH)2D3 increased fat droplet accumulation, whereas high physiological (10⁻9  M) and supraphysiological concentrations (≥10⁻7  M) inhibited fat accumulation. This increased accumulation of fat with low physiological concentrations (10⁻¹³ and 10⁻¹¹  M) was associated with a sequential up-regulation of PPARγ2 (PPARG) and FABP4 mRNA, indicating formation of adipocytes, whereas higher concentrations (≥10⁻9  M) reduced all these effects, and the highest concentration (10⁻5  M) appeared to have toxic effects. This is the first study to demonstrate dose-dependent effects of 1,25(OH)2D3 on the transdifferentiation of muscle cells into adipose cells. Low physiological concentrations (possibly mimicking a deficient state) induced adipogenesis, whereas higher (physiological and supraphysiological) concentrations attenuated this effect.

The Effects of Prenatal Protein Restriction on ß-Adrenergic Signalling of the Adult Rat Heart during Ischaemia Reperfusion.

A maternal low-protein diet (MLP) fed during pregnancy leads to hypertension in adult rat offspring. Hypertension is a major risk factor for ischaemic heart disease. This study examined the capacity of hearts from MLP-exposed offspring to recover from myocardial ischaemia-reperfusion (IR) and related this to cardiac expression of ß-adrenergic receptors (ß-AR) and their associated G proteins. Pregnant rats were fed control (CON) or MLP diets (n = 12 each group) throughout pregnancy. When aged 6 months, hearts from offspring underwent Langendorff cannulation to assess contractile function during baseline perfusion, 30 min ischemia and 60 min reperfusion. CON male hearts demonstrated impaired recovery in left ventricular pressure (LVP) and dP/dt(max) (P < 0.01) during reperfusion when compared to MLP male hearts. Maternal diet had no effect on female hearts to recover from IR. MLP males exhibited greater membrane expression of ß(2)-AR following reperfusion and urinary excretion of noradrenaline and dopamine was lower in MLP and CON female rats versus CON males. In conclusion, the improved cardiac recovery in MLP male offspring following IR was attributed to greater membrane expression of ß(2)-AR and reduced noradrenaline and dopamine levels. In contrast, females exhibiting both decreased membrane expression of ß(2)-AR and catecholamine levels were protected from IR injury.