The Performance of an Artificial Neural Network Model in Predicting the Early Distribution Kinetics of Propofol in Morbidly Obese and Lean Subjects.

BACKGROUND: Induction of anesthesia is a phase characterized by rapid changes in both drug concentration and drug effect. Conventional mammillary compartmental models are limited in their ability to accurately describe the early drug distribution kinetics. Recirculatory models have been used to account for intravascular mixing after drug administration. However, these models themselves may be prone to misspecification. Artificial neural networks offer an advantage in that they are flexible and not limited to a specific structure and, therefore, may be superior in modeling complex nonlinear systems. They have been used successfully in the past to model steady-state or near steady-state kinetics, but never have they been used to model induction-phase kinetics using a high-resolution pharmacokinetic dataset. This study is the first to use an artificial neural network to model early- and late-phase kinetics of a drug. METHODS: Twenty morbidly obese and 10 lean subjects were each administered propofol for induction of anesthesia at a rate of 100 mg/kg/h based on lean body weight and total body weight for obese and lean subjects, respectively. High-resolution plasma samples were collected during the induction phase of anesthesia, with the last sample taken at 16 hours after propofol administration for a total of 47 samples per subject. Traditional mammillary compartment models, recirculatory models, and a gated recurrent unit neural network were constructed to model the propofol pharmacokinetics. Model performance was compared. RESULTS: A 4-compartment model, a recirculatory model, and a gated recurrent unit neural network were assessed. The final recirculatory model (mean prediction error: 0.348; mean square error: 23.92) and gated recurrent unit neural network that incorporated ensemble learning (mean prediction error: 0.161; mean square error: 20.83) had similar performance. Each of these models overpredicted propofol concentrations during the induction and elimination phases. Both models had superior performance compared to the 4-compartment model (mean prediction error: 0.108; mean square error: 31.61), which suffered from overprediction bias during the first 5 minutes followed by under-prediction bias after 5 minutes. CONCLUSIONS: A recirculatory model and gated recurrent unit artificial neural network that incorporated ensemble learning both had similar performance and were both superior to a compartmental model in describing our high-resolution pharmacokinetic data of propofol. The potential of neural networks in pharmacokinetic modeling is encouraging but may be limited by the amount of training data available for these models.

GABA Measurement in a Neonatal Fragile X Syndrome Mouse Model Using 1H-Magnetic Resonance Spectroscopy and Mass Spectrometry.

Fragile X syndrome (FXS) is the leading monogenetic cause of autism spectrum disorder and inherited cause of intellectual disability that affects approximately one in 7,000 males and one in 11,000 females. In FXS, the Fmr1 gene is silenced and prevents the expression of the fragile X mental retardation protein (FMRP) that directly targets mRNA transcripts of multiple GABAA subunits. Therefore, FMRP loss adversely impacts the neuronal firing of the GABAergic system which creates an imbalance in the excitatory/inhibitory ratio within the brain. Current FXS treatment strategies focus on curing symptoms, such as anxiety or decreased social function. While treating symptoms can be helpful, incorporating non-invasive imaging to evaluate how treatments change the brain's biology may explain what molecular aberrations are associated with disease pathology. Thus, the GABAergic system is suitable to explore developing novel therapeutic strategies for FXS. To understand how the GABAergic system may be affected by this loss-of-function mutation, GABA concentrations were examined within the frontal cortex and thalamus of 5-day-old wild type and Fmr1 knockout mice using both 1H magnetic resonance imaging (1H-MRS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our objective was to develop a reliable scanning method for neonatal mice in vivo and evaluate whether 1H-MRS is suitable to capture regional GABA concentration differences at the front end of the critical cortical period where abnormal neurodevelopment occurs due to FMRP loss is first detected. 1H-MRS quantified GABA concentrations in both frontal cortex and thalamus of wild type and Fmr1 knockout mice. To substantiate the results of our 1H-MRS studies, in vitro LC-MS/MS was also performed on brain homogenates from age-matched mice. We found significant changes in GABA concentration between the frontal cortex and thalamus within each mouse from both wild type and Fmr1 knockout mice using 1H-MRS and LC-MS/MS. Significant GABA levels were also detected in these same regions between wild type and Fmr1 knockout mice by LC-MS/MS, validating that FMRP loss directly affects the GABAergic system. Thus, these new findings support the need to develop an effective non-invasive imaging method to monitor novel GABAergic strategies aimed at treating patients with FXS.

The Role of Dopamine in the Collective Regulation of Foraging in Harvester Ants.

Colonies of the red harvester ant (Pogonomyrmex barbatus) differ in how they regulate collective foraging activity in response to changes in humidity. We used transcriptomic, physiological, and pharmacological experiments to investigate the molecular basis of this ecologically important variation in collective behavior among colonies. RNA sequencing of forager brain tissue showed an association between colony foraging activity and differential expression of transcripts related to biogenic amine and neurohormonal metabolism and signaling. In field experiments, pharmacological increases in forager brain dopamine titer caused significant increases in foraging activity. Colonies that were naturally most sensitive to humidity were significantly more responsive to the stimulatory effect of exogenous dopamine. In addition, forager brain tissue significantly varied among colonies in biogenic amine content. Neurophysiological variation among colonies associated with individual forager sensitivity to humidity may reflect the heritable molecular variation on which natural selection acts to shape the collective regulation of foraging.

Plasma anandamide concentrations are lower in children with autism spectrum disorder.

Background: Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by restricted, stereotyped behaviors and impairments in social communication. Although the underlying biological mechanisms of ASD remain poorly understood, recent preclinical research has implicated the endogenous cannabinoid (or endocannabinoid), anandamide, as a significant neuromodulator in rodent models of ASD. Despite this promising preclinical evidence, no clinical studies to date have tested whether endocannabinoids are dysregulated in individuals with ASD. Here, we addressed this critical gap in knowledge by optimizing liquid chromatography-tandem mass spectrometry methodology to quantitatively analyze anandamide concentrations in banked blood samples collected from a cohort of children with and without ASD (N = 112). Findings: Anandamide concentrations significantly differentiated ASD cases (N = 59) from controls (N = 53), such that children with lower anandamide concentrations were more likely to have ASD (p = 0.041). In keeping with this notion, anandamide concentrations were also significantly lower in ASD compared to control children (p = 0.034). Conclusions: These findings are the first empirical human data to translate preclinical rodent findings to confirm a link between plasma anandamide concentrations in children with ASD. Although preliminary, these data suggest that impaired anandamide signaling may be involved in the pathophysiology of ASD.

A drug repositioning approach identifies tricyclic antidepressants as inhibitors of small cell lung cancer and other neuroendocrine tumors.

UNLABELLED: Small cell lung cancer (SCLC) is an aggressive neuroendocrine subtype of lung cancer with high mortality. We used a systematic drug repositioning bioinformatics approach querying a large compendium of gene expression profiles to identify candidate U.S. Food and Drug Administration (FDA)-approved drugs to treat SCLC. We found that tricyclic antidepressants and related molecules potently induce apoptosis in both chemonaïve and chemoresistant SCLC cells in culture, in mouse and human SCLC tumors transplanted into immunocompromised mice, and in endogenous tumors from a mouse model for human SCLC. The candidate drugs activate stress pathways and induce cell death in SCLC cells, at least in part by disrupting autocrine survival signals involving neurotransmitters and their G protein-coupled receptors. The candidate drugs inhibit the growth of other neuroendocrine tumors, including pancreatic neuroendocrine tumors and Merkel cell carcinoma. These experiments identify novel targeted strategies that can be rapidly evaluated in patients with neuroendocrine tumors through the repurposing of approved drugs. SIGNIFICANCE: Our work shows the power of bioinformatics-based drug approaches to rapidly repurpose FDA-approved drugs and identifies a novel class of molecules to treat patients with SCLC, a cancer for which no effective novel systemic treatments have been identified in several decades. In addition, our experiments highlight the importance of novel autocrine mechanisms in promoting the growth of neuroendocrine tumor cells.

Blood basophils from cystic fibrosis patients with allergic bronchopulmonary aspergillosis are primed and hyper-responsive to stimulation by aspergillus allergens.

INTRODUCTION: Fifteen to sixty percent of cystic fibrosis patients harbor Aspergillus fumigatus (Af) in their airways (CF-AC) and some will develop allergic bronchopulmonary aspergillosis (CF-ABPA). Since basophils play a key role in allergy, we hypothesized that they would display alterations in CF-ABPA patients compared to CF-AC or patients without Af colonization (CF). METHODS: Using flow cytometry, we measured CD203c, CD63 and CD123 levels on basophils from CF-ABPA (N=11), CF-AC (N=14), and CF (N=12) patients before and after ex vivo stimulation with Af allergens. RESULTS: Baseline CD203c was increased in basophils from CF-ABPA compared to CF-AC and CF patients. Af extract and recombinant Aspf1 stimulated basophils from CF-ABPA patients to markedly upregulate CD203c, along with modest upregulation of CD63 and a CD123 downward trend. Plasma TARC/CCL17 at baseline and post-stimulation cell supernatant histamine levels were similar in the three groups. CONCLUSIONS: In CF-ABPA, blood basophils are primed and hyperresponsive to Af allergen stimulation.

Neural precursor cell-expressed developmentally down-regulated protein 4-2 (Nedd4-2) regulation by 14-3-3 protein binding at canonical serum and glucocorticoid kinase 1 (SGK1) phosphorylation sites.

Regulation of epithelial Na(+) channel (ENaC)-mediated transport in the distal nephron is a critical determinant of blood pressure in humans. Aldosterone via serum and glucocorticoid kinase 1 (SGK1) stimulates ENaC by phosphorylation of the E3 ubiquitin ligase Nedd4-2, which induces interaction with 14-3-3 proteins. However, the mechanisms of SGK1- and 14-3-3-mediated regulation of Nedd4-2 are unclear. There are three canonical SGK1 target sites on Nedd4-2 that overlap phosphorylation-dependent 14-3-3 interaction motifs. Two of these are termed "minor," and one is termed "major," based on weak or strong binding to 14-3-3 proteins, respectively. By mass spectrometry, we found that aldosterone significantly stimulates phosphorylation of a minor, relative to the major, 14-3-3 binding site on Nedd4-2. Phosphorylation-deficient minor site Nedd4-2 mutants bound less 14-3-3 than did wild-type (WT) Nedd4-2, and minor site Nedd4-2 mutations were sufficient to inhibit SGK1 stimulation of ENaC cell surface expression. As measured by pulse-chase and cycloheximide chase assays, a major binding site Nedd4-2 mutant had a shorter cellular half-life than WT Nedd4-2, but this property was not dependent on binding to 14-3-3. Additionally, a dimerization-deficient 14-3-3ε mutant failed to bind Nedd4-2. We conclude that whereas phosphorylation at the Nedd4-2 major site is important for interaction with 14-3-3 dimers, minor site phosphorylation by SGK1 may be the relevant molecular switch that stabilizes Nedd4-2 interaction with 14-3-3 and thus promotes ENaC cell surface expression. We also propose that major site phosphorylation promotes cellular Nedd4-2 protein stability, which potentially represents a novel form of regulation for turnover of E3 ubiquitin ligases.

Azetidine-2-carboxylic acid in the food chain.

Azetidine-2-carboxylic acid (Aze) 1 is a non-protein amino acid present in sugar beets and in table beets (Beta vulgaris). It is readily misincorporated into proteins in place of proline 2 in many species, including humans, and causes numerous toxic effects as well as congenital malformations. Its role in the pathogenesis of disease in humans has remained unexplored. Sugar beet agriculture, especially in the Northern Hemisphere, has become widespread during the past 150 years, and now accounts for nearly 30% of the world's supply of sucrose. Sugar beet byproducts are also used as a dietary supplement for livestock. Therefore, this study was undertaken as an initial survey to identify Aze-containing links in the food chain. Herein, we report the presence of Aze 1 in three sugar beet byproducts that are fed to farm animals: sugar beet molasses, shredded sugar beet pulp, and pelleted sugar beet pulp.

Azetidine-2-carboxylic acid in garden beets (Beta vulgaris).

Azetidine-2-carboxylic acid (L-Aze) is a toxic and teratogenic non-protein amino acid. In many species, including man, L-Aze is misincorporated into protein in place of proline, altering collagen, keratin, hemoglobin, and protein folding. In animal models of teratogenesis, it causes a wide range of malformations. The role of L-Aze in human disease has been unexplored, probably because the compound has not been associated with foods consumed by humans. Herein we report the presence of L-Aze in the garden or table beet (Beta vulgaris).

Direct proteomic mapping of the lung microvascular endothelial cell surface in vivo and in cell culture.

Endothelial cells can function differently in vitro and in vivo; however, the degree of microenvironmental modulation in vivo remains unknown at the molecular level largely because of analytical limitations. We use multidimensional protein identification technology (MudPIT) to identify 450 proteins (with three or more spectra) in luminal endothelial cell plasma membranes isolated from rat lungs and from cultured rat lung microvascular endothelial cells. Forty-one percent of proteins expressed in vivo are not detected in vitro. Statistical analysis measuring reproducibility reveals that seven to ten MudPIT measurements are necessary to achieve > or =95% confidence of analytical completeness with current ion trap equipment. Large-scale mapping of the proteome of vascular endothelial cell surface in vivo, as demonstrated here, is advisable because distinct protein expression is apparently regulated by the tissue microenvironment that cannot yet be duplicated in standard cell culture.

Subtractive proteomic mapping of the endothelial surface in lung and solid tumours for tissue-specific therapy.

The molecular complexity of tissues and the inaccessibility of most cells within a tissue limit the discovery of key targets for tissue-specific delivery of therapeutic and imaging agents in vivo. Here, we describe a hypothesis-driven, systems biology approach to identifying a small subset of proteins induced at the tissue-blood interface that are inherently accessible to antibodies injected intravenously. We use subcellular fractionation, subtractive proteomics and bioinformatics to identify endothelial cell surface proteins exhibiting restricted tissue distribution and apparent tissue modulation. Expression profiling and gamma-scintigraphic imaging with antibodies establishes two of these proteins, aminopeptidase-P and annexin A1, as selective in vivo targets for antibodies in lungs and solid tumours, respectively. Radio-immunotherapy to annexin A1 destroys tumours and increases animal survival. This analytical strategy can map tissue- and disease-specific expression of endothelial cell surface proteins to uncover novel accessible targets useful for imaging and therapy.