bluuebunny/crossref_metadata_2025 huggingface dataset:
Co‐modulation of the Na+/K+ pump and hyperpolarization‐activated currents as a mechanism for robust neuromodulation
Authors: Ellingson Parker , Barnett William Halbert , Vargas Alex , Kueh Daniel , Calabrese Ronald , Cymbalyuk Gennady
Published: April 2020

Specialized oscillatory circuits, central pattern generators (CPGs), control rhythmic motor behaviors such as locomotion and breathing. To accommodate a motor pattern to environmental changes and behavioral goals, neuromodulators adjust the dynamics of CPGs by orchestrating changes in various ionic currents in a wide range of their biophysical parameters to expand temporal characteristics of the functional pattern. Recent studies provide evidence that the Na+/K+ pump contributes to the dynamics of CPGs and is controlled by neuromodulation
[1–
4]. In the leech heartbeat CPG, the neuropeptide myomodulin reduces the period of bursting activity by increasing the hyperpolarization‐activated (h)‐current and decreasing the Na+/K+ pump current
[4]. The application of myomodulin reduces the period of oscillatory activity by 17%. Application of Cs+, which is an h‐current blocker, increases the period of bursting by 24% relative to control. The application of myomodulin along with Cs+ decreases the period by 12% relative to treatment with Cs+
[4]. Here we investigate how the period of a bursting can be controlled in a wide range while functional bursting is maintained with a focus on the role of the Na+/K+ pump. We also investigate the risk associated with multistability in neuronal dynamics.

We optimized a model of the leech heart interneuron (HN), which includes the Na+/K+ pump current and intracellular Na+ dynamics
[1] and investigated the activity regimes of pairs of mutually inhibitory coupled HNs forming half‐center oscillators (HCOs). HCOs form the kernel of the leech heartbeat CPG. We investigated eight model variants representing combinations of three experimental treatments: the blockade of chemical synapses representing the application of bicuculline, the blockade of h‐current representing the application of Cs+, and the enhancement of the h‐current and inhibition of the Na+/K+ pump current representing the application of myomodulin. The model captures the qualitative trends in change of cycle period observed in experiments with myomodulin and Cs+. We found ranges of parameters where neurons showed functional bursting. The coordinated changes of maximal conductance of h‐current (Gh) and maximal pump activity (IPumpMax) increases the range of period of functional bursting as well as the range of parameters Gh and IPumpMax. We hypothesize that myomodulin co‐modulates h‐ and pump currents to expand the domain of the functional activity.

Support or Funding Information

Supported by NINDS 1 R01 NS085006 to RLC and 1 R21 NS111355 to RLC and GSC.
Mechanistic Insights Into RNA Splicing As A Trigger For Arrhythmogenic Right Ventricular Cardiomyopathy
Authors: Bradford William H. , Liang Yan , Mataraarachchi Nirosh , Gu Yusu , Dalton Nancy D. , Peterson Kirk L. , Sheikh Farah
Published: April 2020

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic cardiac disease that causes sudden death in young adults and athletes. ARVC is termed a “disease of the desmosome” as 40% of mutations in ARVC patients are found in the cardiac desmosomal components, with plakophilin‐2 (PKP2) being the most frequently mutated desmosomal gene in ARVC. Evidence from humans suggests that altered RNA splicing may be a critical mechanism through which PKP2 patient genetics drive ARVC. Studies have suggested that approximately one third of all human disease‐causing mutations are a result of defects in RNA splicing; highlighting the importance of testing the relevance of this mechanism in human ARVC. However, there are no models and limited mechanistic insights into how mutations in RNA splicing impact ARVC disease pathogenesis. We hypothesize that RNA splicing mutations in PKP2 are sufficient to recapitulate classic disease features associated with human ARVC and can provide valuable mechanistic insight into how altered RNA splicing impacts disease. Through CRISPR‐Cas9 we generated a novel knock‐in mouse model harboring a human equivalent PKP2 mutation (IVS10‐1 G>C) that impacts RNA splicing and is sufficient to recapitulate all classic ARVC disease features. PKP2 homozygous mutant (PKP2 Hom) mice are viable at birth yet display adult hallmarks of ARVC including ventricular arrhythmias, right and left ventricular dysfunction, and fibro‐fatty replacement of myocardium leading to sudden death. RNA and sequencing analyses of exons spanning the PKP2 mutation site reveals a larger PKP2 transcript that retains a 54 base pair portion of the intronic sequence in PKP2 Hom hearts. However, RNA analysis of exons outside the PKP2 mutation site reveals PKP2 RNA transcripts at similar levels to wild type PKP2, suggesting that total RNA levels are not impacted by the mutation. Instead, we show that PKP2 Hom hearts express a higher molecular weight mutant PKP2 protein in the absence of endogenous PKP2. Analysis of PKP2 Hom neonatal hearts and cardiomyocytes reveal early desmosomal protein loss coupled with a susceptibility to baseline arrhythmias in the absence of overt cardiac disease features at this stage (e.g., no changes in heart weight/body weight ratios, no upregulation of cardiac stress and fibrotic markers). We provide a novel mouse model that highlights the sufficiency and molecular consequences of a PKP2 RNA splicing mutation that triggers all of the classic early and adult onset disease features associated with human ARVC.

Support or Funding Information

National Science Foundation (Graduate Research Fellowship Program), National Institutes of Health (NIH R01, HL142251‐A1)
Neuromodulation of afferent fibers for Postural Orthostatic Trachycardia Syndrome (POTS)
Authors: Rhoads Marie , Harper Rebecca K. , Snodgrass Danielle , Yan-Go Frisca , Sauerland Eberhardt K. , Harper Ronald
Published: April 2020

Postural Orthostatic Trachycardia Syndrome, or POTS, is characterized by low arterial blood pressure, inappropriate cardiovascular and breathing responses to tilt, dizziness and syncope to sudden standing, and a range of other autonomic signs, including thermoregulatory issues in the periphery and frequent manifestations of migraine pain. Interventions are few, and typically include volume repletion by saline and enhanced fluid intake. In the course of evaluating neuromodulatory procedures for migraine pain (n=62), several subjects showed classic POTS symptoms, many of which spontaneously resolved with the intervention for migraine. Non‐invasive neuromodulation of two different areas, one being the auditory canal containing mandibular nerve V3, cranial nerves VII, IX, and X and cervical nerves C2 and C3; and the other being regional stimulation of V1 and V2, together with C2 and C3 sensory areas, caused significant declines in dizziness, hand cooling, and other POTS symptoms. The neuromodulatory device stimulating the auditory canal consisted of bilateral silicon impressions of the auditory canals, each containing a mechanical vibratory device which reached a range of cranial nerves, and especially the IXth (glossopharyngeal) nerve to the carotid baroreceptors. The other neuromodulatory device applied vibrating signals to the cutaneous surface over areas innervated by V1 and V2. The trials consisted of a 10‐min control period, followed by 25‐min stimulation, and finished with a 10‐min control period. Continuous thoracic wall movement, pulse oximetry, and ECG were collected, and beat‐by‐beat blood pressure values were inferred from the pulse transit time of the cardiac R wave and oximetry signal. All procedures were approved by the UCLA Institutional Review Board. Subjects who began the trial with low blood pressure increased systolic and diastolic pressures. In two subjects, repeated trials benefitted critical signs (hand warming, dizziness, syncope to sudden movement). These findings suggest that repeated activation of afferent input to baroreceptors can elicit both short‐ and long‐term effects on blood pressure control. The means by which this “learning" or adaptation of cardiovascular regulatory systems remain unknown, but raise the possibility that a low‐cost, non‐invasive intervention can provide another means to restore effective blood pressure control in a syndrome with few options for treatment approaches.

Support or Funding Information

This research was supported in part by the Fidelity Charitable Nancy Adams and Scott Schoen Fund and the Kraig and Linda Kupiec Family Trust.
Assessment of CYP2A6 and CYP2C19 Genetic Variation on Cotinine‐N‐Oxide Formation and their Potential Effect on the Nicotine Metabolic Ratio (NMR)
Authors: Perez Paramo Yadira X. , Watson Christy J.W. , Chen Gang , Natesan Senthil , Lazarus Philip
Published: April 2020

Significance

The nicotine metabolic ratio (NMR) of 3‐hydroxycotinine (3HC)/cotinine in smokers is a determinant of CYP2A6 activity and is widely used in clinical practice to help select smoking cessation pharmacotherapy. The major mode of metabolism of nicotine is via the formation of cotinine and subsequently to 3HC by the enzyme CYP2A6. Cotinine is also metabolized to cotinine‐N‐glucuronide and to cotinine‐N‐oxide (COX), accounting for ~7 and 5% of urinary nicotine metabolites, respectively, in smokers. The goal of the present study was to investigate the enzymes that catalyze COX formation and determine whether genetic variation in these enzymes may affect this pathway.

Methods

We used specific inhibitors of the major hepatic cytochrome P450 (CYP) enzymes including CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 in cotinine‐N‐oxidation reactions using pooled human liver microsomes (HLM). COX formation was monitored by UPLC‐MS/MS, and enzyme kinetic analysis was performed using microsomal fractions from CYP‐overexpressing HEK293 cell lines. Additionally, genotype‐phenotype analysis was performed for a panel of 113 HLM specimens.

Results

Inhibition of COX formation was only observed when using inhibitors of CYPs 2A6, 2B6, 2C19, 2E1, and 3A4. CYPs 2A6 and 2C19 exhibited similar oxidation activity against cotinine with Vmax/KM values of 4.4, and 4.2 nL•min−1•mg−1, respectively; the Vmax/KM value for the 2B6 isoform was of 2.2 nL•min−1•mg−1. The activity observed for both CYPs 2E1 and 3A4 were low, with KM’s greater than 10 mM. We measured the levels of COX formation in 113 HLM specimens and genotyped variants in CYPs 2A6 (*2, *9, and *14), 2B6 (*2, *5, *9, rs4803419 and rs8109525) and 2C19 (*2 and *17) using qPCR. An association between decreased levels of COX formation and CYP variants in the 2C19 (up to 35% reduction) and 2A6 (up to 38% reduction) isoforms was observed. No statistically significant correlation was observed with variants in the 2B6 isoform.

Conclusions

These results demonstrate that genetic variants in CYPs 2A6 and 2C19 are associated with decreased COX formation, potentially affecting the levels of free cotinine and consequently the calculated NMR in smokers and clinical practices surrounding smoking cessation therapies.

Support or Funding Information

This work is supported by grants from NIH, National Institutes of Environmental Health Sciences (grant R01‐ES025460; to P. Lazarus), the Fulbright‐Garcia Robles Program and CONACyT I2T2 (to Y. Perez‐Paramo), Health Sciences and Services Authority of Spokane, Washington (grant WSU002292 to College of Pharmacy and Pharmaceutical Sciences, Washington State University)
Hyperoxic Lung Injury in Newborn Mice is Abrogated by Neutralizing mAb 2A11 to Gastrin‐Releasing Peptide (GRP)
Authors: Sunday Mary E. , Cuttitta Frank , Cambey Colette
Published: April 2020

Rationale

Gastrin‐releasing peptide (GRP) promotes fetal lung development in mice and baboons. Postnatally, GRP levels in lung normally drop, but rise in newborns who later develop bronchopulmonary dysplasia (BPD), chronic lung disease of newborns, with interstital fibrosis and arrested alveolarization. In premature baboons, blocking anti‐GRP mAb2A11 abrogated BPD. Rodent BPD models could help to clarify mechanisms by which GRP mediates BPD. Mice are born at the canalicular stage of lung development, ~24–28 wks human gestation, when BPD peaks.

Methods

Newborn (NB) timed‐pregnant Swiss Webster mice were treated with continuous 75% O2 and twice weekly injections of mAb MOPC21 (negative control IgG1) or mAb2A11. On day 18, right lungs were inflation‐fixed and paraffin embedded. Myofibroblasts were immunostained with alkaline‐phosphatase‐conjugated mAb1A4 (Sigma‐Aldrich), developed with Vector Red. Eight‐ten random lung 20x images were captured per mouse. Using ImageJ, the area‐percent of SMA+ alveolar tissue was determined as: (area of SMA+ alveolae)/(alveolar tissue) ×100%. Mean linear intercept (MLI) was used to determine alveolar size with a fixed grid with lines totaling 1‐mm: 1/(#air‐tissue interfaces)×1000 = mean distance between two air‐tissue interfaces in microns. Data are shown as mean ±SEM, with significance at p < 0.05 (one‐way t‐test).

Results

Normoxic (NO) mice ± MOPC or 2A11, had area‐percent SMA+ alveoli unchanged (5.1–6.3 ±0.5–1.4). Hyperoxia+MOPC led to 3‐fold increased SMA (15.6±2%). Hyperoxia+2A11 reduced SMA to 10.3±1.2%; P<0.01 comparing hyperoxia+2A11 vs. hyperoxia+MOPC. 2A11 had no effect on MLI with either normoxia or hyperoxia, possibly due to insufficient time. We performed Affymetrix cDNA analysis of lung RNA from hyperoxic mice +2A11 or MOPC for 18d. HO+2A11 vs. HO+MOPC altered expression of >1000 genes (FC>10, P<0.05), many potentially protective vs. lung injury/fibrosis.
Increased gene expression included: nucleoredoxin, an antioxidant related to thioredoxin (FC x78); surfactant protein A (FC x140); Tom20, mitochondrial transport protein that reduces lung injury (x74), and multiple other mitochondrial genes (>20x).
Reduced gene expression included: Adam10, profibrotic; amphiregulin, elevated in BPD; amyloid beta A4, toxic fragment; laminin B1, which usually declines by birth; Tgfb1i4, profibrotic; and Stat1, profibrotic.

Conclusions

After 18d exposure to 75% O2, newborn mice have >3‐fold more interstitial myofibroblasts that are significantly reduced by mAb2A11. Many pulmonary genes are up‐ or down‐regulated by mAb2A11 consistent with a multigenic downstream effect on reducing interstitial fibrosis via interacting signaling pathways. Mice can provide a useful model for carrying out preclinical investigations.

Support or Funding Information

This work was supported by NIH grants #R01‐HL44984 (M.E.S.), U01‐HL52638 (M.E.S.), and a Scientific Research Bequest from the Jenny Lillian Semans Koortbojian Trust Fund
Cerebro, the Virtual Lab ‐ a mobile neuroanatomy game that is both fun and educational
Authors: Marais Olivia , Tourigny Katherine , Lellis-Santos Camilo , Burke Kim , Pucci Niccolo , Riisager Oliver , Jeon Sean , Zhang Weiyi , Lastovikova Julia , Kopak Jeannette , Krebs Claudia
Published: April 2020

Neuroanatomy remains one of the more complex disciplines in anatomical sciences education. It requires both a deep understanding of 3D anatomical relationships within the brain and a translation of that knowledge to 2D slices through the brain – an important skill, which enables the interpretation of clinical imaging studies of the brain. An additional challenge for learners is to understand the anatomy of the main ascending and descending tracts through the brainstem as well as the location of the crossing to the contralateral side for these pathways. Mastery of neuroanatomy requires both a conceptual understanding and practice in identifying structures with confidence on slices through the brainstem and the forebrain.

Gamification of learning is a concept that aims to harness the fun and rewarding qualities of a game to make learning appear effortless.

In this project we put together a multidisciplinary team of neuroanatomy content experts, game & software designers, and artists to develop a neuroanatomy game that is easy to use and yields learning results for students. The target audience was identified and characterized and the user experience and user interface were designed for this target audience. Iterative, agile design cycles with multiple sessions of user testing resulted in a final product that serves the needs of undergraduate students seeking a high yield return on their time investment for learning. A narrative arc was developed for the game and chapters with three levels from beginner to expert were created based on the learning objectives of typical neuroanatomy lab sessions. Principles of design thinking and iterative software design were applied in the creation of this game so that the final product would meet both the learning objectives of the course and the objective to keep the game fun and competitive.

The game was designed to fit into the existing learning ecosystem for neuroanatomy, which includes interactive online resources, 3D experiences, and lab sessions that are integrated with clinical sciences.
The Role of Magnesium Biodegradable Material in Peripheral Nerve Repair
Authors: Abu-Niaaj Lubna F. , Harris Greg , Pixley Sarah
Published: April 2020

Peripheral nerve injuries can greatly diminish a patient’s quality of life. The use of permanent metallic material implants, including stainless steel, introduce a toxicity risk, due to release of allergenic particles upon chronic use, The significant difference in hardness versus bodily tissues is also unsatisfactory for long‐time placement. Biodegradable biomaterial scaffolds are an innovative alternative therapeutic methodology to enhance tissue repair. Magnesium is a biodegradable metal which has received significant interest recently because it is both closer in hardness to tissues and it biodegrades safely, releasing Mg ions, playing many beneficial roles in the human body. Mg ions regulate ion channel opening, DNA replication, protein synthesis and are a crucial cofactor for >300 enzymes. Serum deficiencies of Mg result in general inflammation and are implicated in disorders such as Parkinson’s and Alzheimer’s diseases. Thus, Mg metal biodegradable implants have great promise as a new generation of tissue repair materials. In vivo studies in the Pixley lab have shown that peripheral nerve axons regenerating across an injury gap, react positively to the presence of the Mg, including giving indications that they may be drawn towards the magnesium wire within the tissue. The long term goal is to understand cellular reactions to magnesium (Mg) metal, which will aid the development of a clinical application in peripheral nerve repair.

Objectives

Toward investigating the underlying cellular mechanisms, this study sought to 1) design in vitro cell culture systems to determine the effects of selected types of Mg metal wires being tested for use in clinical practice. Then, 2) we sought to determine the in vitro responses to Mg metal by two cell types that are important in peripheral nerve repair: Schwann cells and the class of immune cells, macrophages, that are the first to attach to any implant material and that also play a large role in nerve repair.

Materials and Methods

Cellular interactions with the selected Mg wires were studied in vitro using immunostaining and phagocytosis assays. The cells analyzed were mouse Schwannoma cells (RT4‐D6P2T) and a mouse macrophage cell line (RAW264.7).

Results

Schwannoma cells showed aggregates around the edge of the immobilized Mg wires, though, no significant migration towards/away from Mg wires, but macrophages appear to react to the Mg wires. The research is continuing to determine the mechanism of cellular interactions with the Mg metal, and whether these interactions would influence the quality of the peripheral nerve repair.

Support or Funding Information

Funding was provided by the NSF ERC RMB EEC‐0812348
Studies of Continuous Lorcaserin Plus Buprenorphine in Rat Fentanyl Self‐Administration
Authors: Merritt Christina , Stutz Sonja , Fox Robert , Anastasio Noelle , Moeller F. Gerard , Cunningham Kathryn
Published: April 2020

Aims

Opioid overdose deaths and the rise in problematic opioid use patterns that indicate the development of opioid use disorder (OUD) have reached crisis levels in the United States. Medication‐assisted treatment (MAT) with therapeutics such as the partial μ‐opioid receptor agonist buprenorphine mitigate withdrawal, reduce mortality, and reduce intake of abused opioids (e.g., fentanyl, heroin). Still, the opioid epidemic has crystalized the need to identify adjunctive therapeutics to substantially improve the odds of successful recovery. In the present study, we tested the hypothesis that treatment with the FDA‐approved, 5‐HT2CR agonist lorcaserin (Belviq®), previously shown to reduce opioid intake in the preclinical self‐administration (SA) model, may serve as an efficacious adjunct to suppress opioid intake and/or prevent escalation of opioid use after cessation of MAT. We employed osmotic minipumps to chronically administer buprenorphine and/or lorcaserin to achieve stable levels and mimic a clinical dosing regimen.

Methods

Male Sprague‐Dawley rats (n=34) were implanted with osmotic minipumps [sham, lorcaserin (1 mg/kg/day), buprenorphine (3 mg/kg/day), or the combination] following stable acquisition of fentanyl self‐administration. Rats resumed fentanyl self‐administration for 10 days and were then monitored for an additional ten days after removal of osmotic pumps. During this phase, the efficacy of acute lorcaserin (1 mg/kg) to impact fentanyl intake was evaluated.

Results

Chronic treatment with buprenorphine or the combination, but not lorcaserin alone, attenuated fentanyl intake relative to sham‐operated rats [F3,30=15.3, p<0.05]. Acute treatment with lorcaserin attenuated fentanyl intake relative to saline‐treated rats [F1,12=51.81, p<0.05].

Conclusions

The efficacy of acute lorcaserin to suppress opioid intake extended to the illicit opioid fentanyl. Further, chronic buprenorphine, with or without lorcaserin, resulted in sustained inhibition of fentanyl intake. Taken together, these studies provide support for further interrogation of the efficacy or lorcaserin as a viable adjunct pharmacotherapy for MAT.

Support or Funding Information

Supported by the NIDA U54 DA038999
Cardiac TRPV1‐Afferent Signaling Promotes Arrhythmogenic Ventricular Remodeling after Myocardial Infarction
Authors: Yoshie Koji , Rajendran Pradeep A. , Massoud Louis , Mistry Janki , Swid M. Amer , Wu Xiaohui A. , Sallam Tamer A. , Zhang Rui , Goldhaber Joshua I. , Salavatian Siamak A. , Ajijola Olujimi A.
Published: April 2020

Chronic sympathoexcitation is implicated in ventricular arrhythmogenesis (VAs) following myocardial infarction (MI), but the critical neural pathways involved are not well understood. Cardiac adrenergic function is partly regulated by sympathetic afferent reflexes, transduced by spinal afferent fibers expressing the TRPV1 channel. The role of chronic TRPV1 afferent signaling in VAs is not known. We hypothesized that persistent TRPV1 afferent neurotransmission promotes VAs after MI. Using epicardial Resiniferatoxin (RTX) to deplete cardiac TRPV1‐expressing fibers, we dissected the role of this neural circuit in VAs after chronic MI in a porcine model. We examined the underlying mechanisms using molecular approaches, immunohistochemistry, in vitro and in vivo cardiac electrophysiology, and simultaneous cardio‐neural mapping. Epicardial RTX depleted cardiac TRPV1 afferent fibers and abolished functional responses to TRPV1 agonists. Ventricular tachycardia/fibrillation (VT/VF) was readily inducible in MI subjects by programmed electrical stimulation or cesium chloride administration, however, TRPV1 afferent depletion prevented VT/VF induced by either method. Mechanistically, TRPV1 afferent depletion neither altered cardiomyocyte action potentials and calcium transients; nor the expression of ion channels and calcium handling proteins. However, it attenuated fibrosis and mitigated electrical instability in the scar‐border zone. In vivo recordings of cardiovascular‐related stellate ganglion neurons (SGNs) revealed that MI enhances SGN function and disrupts integrated neural processing. Depleting TRPV1 afferents normalized these processes. Taken together, these data indicate that after MI, TRPV1 afferent‐induced adrenergic dysfunction promotes fibrosis, adverse cardiac remodeling, and worsens border zone electrical heterogeneity, resulting in electrically unstable ventricular myocardium. We propose targeting TRPV1‐expressing afferent to reduce VT/VF following MI.

Support or Funding Information

NIH/NHLBI DP2 OD024323‐01
Amplified Protein Kinase C Signaling in Alzheimer's Disease
Authors: Lorden Gema , Wosniak Jacob M. , Dozier Lara E. , Patrick Gentry N. , Gonzalez David J. , Roberts Amanda J. , Newton Alexandra C.
Published: April 2020

Protein kinase C (PKC) isozymes are tightly regulated kinases that transduce signals from receptor‐mediated hydrolysis of membrane phospholipids. Whereas loss‐of‐function mutations in PKC are associated with cancer, germline mutations that enhance the activity of one isozyme, PKCα, are associated with Alzheimer’s Disease (AD). Here we show that introducing a highly penetrant AD‐associated variant of PKCα (M489V; increases catalytic activity by 30%) into a mouse model accelerates brain pathogenesis and results in a cognitive defect compared to wild‐type mice. Specifically, behavioral studies reveal a cognitive impairment in mice harboring this mutation compared to WT mice at 12 months of age, as assessed by performance on a Barnes Maze test, which measures spatial learning and memory. Additionally, the presence of the PKCα mutation in an AD mouse model overexpressing the amyloid precursor protein with the Swedish mutation, significantly exacerbated the cognitive decline as early as 6 months of age. To better understand the molecular basis for the cognitive defect, we analyzed the phosphoproteome and synapse morphology of mice harboring the PKCα M489V mutation. Analysis of the brain phosphoproteome of PKCα M489V mice vs wild‐type mice identified a variety of substrates with altered phosphorylation including known substrates of PKC involved in synaptic depression. Phosphorylation of one such protein, MARCKS, was increased both in mice harboring the PKCα mutation as well as human brains from patients with AD. Analysis of spine density revealed a modest (approximately 10%) but highly significant reduction in the number of spines per micron in the homozygous M489V mice compared to the wild‐type mice. Reduced spine density is consistent with the enhanced phosphorylation of MARCKS observed in brains from the M489V mice. In summary, this work reveals that a single amino acid change in the catalytic domain of PKCα is sufficient to cause increased phosphorylation of PKC substrates, neurite degeneration, and cognitive decline. These data strongly support inhibition of PKCα as a potential therapeutic target in AD.

Support or Funding Information

Cure Alzheimer's Fund
SPARC‐Network‐Scale Interactions Among Simultaneously Recorded Brainstem Neurons In The Dorsal Medulla, Ventrolateral Medulla, And Pons: A Substrate For Central Autonomic Control And Processing Of Cardiovascular Afferent Feedback
Authors: Morris Kendall F. , Segers Lauren S. , Nuding Sarah C. , Lindsey Bruce G. , Pitts Teresa G. , Iceman Kimberly E. , Bolser Donald C.
Published: April 2020

The central autonomic control system provides parasympathetic and sympathetic drive to peripheral ganglia that regulate a host of visceral functions. Detailed knowledge of the network scale organization of this central circuit is incomplete. We hypothesized that functional synaptic interactions between many simultaneously recorded neurons in and around the nucleus of the tractus solitarius (NTS), the retrotrapezoid nucleus (RTN), ventral respiratory column (VRC) and pons would yield information about the network‐scale strategy that is employed by this system to regulate autonomic drive. Experiments were conducted in 73 decerebrated, paralyzed, and artificially ventilated cats. Multi‐electrode arrays including up to 100 channels were placed in the areas of the NTS, RTN, and VRC. A total of 2,643 neurons were challenged with alterations in blood pressure (BP) and cardiac modulation was assessed in 3,531 neurons. The spike activity patterns of many simultaneously recorded single neurons were evaluated during breathing, the cardiac cycle, increased activation of baroreceptors by inflation of a balloon in the abdominal aorta and unloading of baroreceptors by inflation of a balloon in the vena cava. There was no apparent anatomical segregation of neurons in these areas with regard to cardiac or BP modulation based on stereotaxic coordinates. Cross correlation analysis revealed robust excitatory and inhibitory functional interactions between neurons in the areas of the NTS, VRC, RTN, and pons. Plots customized to reveal patterns of information transfer between neurons in different areas provided evidence that units in the RTN and VRC were predominantly exciting those in the NTS area, while these same NTS neurons were exciting and inhibiting different neurons in the RTN and VRC. Specific evidence was found for putative intermediate ventrolateral medulla (iVLM) neurons that were excited by BP and involved in inhibitory interactions with putative rostral ventrolateral medulla (RVLM) neurons that were inhibited by increased BP. However, this mechanism was a low frequency feature of the dataset. Evidence for synaptic inhibition between neurons that responded to increased BP was a predominant finding. Among neurons that responded to decreased BP, evidence for synaptic excitation was more common. These results support differential organization of brainstem networks that mediate autonomic responses to increased BP relative to those that mediate decreased BP. The brainstem network that mediates sympatho‐inhibition in response to increased blood pressure is sparsely excitatory and may depend on abundant inhibitory mechanisms to enhance signal/noise ratios in support of information transmission from baroreceptors.

Support or Funding Information

Supported by 3OT2OD023854
Sex differences in salt‐responsive modulation of bile acids by microbiota regulates Hypertension
Authors: Chakraborty Saroj , Lulla Anju , Cheng Xi , McCarthy Cameron G. , Yeo Ji-Youn , Alimadadi Ahmad , Mandal Juthika , Saha Piu , Yeoh Beng San , Louis Evangelina F , Mell Blair , Jia Wei , Wenceslau Camilla F. , Kumar Matam Vijay , Meyer Katie
Published: April 2020

Men and women are differently prone to cardiovascular diseases. Salt‐sensitive women are more likely to develop aggravated hypertension in response to an overloaded salt diet than men. Men and women also differ in gut microbiota, which regulates both bile acid composition and hypertension; however, whether sex may differentially influence the gut microbial production of bile acids, with attendant effects on hypertension, is not known. We hypothesized that reshaping of gut microbiota by dietary salt is sex‐dependent and adversely affects bile acids to promote hypertension. To test this hypothesis, groups of male and female salt‐sensitive hypertensive (Dahl S) and salt‐resistant normotensive (Dahl R) rats (n=6/gp) on low (0.3% NaCl) or high (2% NaCl) diets were profiled for fecal microbiota by 16S sequencing and serum bile acids by targeted GC‐TOF‐MS metabolomics. Hierarchical clustering analysis of bile acids with microbiotal profiles revealed that glycine‐ and taurine‐conjugated bile acids were prominently negatively correlated with blood pressure (BP) in female rats, while only taurine‐conjugated bile acids were negatively correlated with BP in male rats. These observations were corroborated by microbiotal and bile acid metabolomic profiling of human hypertensive samples from a population‐based cohort (CARDIA). Next, to test whether hypertension can be prevented by supplementing substrates for bile acid conjugation, high salt fed S rats were administered with either taurine (3% v/v in drinking water) or glycine (1% v/v in drinking water) in drinking water, monitored for BP by telemetry, and profiled for bile acid metabolites. Both taurine and glycine feeding prevented hypertension in females (Systolic BP: taurine:176±7*, glycine: 182±4*, control: 197±4 mmHg, n=6–9/gp, *p<0.05) likely by rescuing levels of levels of respective conjugated bile acids, whereas only taurine, but not glycine, prevented hypertension in males (Systolic BP: taurine: 166±2*, glycine:184±5, control: 184±8 mmHg, n= 5–6/gp, *p<0.05). Our results connect sex‐specific differences in salt‐sensitive hypertension to the gut‐liver axis and suggests that nutritional restoration of host taurine‐ or glycine‐conjugated bile acids for women and host taurine‐conjugated bile acids for men as potential therapeutics for salt‐sensitive hypertension.

Support or Funding Information

National Institutes of Health (R01HL143082)
Utilizing BASIL Consortium Modules to Characterize Putative Kinases of Unknown Function
Authors: Wheeler Baleigh , Memic Nejla , Dollen Julia , Duplan Amanda , Hall Bonnie L.
Published: April 2020

The Protein Data Bank (PDB) contains 157,712 solved protein structures, with 4,465 of these listed as having an unknown function. We are interested in identifying novel kinases, and screened for them using modules from the BASIL Consortium’s curriculum. BLASTp was initially used to analyze almost 1,500 protein structures of unknown function, narrowing the list to 12 putative kinases. Next, in silico tools including Pfam, DALI, PANNZER, SANS, and ProMOL (a PyMOL plug‐in) were used to further characterize these putative kinases. Two structures were identified as likely kinases: 1ZBS and 3DNU. The 1ZBS protein is predicted to be an N‐acetyl‐glucosamine (NAG) kinase. The 3DNU protein appears to be a toxin‐antitoxin type 2 protein that functions as a serine‐threonine kinase. To further confirm these assignments, docking simulations were performed with the PyRx plug‐in for PyMOL. NAG plus ATP could be docked into the 1ZBS protein with a good calculated binding affinity, supporting its function as a NAG kinase. Similarly, ATP and the tri‐peptide RTV were stably docked with the 3DNU protein. In vitro confirmation of these in silico results is now underway for both 1ZBS and 3DNU. Protein over‐expression and purification are being optimized, and the purified proteins will be used to confirm the identification of NAG/ATP and RTV/ATP respectively as viable substrates for these enzymes.

Support or Funding Information

This project is supported by NSF IUSE 1709170
Essential Anatomy for Obstetrics and Gynecology in the Undergraduate Medical Curriculum
Authors: Harrell Kelly , Davies David , Topping Daniel , Keim Sarah , Marzban Hassan , Latacha Kimberly , Mork Amy , Wineski Lawrence , Lopez Lisa , Kirera Francis , McNary Thomas , McWhorter David , Zumwalt Ann , Carpenetti Tiffany , Downs Mary Beth , Sanky Charles , Laitman Jeffrey , Reidenberg Joy S , Pratt Rebecca , Lewis Steven , Farias Anna , Brooks William , Royer Danielle , Cotter Meghan , Martindale Jim , Harmon Derek , Hankin Mark
Published: April 2020

Introduction

To prepare medical students for clinical training and practice, it is critical to understand the anatomical knowledge considered most important for different clinical specialties.

Aim

To address this issue, a consortium of anatomists in the US and Canada is collecting data from clinical educators in Obstetrics and Gynecology (ObGyn) clerkships and electives to identify the anatomy they consider essential.

Methods

An IRB‐approved, online survey (Qualtrics, Seattle, WA) was used to assess the importance of 98 anatomical topics in seven body regions. The study first examined the percentage of ObGyn clinical educators (clerkship/elective directors and attending physicians) that considered each anatomical region important to their specialty. Second, the study examined the rank assigned to each anatomical topic using an ordinal scale from 1 (not important) to 4 (essential).

Results

At the time of abstract submission, data had been collected from 71 ObGyn clinical educators at 22 medical schools. The percentage of ObGyn clinical educators that considered each anatomical region important to their specialty were (highest‐to‐lowest): Pelvis & Perineum (100%), Abdomen (96%), Lower Limb (47%), Back (34%), Thorax (26%), Head & Neck (18%), and Upper Limb (13%). Further data analysis has identified the highest ranked anatomical topics in each body region for the ObGyn clerkship/elective.

Discussion and Conclusion

This database provides detailed information regarding the most clinically relevant anatomical topics as identified by ObGyn clinical educators. This information can aid in focusing preclinical learning to best prepare medical students for success in their undergraduate and graduate clinical experiences.
A Nonperfusion‐Based Method of Hepatic Cell Isolation and Development of Fatty Liver Disease Model for Dairy Cattle
Authors: LaCasse Michelle , Choudhary Shanti , Choudhary Ratan , de Avila Jeanene , Beitz Don , Du Min , Testroet Eric
Published: April 2020

Fatty liver disease (FLD) is a major metabolic disorder of over‐conditioned periparturient dairy cattle and can precede many costly comorbidities, such as mastitis and ketosis. While management practices can decrease the incidence rate of FLD, the etiology is not fully understood. In vivo studies using large animals are expensive, difficult to perform, and time‐consuming; so, an in vitro model of FLD would be valuable for probing the biochemical mechanisms of the disease. Available methods for the isolation of bovine liver cells are mostly perfusion‐based and are often time‐consuming, require specialized equipment, or tend to favor the isolation of vascular epithelial cells rather than parenchymal cells. With these considerations in mind, our objective was to develop an in vitro model of FLD, optimizing a non‐perfusion method of hepatic cell isolation from adult cows. Hepatic tissues (~10 g) of cows from the abattoir were immediately collected following slaughter and transported to the laboratory on ice. After several washings with PBS, tissues were chopped, washed, and digested in 200 U/mL of collagenase II for ~30 min with shaking at 37 °C. When hepatic cells were grown, an overnight serum deprivation was done before the treatment with 0.4 mM palmitate, 20 ng/mL TNFα, and a cocktail of 0.4 mM palmitate and 20 ng/mL TNFα. Control cells were grown in growth medium alone. Identification of hepatic cells in culture was done by using immunolocalization of two epithelial cell markers namely, e‐cadherin and mesenchymal marker alpha smooth muscle actin (αSMA). Cell viability, apoptosis, and lipid accumulation were estimated by using lactate dehydrogenase (LDH) assay, caspase 3/7 assay, and oil red O staining, respectively. Hepatic cells were grown after 7–10 days of culture. Mixed cultures of hepatic cells showed presence of e‐cadherin positive hepatocytes and αSMA‐positive stellate cells. Consistent with FLD in vivo, presence of palmitate and palmitate along with TNFα increased (p < 0.05) LDH leakage in comparison to control culture. Likewise, TNFα and palmitate increased (p < 0.05) accumulation of lipid within the cell. In comparison to control cells, presence of palmitate and TNFA enhanced (3–3.5‐fold; p < 0.05) expression of caspase 3/7. Based on preliminary data presented herein, it appears that the non‐perfusion method of hepatic cell isolation and treating primary hepatic cells with palmitate and TNFα is an in vitro model for FLD.
Cardiovascular Deconditioning Enhances Cardiovascular and Autonomic Response to Arterial Chemoreflex Stimulation
Authors: De Andrade Ozahyr , Kline David D. , Hasser Eileen M.
Published: April 2020

Cardiovascular deconditioning (CVD) due to bedrest, inactivity or exposure to microgravity impairs cardiovascular regulation and circulatory homeostasis. Cardiovascular changes associated with CVD, including resting tachycardia, decreased exercise capacity and autonomic impairments are reproduced in an animal model of CVD, hindlimb unloaded (HU) rats. Autonomic dysfunction, including blunted baroreflex function, in CVD is associated with mechanisms involving the central nervous system. We hypothesized that CVD augments the arterial chemoreflex. Male Sprague‐Dawley rats were subjected to 2 weeks hindlimb unloading (HU) to simulate CVD, or maintained at normal postural position (cage control, CC). Under inactin anesthesia, baseline heart rate (HR) was elevated in HU vs CC (381±8 bpm vs 334±6 bpm). To examine the chemoreflex, HU and CC rats (n=5–6 each) were given bolus NaCN (25, 50, 75ug/kg, iv) or acute hypoxia (12% O2, 45 seconds). NaCN dose dependently increased HR and mean arterial pressure (MAP) in HU but not CC. At the highest NaCN concentration, HU increased MAP (22±4 mmHg) and HR (16±3 bpm) more than CC MAP (8±1 mmHg) and HR (6±2 bpm). NaCN also increased splanchnic sympathetic and phrenic nerve activity (SSNA and PhrNA) in both groups, and responses tended to be greater in HU rats. Likewise, the peak hypoxic response of SSNA and PhrNA were greater in HU than CC rats (SSNA, HU 143 ± 26% vs CC 111 ± 9%; PhrAmp%; HU 382 ± 208 vs CC 300 ± 103). Exposure to NaCN or hypoxia did not change PhrFreq in either group. These data suggest that HU enhances cardiovascular responses to chemoreflex activation.

Support or Funding Information

Funding: HL132836
Crystallographic Structural Elucidation of E. coli HU and a Four‐Way Junction
Authors: Khun Charya , Mukerji Ishita
Published: April 2020

Histone‐like protein HU binds with high affinity (Kd = 30 nM in gel and Kd = 10 nM in solution) to a Holliday or Four‐Way junction (HJ), a DNA recombination intermediate. While previous Förster resonance energy transfer (FRET) studies have determined possible structural models for the HU‐junction interaction, X‐ray crystallographic data is needed to confirm these models. In our studies, we are using J20, a version of the well‐characterized J3 Holliday junction, that has been modified to have only ten base pairs per arm. Based on our anisotropy measurements, the shorter arms reduce the relative flexibility of the junction, potentially making this junction a better substrate for our crystallization studies. Our investigation aims to determine the X‐ray crystal structure of the HU‐HJ interaction by elucidating the ideal conditions for generating HU‐J20 crystals. Initial investigations of crystallization conditions have produced crystals that diffract to 20 Å resolution, therefore, our current efforts are focused on generating crystals that will diffract at higher Angstrom resolution. In order to do so, we will screen around our successful conditions and slightly vary the pH, viscosity, and protein concentrations of our crystallography solutions. Conditions for optimizing crystal generation and diffraction will be discussed.
Environmental Chemicals Disrupted Lipid/Fatty Acid Contents of Rat Organs and Reversed by Treatment With Citrus Lime Nano‐particle
Authors: Gonnabathula Pavani Kumar , Yakubu Momoh A.
Published: April 2020

The environment is rich with trace concentrations of toxicants, these toxicants in the living system can disrupt biological homeostasis. We had reported that trace concentrations of emerging contaminants dysregulate antioxidant systems. In the present study we investigated the effects of these cellular defense disrupting chemicals on organs’ fatty acids and lipids contents and evaluated effects of Citrus lime conjugated nano‐particles. Rats were treated with environmental chemicals (EC) consisting of mixture of atrazine, dieldrine, endrine, endosulfan, anthracene, 17‐alpha ethynyl estradiol, diethylstilbestrol, estradiol, estrone, testosterone and estriol at a concentration of 1–10 ug/L in drinking water for 6 weeks along with control. Another group was treated with synthesized 2 μM Citrus lime nano‐particle alone or in combination with EC. At the end of the treatment, rats were euthanized, and organs collected. Samples were extracted for determination of fatty acids and lipids using Agilent 7890B/5977B GC/MSD System with SQ detector with HP‐5 MS capillary column. Results shows treatment with environmental toxicants dysregulated fatty acid species in the heart, liver, lung, spleen, brain, and fat with changes that varied up to 7.0×108 % when compared to the control. Similarly, cholesterol contents were severely dysregulated with the range of changes varying from −68 to 2.0×1010 %. The highest dysregulation occurred in the fat tissues. All these changes were reversed in the group treated with synthesized Citrus lime nano‐particle. Our results indicate that environmental chemicals in trace concentrations disrupts fatty acids species and cholesterol contents of different organs. The disrupted fatty acids are Hexadexcanoic acid methyl ester; n‐Hexadecanoic acid, 10‐methyl‐, methyl ester; 9,12‐Octadecadienoic acid; Heptadecanoic acid; Eicosatetraenoic acid, methyl ester, Methyl stearate. These disruptions by environmental chemicals were reversed by nano‐particle conjugated to alkaloid from Citrus lime. These fatty acid species possess antioxidants, anti‐inflammatory, and other cellular defense and regulatory functions. Thus, our results suggest that low concentrations of environmental chemicals’ disruptions of these functions is the hallmark of slow and long‐term developing health altering conditions such as diabetes, Alzheimer’s, rheumatoid arthritis etc. possibly mediated by inflammatory processes and induced oxidative stress. Our results further suggest that nano‐particle conjugated alkaloid could serve as an antidote to long term effects of exposure to trace concentrations of environmental chemicals.

Support or Funding Information

This research is supported by Tittle III under the Award Number P031B090216;Title III, Part B, HBGI (CFDA No. 84.031B).