bluuebunny/crossref_metadata_2025 huggingface dataset:
AMPK is Activated During Sleep Deprivation in Caenorhabditis elegans Ryan Schuck, Edwin Li, Matthew D. Nelson Saint Joseph’s University, Department of Biology, Philadelphia, PA 19131
Authors: Schuck Ryan James , Li Edwin , Nelson Matthew D.
Published: April 2020

Sleep conserves energy and allows for its reallocation towards biosynthetic pathways, suggesting that one of sleep’s functions is cellular restoration. During times of sickness, cells are damaged and sleep is heightened, supporting an additional immune function for sleep. In some cases, sleep is directly induced by signaling mechanisms of the immune response. Work in model systems, such as Caenorhabditis elegans and Drosophila melanogaster, have identified a protective sleep state called stress‐induced sleep (SIS), which resembles sleep during sickness. SIS occurs following exposure to environmental stressors such as noxious temperatures, osmotic imbalances, physical damage and UV irradiation. While the signaling molecules regulating SIS have begun to be elucidated, the mechanisms linking sleep to cellular restoration are still unknown. A master regulator of nutrient/energy sensing, adenosine monophosphate‐activated protein kinase (AMPK), which is highly conserved from invertebrates to mammals, is a candidate for such a link. In mammalian cells, AMPK is activated by low levels of ATP and oxidative stressors, the latter occurring through the overproduction of reactive oxygen species. In both cases, activation of AMPK requires phosphorylation of a threonine residue (T172). We used CRISPR to make an AMPK mutant in C. elegans in which the predicted, homologous T172 site is changed to an alanine, to determine if this amino acid is conserved functionally. We find that these mutants live significantly shorter lives than wild type animals following exposure to paraquat, a known inducer of oxidative stress. Moreover, these animals provide us with an important control for testing our broader hypothesis that AMPK links sleep to cellular repair during SIS. To test this, we conducted a temporal western blot analysis, assessing AMPK’s phosphorylation levels during SIS in both wild type animals and those that are genetically sleep deprived. We find that AMPK is not significantly activated during times of normal SIS, however, mutant animals that lack sleep‐promoting neuropeptides, display increased AMPK activation 24 hours after stress exposure. These mutants also display reduced longevity in response to oxidative stress. We hypothesize that sleep deprivation promotes an increase in AMPK phosphorylation in an attempt to provide cellular protection/repair during SIS.

Support or Funding Information

NSF Award #1845020 (PI Nelson)
Exercise Pressor Reflex in UC Davis Type‐2 Diabetes Mellitus (UCD‐T2DM) Rats Prior to the Onset of Diabetes
Authors: Huo Yu , Grotle Ann-Katrin , Lee Junghoon , Ybarbo Kai M. , Graham James , Stanhope Kimner L. , Havel Peter J. , Harison Michelle L. , Stone Audrey J.
Published: April 2020

Both the exercise pressor reflex and mechanoreflex are exaggerated after the development of T2DM in UCD‐T2DM rats. Whether the pressor reflex is also exaggerated before UCD‐T2DM rats developed diabetes is not known. Therefore, the purpose of this study was to determine if both the exercise pressor reflex and mechanoreflex are exaggerated in the UCD‐T2DM rats before the onset of diabetes.

Methods

For this study, we used young (8–12 wk old), male (n=12) and female (n=10), non‐diabetic UCD‐T2DM rats and age‐matched, male (n=11) and female (n=7), healthy Sprague Dawley rats. The exercise pressor reflex was evoked by statically and intermittently contracting the hindlimb muscle for 30s, and the mechanoreflex was evoked by passively stretching the Achilles tendon for 30s. Changes in mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA) were continuously measured during both contraction and stretch.

Results

We found that fasting blood glucose for both groups was below the threshold for diagnosis of diabetes (200mg/dl). Statically contracting the muscle did not significantly increase peak MAP (CTL: 11 ± 1 mmHg, n=12; UCD‐T2DM: 14 ± 2 mmHg, n=13, p>0.05), HR (CTL: 12 ± 4 bpm, n=12; UCD‐T2DM: 14 ± 5 bpm, n=13, p>0.05), or RSNA (CTL: 40 ± 20 %, n=4; UCD‐T2DM: 39 ± 23 %, n=3, p>0.05) in UCD‐T2DM rats compared to control rats with similar developed tensions. Likewise, intermittently contracting the muscle did not significantly increase peak MAP (CTL: 14 ± 2 mmHg, n=13; UCD‐T2DM: 14 ± 2 mmHg, n=6, p>0.05), HR (CTL: 16 ± 3 bpm, n=13; UCD‐T2DM: 14 ± 5 bpm, n=6, p>0.05), or RSNA (CTL: 73 ± 42 %, n=5; UCD‐T2DM: 34 ± 14 %, n=2, p>0.05) in UCD‐T2DM rats compared to control rats with similar developed tensions. Moreover, stretching the Achilles tendon did not significantly increase peak MAP (CTL: 15 ± 3 mmHg, n=11; UCD‐T2DM: 13 ± 1 mmHg, n=14, p>0.05), HR (CTL: 11 ± 4 bpm, n=11; UCD‐T2DM: 14 ± 2 bpm, n=14, p>0.05), or RSNA (CTL: 60 ± 12 %, n=3; UCD‐T2DM: 24 ± 19 %, n=4, p>0.05) in UCD‐T2DM rats compared to control rats with similar developed tensions.

Conclusion

We conclude that both the exercise pressor reflex and the mechanoreflex are not exaggerated in UCD‐T2DM rats before the onset of diabetes. This study suggests that young UCD‐T2DM rats have similar MAP, HR, and RSNA responses to both muscle contraction and tendon stretch as seen in age‐matched Sprague Dawley rats prior to the onset of diabetes.

Support or Funding Information

This project was supported by NIH R01 HL144723.
Hypoxia and Hypercapnia Diminish Intestinal Barrier Integrity in a Human Enteroid Model System
Authors: Weber Gregory J. , van Sambeek Dana M. , Pearce Sarah C.
Published: April 2020

Warfighters are commonly exposed to unique physiologic stressors that affect their physical and mental performance, as well as their overall health. The adverse impact of these stressors may be in part due to effects on the microorganisms residing in the human gut, collectively known as the gut microbiome. Numerous studies have revealed the gut microbiome can influence multiple physiological process, including immunity, nutrient metabolism, and cognition. Hypoxia refers to a lack of oxygen in a given system and exists as a gradient in intestinal tissue, however, the intestine is sensitive to changes in oxygenation and can alter normal cellular process, such as inflammation and barrier function. Similarly, an excess of carbon dioxide (CO2), or hypercapnia, also affects intestinal tissue, as levels of CO2 are inversely coupled with the level of oxygen and are found to be elevated in hypoxic tissue. The GI tract is thought to have a low‐grade, albeit constant, level of inflammation and is home to multiple immune cell types. In addition, both epithelial and immune cells in the intestine can sense and respond to various stimuli including pathogens, and compromises to intestinal barrier integrity due to injury or stress can result. In this study, we sought to determine the extent to which hypoxia and hypercapnia degrade intestinal cellular integrity through inflammation‐mediated mechanisms. Intestinal epithelial organoids (enteroids) derived from the duodenum were exposed to hypoxic (0.5% O2) or hypercapnic (10%, 20% CO2) conditions for a period of 72 hours. Initial results indicated intestinal barrier integrity was compromised in hypoxia and 20% CO2 conditions as evidenced by transepithelial electrical resistance (TEER), with hypoxia (0.7 Ω) and 20% CO2 (0.85 Ω) being significantly lower than control (1.19 Ω; P < 0.05). The Quantigene Plex array system was used to interrogate a panel of intestinal and inflammatory markers. Preliminary results suggest alterations in tight‐junction markers as well as in genes involved in inflammatory response pathways. In addition, IL‐8, a macrophage‐mediated chemokine produced in intestinal epithelial cells and involved in the innate immune response, was found to be significantly downregulated in hypoxia (58.7 pg/mL) and hypercapnia (42.5 pg in 10% CO2, 49.7 pg/mL in 20% CO2) compared to control (190.7 pg/mL; P < 0.05). In sum, our data indicate hypoxic and hypercapnic conditions lead to alterations in molecules involved in the innate immune response pathway, tight‐junction markers, as well as compromised epithelial barrier integrity. Future experiments will be aimed at identifying the mechanism(s) responsible for diminished intestinal barrier function as well as identify potential preventative strategies.

Support or Funding Information

Funding support provided by the Office of Naval Research award #N0001419MP00144 to SP FY18‐FY20.
MDMA and TAAR1‐mediated RhoA Activation in Serotonin Neurons
Authors: Underhill Suzanne , Amara Susan
Published: April 2020

We have recently described the biochemical cascade through which the psychostimulant amphetamine leads to internalization of the dopamine and norepinephrine transporters, (DAT and NET). Amphetamine is a relatively high affinity substrate for DAT and NET and they both serve as efficient conduits for amphetamine entry into the cell. Once inside the cell, amphetamine activates the trace amine associated receptor 1 (TAAR1), a GPCR that we have shown couples to both GS and G13 signaling pathways. TAAR1 signaling through G13 increases the activity of RhoA, a small GTPase that regulates trafficking of the catecholamine carriers and increases their internalization. We wanted to investigate if similar G‐protein coupled signaling events also occur in serotonergic neurons. Although amphetamine does not have a particularly high affinity for the serotonin transporter (SERT), other TAAR1 ligands do, including 3,4‐methylenedioxymethamphetamine (MDMA).

We developed primary cultures from the raphe nuclei of E15 mice. 3H‐serotonin uptake mediated by these cultures was largely citalopram sensitive, indicating SERT‐mediated serotonin transport. Pre‐treatment of these cultures with MDMA resulted in a decrease of SERT activity that could be blocked by an inhibitor of RhoA. Similarly, by total internal reflection fluorescence microscopy in HEK293 cells we found internalization of GFP‐tagged SERT in response to MDMA was blocked by a RhoA inhibitor. In HEK293 cells in which the TAAR1 gene has been deleted, MDMA did not trigger SERT internalization. In noradrenergic neurons cultured from the locus coeruleus or dopamine neurons from the midbrain, only higher doses of MDMA pretreatment could decrease transporter activity at the cell surface, likely reflecting the differences in affinity of the drug for the different monoamine transporters.

These data indicate that pharmacological agents can regulate TAAR1 signaling in serotonergic, as well as catecholaminergic neurons and that the selectivity of TAAR1 agonists depends not only on affinity for the GPCR but also on their transport efficiency. Moreover, these findings suggest that the activation of TAAR1 in serotonin neurons and the subsequent internalization of surface SERT likely contribute to the modulation of serotonin signaling observed in response to MDMA.

Support or Funding Information

This work was supported by Intramural Research Program at the NIH, NIMH (ZIAMH002946).
Neuropeptide Y and Pial Artery Vasomotor Control: An Ordered Affair
Authors: Morse Cameron , Boerman Erika M. , Thorne Pamela K. , Emter Craig A. , Padilla Jaume , Olver T. Dylan
Published: April 2020

Evidence indicates sympathetic neurotransmitters influence cerebral vasomotor tone in a branch order‐dependent manner. Further, in addition to eliciting vasoconstriction, sympathetic co‐transmitter neuropeptide Y (NPY) may also elicit dilation in the cererbrovasculature depending on whether it acts on vascular smooth muscle or the endothelium. Herein, we investigated vasomotor responses to NPY in pial arteries (intracranial arteries on the surface of the brain) along the cerebrovascular tree. Female pigs (n=15, total arteries=88; age=2±0 months) were euthanized and their brains were harvested. First order (1A; n=7–8 per experiment), second order (2A; n=7–8 per experiment), and third order (3A; n=7–8 per experiment) pial arterial branches from the middle cerebral artery were dissected for pressure myography experiments. Following stabilization, NPY was administered either extra‐luminally (1e‐10 ‐‐to 1e‐6 M; half log doses, n=8 pigs) or intra‐luminally (1e‐9 M, n=7 pigs). To evaluate the contribution of nitric oxide (NO) on vasomotor responses to NPY, a subset of arteries were pre‐treated with a NO synthase (NOS) inhibitor (L‐NAME 3e‐4 M) 30 minutes prior to NPY administration. Whereas extra‐luminal NPY elicited vasoconstriction (24/24 arteries), intra‐luminal NPY tended to elicit vasodilation (16/21 arteries). Maximal vasoconstriction (% of baseline diameter) in response to extra‐luminal NPY was greater in 3a vs. 2a and 1a pial arteries (1a=13±1%; 2a=15±2%; *3a=23±4%; p≤0.01). Differences between 1a and 2a pial arteries were not significant (p=0.17). Pre‐treatment with a NOS inhibitor enhanced the maximal vasoconstriction in 1a, 2a and 3a pial arteries (grouped data; untreated=17±1%; *following NOS inhibition=29±1%; p<0.01). Dilation (% of baseline diameter) in response to intra‐luminal NPY was greater in 1a and 3a vs. 2a pial arteries (*1a=4±4%; 2a=−3±6%; *3a=9±4%; p≤0.01). Of note, whereas 86% of 1a and 3a pial arteries dilated, only 57% of 2a pial arteries dilated following transient exposure to intra‐luminal NPY. NOS inhibition abolished NPY‐induced dilation and promoted an NPY‐mediated vasoconstriction (grouped data; untreated=4±3%; *following NOS inhibition=−15±3%; p<0.01). In summary, NPY regulates cerebral vasomotor control through both vasoconstriction and dilation. These results indicate vasomotor responses to NPY are branch order‐dependent and most apparent in 3a pial arteries. NOS inhibition exacerbated NPY‐induced constriction and ablated NPY‐induced dilation, implicating NO signaling as a regulator of NPY‐dependent effects on cerebrovascular tone.

Support or Funding Information

This work was supported by a MU CVM COR grant and a SHRF Establishment grant (#4522).
Integration of Drug Perturbed Dose Dependent Response Data in Predictive Computational Models of Chemotherapeutic Response Using Multiple‐Kernel‐Learning
Authors: Mannheimer Joshua David , Prasad Ashok , Gustafson Daniel
Published: April 2020

Recently there has been a great deal of work to understand the dynamics of genome expression under small molecule perturbation. This has been demonstrated by the recent expansion of the Connectivity Map (CMAP) (
www.broadinstitute.org/connectivity‐map‐cmap) with roughly 5000 small molecule signatures. While this has proven to be invaluable resource it is still limited due to many drugs being constrained to a handful of cancer cell genomes, because of this, it is difficult to leverage these datasets to make an accurate and robust predictive model. Outside CMAP there is limited data that systematically characterize small molecule induced genomic changes, however, recently Monks et al. (Cancer Res 2018 DEC 15) has made available data in the NCI60 cell line panel for treated and untreated cell lines at 2, 6, and 24hr and two different dosing concentrations for 15 anti‐cancer agents. In our work we explore how each dataset can be optimally leveraged to build a predictive model of cell IC50 within each of the 15 drugs using a multiple kernel learning approach. With this method we can reach correlations as high as 0.88 (p ~1e−6) between predicted and measured IC50 with an average correlation of 0.57 on an independent test set. Additionally, perturbation with higher drug dose yields better individual models with average correlation around 0.451 compared to non‐treated data of 0.391 suggesting drug induced genomic changes are an important predictor of drug performance. Furthermore, we present a novel method that derives signatures present in perturbed data that can be captured in basal expression data. This approach to selecting features yields model performance that is equal or better than a signature derived basally alone. Our work demonstrates the importance of both basal and dynamic gene expression in modeling and understanding of drug response. We hope this work motivates further experiments to characterize drug induced gene dynamics for both modeling applications and understanding the underlying genomic influences in drug response.

Support or Funding Information

Shipley Chair in Comparative Oncology
Phorbol 12‐myristate 13‐acetate Dedifferentiates Human Cardiac Myofibroblasts to Fibroblasts
Authors: Luu Vy Tran , Phan Sang , Jin Zhuqiu
Published: April 2020

Fibroblast plays an important role in keeping heart shape and elasticity as well as maintaining normal cardiac function. The differentiation of fibroblast to myofibroblast is generally considered as an irreversible conversion which represents a critical step in pathogenesis of fibrosis. Transforming growth factor (TGF)‐β1 is a major cytokine that mediates the conversion of fibroblast to myofibroblast, defined by the overly expression of α‐smooth muscle actin (α‐SMA)‐ a biomarker of myofibroblast with little to no appearance of fibroblast specific protein 1 (FSP‐1) – a biomarker of fibroblast. The cellular and molecular mechanisms of myofibroblast differentiation from fibroblasts have been studied extensively. Reversal of myofibroblast differentiation to fibroblasts remains unclear and incompletely understood. Phorbol 12‐myristate 13 acetate (PMA) is involved in multiple cellular functions such as cell growth, differentiation, programmed cell death via protein kinase C (PKC) signaling pathways. To investigate whether PMA dedifferentiates the formed myofibroblasts, NIH 3T3 fibroblasts and human cardiac fibroblast (HCF), cultured in DMEM and fibroblast medium (FM)‐2 respectively, were induced to convert into myofibroblasts in the presence of 2 ng/ml of TGF‐β1 for 24‐ or 48‐hour incubations. Expression of α‐SMA and FSP‐1 in both cell lines was detected by using western blotting and immunofluorescence. Collagen gel contraction induced by cardiac fibroblasts was determined as well. After incubation with TGF‐β1, morphology changes in the shape and the size of NIH 3T3 and HCF cells were observed by the presence of large nuclei and cytoplasm. The levels of expression of α‐SMA were significantly increased whereas expression of FSP‐1 was reduced after 48‐hour incubation with TGF‐β1. NIH 3T3 and HCF cells were then treated with 50 ng/ml of DMSO which used as control groups and with various PMA concentrations (10 ng/ml, 50 ng/ml and 100 ng/ml) for additional 24‐ and 48‐ hour incubations. Chemiluminescence detection of Western blot confirmed the reduction in expression of α‐SMA. The shape and the size of the both cell lines were recovered under the presence of PMA. The results indicated that the reduction in expression of α‐SMA was directly proportional to the concentration of PMA. As PMA concentration increased, the expression of α‐SMA remarkably decreased. PMA also reduced TGF‐β1‐induced collagen gel contraction. These data unambiguously elucidate the reversal of myofibroblast differentiation induced by PMA. Under basic condition, TGF‐β1 induces myofibroblast conversion from fibroblast, and myofibroblast de‐differentiates back into fibroblast in the presence of PMA. Although the mechanism remains to be identified, the novel findings of this study shed light on future development of novel agents to treat fibrotic diseases.

Support or Funding Information

This project was supported by the Seed Grant from California Northstate University, College of Pharmacy.
Optogenetic stimulation of respiratory related neurons modulates swallow and swallow‐breathing coordination
Authors: Huff Alyssa D. , Karlen-Amarante Marlusa , Baertsch Nathan Andrew , Pitts Teresa , Ramirez Jan Marino
Published: April 2020

Swallow and breathing are tightly coordinated behaviors in which disruption of this coordination leads to aspiration pneumonia, the leading cause of death in many adult and pediatric diseases. Little is known about the neural coordination of these two behaviors. Previous work in the respiratory column has shown activity in swallow‐related and breathing‐related neurons, as well as neurons active during both swallow and breathing, suggesting coordination at a central network level. Evidence in the intermediate reticular nucleus (postinspiratory Complex, PiCo) suggest involvement in swallow behavior but not swallow‐breathing coordination. We hypothesize that swallow predominately occurs during the expiratory phase of breathing due to inspiratory related neurons suppressing swallow activity. Thus, inhibition of excitatory neurons within the Pre‐Bötzinger complex will increase swallow excitability, while exciting glutamatergic neurons within this network will have the opposite effect. To evaluate swallow circuitry and its coordination with respiratory related networks we use optogenetic approaches to manipulate the activity of inhibitory (Vgat‐Cre) and excitatory (Dbx1‐ERT2Cre, Vglut2‐Cre) neuron populations within the ventral respiratory centers as well as modulation of PiCo (ChAT‐Cre) in the presence of light stimulation. Optogenetic activation (blue laser, 447nm) of Dbx1 cells was performed in adult Dbx1‐ERT2Cre/Ai32(ChR2) mice after exposing the ventral surface of the brainstem in vivo, and placing bilateral glass fiber optics over the ventral surface in the region of the Pre‐Bötzinger complex. Swallow activity was measured by monopolar suction electrodes of the hypoglossal (XII) and vagus (X) nerves as well as cervical sympathetic nerve (cSNA). Phases of breathing was measured via monopolar electromyogram (EMG) of the diaphragm and swallow was stimulated orally by injection of 0.1cc of 2.5% citric acid water. Our preliminary data suggest that stimulating Dbx1 cells decreased the frequency and duration of swallows. Interestingly, we observed in the presence of light stimulation multiple swallow attempts (oral phase without the pharyngeal phase) resulting in hyoid elevation but failure to induce pharyngeal phase. The average swallow in the absence of light stimulation had a duration of 1025ms whereas swallow during light stimulation was 809ms. We believe that activation of Dbx1 neurons in the Pre‐Bötzinger complex suppresses swallow activity and decouples oral and pharyngeal phases of swallow.

Support or Funding Information

NIH: P01 HL090554, R01 HL144801 and R01 HL126523
Recognition Motif of Protein Kinase C and Protein Phosphatase 2A
Authors: Jones Alexander Curtis , Newton Alexandra C.
Published: April 2020

Protein phosphorylation plays a central role in transducing intracellular signaling and the balance between phosphorylation and dephosphorylation must be exquisitely controlled to maintain cellular homeostasis. Two central effectors of protein phosphorylation, protein kinase C (PKC) and protein phosphatase 2A (PP2A), are themselves regulated by protein phosphorylation but the mechanism of their interaction is not known. Here we identify a putative binding motif on the PKC kinase domain that is predicted to recruit PP2A via its B56 regulatory subunit. These two enzymes regulate the phosphorylation state of each other, with PP2A opposing constitutive phosphates on PKC required for structural integrity, and PKC proposed to phosphorylate B56, controlling its interaction with PP2A substrates. While many kinase consensus phosphorylation sequences are well‐characterized, emerging research into phosphatase‐recognition of substrates via short linear motifs (SLiMs) has uncovered a PP2A‐B56 SLiM comprised of L/F‐x‐x‐I/V‐x‐E. Sequence analysis of PKC reveals a potential PP2A‐B56 binding site that is conserved in conventional PKC isozymes (α, β, and γ); indeed the Glu is required for binding PP2A‐B56 and is present in a majority of the PKC family. This sequence is located on the C‐lobe of the kinase in a region involved in substrate binding. This study characterizes the effect of mutation on this motif on PP2A‐B56 binding and function, and on the phosphorylation state of PKC. Attesting to the importance of this motif, several cancer‐associated mutations have been identified in the motif, including PKCα‐E552Q and PKCβ‐E555K. Our study demonstrates that phosphatase SLiMs are able to inform new kinase/phosphatase relationships and may be a valuable predictive tool for studying signaling cascades in disease.

Support or Funding Information

This work was supported in part by the UCSD Graduate Training Program in Cellular and Molecular Pharmacology (T32 GM007752), National Science Foundation Graduate Research Fellowship Program, and NIH R35 GM122523.
“Detection of SNP rs2209972 in Insulin Degrading Enzyme Gene as a Predisposition Method for Type II Diabetes in Hispanic Populations”
Authors: Curiel Alejandro , Dominguez Jhonathan , Silverio Rachel , Calleja-Macias Itzel Evelyn
Published: April 2020

More than 30 million Americans have diabetes and about 90 to 95 percent have type II diabetes. Of those with type II diabetes roughly 12.1 percent are of Hispanic populations. With the rising of Hispanic populations, the potential rise of type II diabetes could lead to the rise of financial burdens and premature deaths. However, genetics can help in determining the risk of low or high predisposition of developing type II diabetes in Hispanic populations. Studies have shown a correlation of the Insulin Degrading Enzyme gene (IDE) in type II diabetes and Alzheimer’s disease. In the IDE there is a Single Nucleotide Polymorphism (SNP) called rs2209972 that has been linked to risk of developing type II diabetes in Hispanic populations. Our goal is to find the prevalence of the rs2209972 in IDE gene in Hispanic/Latino subjects and the association of risks with Type II diabetes. After DNA extraction of collected buccal samples, the samples were subjected to polymerase chain reaction (PCR) and confirmed by gel electrophoresis. An enzymatic digestion was conducted using BsaH1. The presence of a T allele produces a band at 424bp while the C allele produce two bands at 212bp. The results of this research show the C allele is present in both diseased and control patients. More research must be done in order to conclude the correlation of this SNP with the disease.
Creating Educator Scholar Leaders – Effectiveness of Robust Curriculum Development of a New PhD Program in Anatomy Education.
Authors: Terrell Mark , Kulesza Randy
Published: April 2020

INTRODUCTION

There is a current deficit in the number of high quality anatomy educators. This shortage has developed as well‐experienced anatomists move into administrative roles or retire and health profession schools expand course offerings and open new campuses. Curricular hour reductions, new technological innovations, and a focus on improved board performances forces anatomists to accomplish more with less. Although many anatomists make education a keystone in their careers, few PhD programs in anatomy provide formalized training in educational practice. AIM. To evaluate the effectiveness of a new PhD program in Anatomy Education designed to graduate educational‐scholar‐leaders as educational specialists.

METHODS

Applying Kern’s six step approach to curriculum development in medical education, we successfully merged the strengths existing educational programs at LECOM to create a new PhD program in Anatomy Education.

RESULTS

A new curriculum was planned, implemented, and evaluated. Program strengths include emphasis on the practice of teaching skills, educational research/scholarship, leadership, while developing robust knowledge in the anatomical sciences. Evidence of effectiveness of the innovative and unique curriculum includes, the number of applications, successful obtainment of accreditation, credit hours devoted to educational training, current evidence‐based dissertation research projects in anatomy education, institutional support of the program, and current PhD student evaluations.

CONCLUSION

This program has a strong commitment to producing a unique breed of new anatomists as education specialists who have the knowledge, skills and behaviors needed to lead health professions education development and research, thereby enhancing academic programs, improving health care outcomes, and strengthening the anatomical profession.
Regulation of GLS1 Expression by Scleraxis in Cardiac Fibroblasts
Authors: Chattopadhyaya Sikta , Nagalingam Raghu , Narhan Pavit , Czubryt Michael
Published: April 2020

Cardiac fibrosis is characterized by the excessive deposition of extracellular matrix, including fibrillar collagens, proteoglycans, and fibronectin. This process is energy‐intensive for cardiac fibroblasts, however it remains unclear how these energy metabolic requirements are met. Liver fibrosis, and in particular collagen synthesis, has been shown to be dependent on glutaminolysis, in which glutamine is converted to glutamate by the enzyme glutaminase (GLS) with further conversion to a‐ketoglutarate to fuel oxidative metabolism via the tricarboxylic acid cycle, but a role for glutaminolysis in cardiac fibrosis remains to be demonstrated. We previously showed that scleraxis (Scx) is a transcription factor that is critically required for the conversion of cardiac fibroblasts to myofibroblasts, but its potential role in glutaminolysis in these cells is unknown. We report here that freshly‐isolated rat cardiac fibroblasts (P0 – unpassaged) passaged twice to become myofibroblasts (P2) show concomitant up‐regulation of Scx by 5‐fold and GLS1 by 4‐fold in P2 versus P0. Scx over‐expression induced a 20‐fold increase in GLS1 expression. To determine whether GLS1 expression is dependent on Scx, we examined GLS1 expression in activated mouse cardiac fibroblasts (P1) isolated from wild type or Scx knockout mice. Scx knockout attenuated GLS1 expression by almost 90%. TGFb is a potent inducer of cardiac fibrosis that both dramatically up‐regulates Scx expression, and requires Scx for many of its downstream effects. TGFb treatment of wild type mouse P1 activated fibroblasts doubled the expression of GLS1, but had no effect in Scx KO cells. In silico analysis revealed four putative Scx binding sites in the GLS1 promoter. While TGFb significantly up‐regulated expression of the myofibroblast marker periostin in cardiac fibroblasts, both scleraxis knockout and the glutaminase inhibitor CB‐839 attenuated this induction, indicating a failure to convert to myofibroblasts. These findings suggest that Scx regulates GLS1 expression to facilitate increased energy metabolism during cardiac fibroblast to myofibroblast conversion.

Support or Funding Information

Canadian Institutes of Health Research (PJT‐162422).
Coupling Electrophysiology with Single Cell RNA Sequencing to Uncover Transcriptional Markers of Chemosensitive Serotonergic Neurons
Authors: Mouradian Gary Charles , Duffy Erin , Liu Pegnyuan , Hodges Matt
Published: April 2020

Central respiratory chemoreceptors are specialized neurons with intrinsic sensitivity to hypercapnia and/or acidosis that couple breathing and pH/CO2 levels. Previous data support the hypothesis that a sub‐population of brainstem serotonin (5‐HT) neurons serve as central respiratory chemoreceptors, where they become sensitive to pH/CO2 on or after post‐natal day 12 (P12) in rats. However, to date it remains unclear which 5‐HT neurons develop chemosensitivity and what molecular markers may identify this unique sub‐population of 5‐HT neurons. To address these questions, we employed a “patch‐to‐seq” technique to first distinguish 5‐HT neurons as chemosensitive (or insensitive) using electrophysiologic recording, and then to isolate each neurons’ RNA for single cell RNA Sequencing to identify potential cellular sensing mechanisms and/or unique transcriptional markers in each phenotypically‐identified 5‐HT neuron population. Acute brainstem slices (200 μm) were made from young (P18–23) transgenic rats which express eGFP in all 5‐HT neurons (SSeGFP), and action potential firing rates measured via a cell‐attached patch configuration in eGFP+ 5‐HT neurons during superfusion with artificial CSF (aCSF) containing inhibitors for synaptic blockade (10 mM CNQX, 50 mM D‐AP5, 20 mM Gabazine) bubbled with either 5% CO2 (bal. O2; pH= 7.36; 5 min) or 15% CO2 (bal. O2; pH= 7.10). Cellular phenotypes were assessed by calculating a Chemosensitivity Index (C.I.). pH‐sensitive eGFP+ neurons (n=7) had an average C.I. of 177.0±14.6 whereas pH‐insensitive eGFP+ neurons (n=9) had an average C.I. of 98.0 ± 2.9 (P < 0.0001). Cellular contents of each recorded cell were collected and subjected to single cell RNA Sequencing (scRNA‐Seq), from which an average of 19.3M reads were generated with a quality score of ~33.7 and ~83% mapping rate per sample (32,883 known genes quantified). Preliminary analyses indicate very few differentially expressed genes (q < 0.05) between pH‐sensitive and ‐ insensitive 5‐HT neuron populations, and of those differentially expressed no gene obviously contributes to pH regulation or sensing. In addition, unbiased hierarchical clustering and principal component analyses failed to clearly separate samples by phenotype. However, a supervised analysis using Support Vector Machine learning and Recursive Feature Elimination (SVM‐RFE) identified several genes in which expression levels significantly predict the predefined phenotype, but the biological/functional significance of these genes in CO2/pH sensing remains unclear and instead these genes may provide a molecular signature to distinguish pH/CO2 sensitive vs insensitive 5‐HT neurons. We conclude from these preliminary data that pH/CO2 sensitivity in 5‐HT neurons is not a transcriptionally regulated phenotype.

Support or Funding Information

Parker B. Francis Foundation

NHLBI ‐ R01 HL122358
Endothelial Mineralocorticoid Receptor Deletion Abrogates Leptin‐Induced Endothelial Dysfunction in Pregnant Mice
Authors: Faulkner Jessica L. , Wright Derrian , Kennard Simone , Antonova Galina , Jaffe Iris , Belin de Chantemèle Eric J.
Published: April 2020

Obesity is a risk factor for endothelial dysfunction and hypertension in pregnancy, both of which are hallmarks of preeclampsia. Our lab has demonstrated that obesity‐associated endothelial dysfunction and hypertension in female mouse models is mediated via leptin‐induced aldosterone production and subsequent mineralocorticoid receptor (MR) activation. We also showed that progesterone increases endothelial MR (ECMR) expression and that high progesterone levels in pregnant mice are associated with highly elevated ECMR expression, potentially rendering pregnant female mice susceptible to leptin‐induced endothelial dysfunction in obese pregnancy. Therefore, we hypothesized that ECMR deletion would prevent leptin‐induced endothelial dysfunction in pregnant mice in late gestation. Endothelial function was experimentally measured by wire myography concentration response curves to acetylcholine in mouse aorta. Chronic leptin infusion (0.9mg/kg/day, s.c. osmotic minipump) throughout late‐gestation (gestational days 11–18) induced endothelial impairment in wild‐type pregnant mice that was impaired both compared to sham‐treated pregnant (2‐way ANOVA with repeated measures, *P<0.05) and non‐pregnant leptin‐infused females (*P<0.05). However, leptin infusion in pregnant ECMR−/− mice did not induce similar endothelial dysfunction (*P<0.05). Relaxation responses to acetylcholine in the presence of LNAME indicated that endothelial dysfunction in pregnant leptin‐infused mice was attributable to reduction in nitric oxide activity. In addition, no changes in endothelial‐independent sodium nitroprusside responses were observed among groups. Placental dysfunction is a characteristic of preeclampsia that precludes the development of endothelial dysfunction and hypertension. In homogenized placentas, mRNA expression of hypoxia‐inducible factor 1α (HIF1α), a marker of ischemia, was increased by leptin infusion in wild‐type (1.9±0.2‐fold change from sham wild‐type, P=0.08), but not in ECMR−/− (1.2±0.2‐fold change from sham ECMR−/−) pregnant mice. These changes in placental function were associated with adverse fetal effects in leptin‐infused wild‐type mice as indicated by decreased pup weight (0.86±0.04g sham wild‐type vs 0.52±0.11g wild‐type +leptin, *P<0.05). However, ECMR deletion rescued pup weight in leptin‐infused pregnant mice (0.79±0.08g, sham ECMR−/− vs 0.86±0.11g, ECMR−/− +leptin). Collectively, these data indicate that high leptin levels in obese pregnancies may predispose to placental dysfunction and endothelial dysfunction, which are characteristics of preeclampsia.

Support or Funding Information

1R01HL130301‐01, 19EIA34760167, Adrenal Center pilot grant, 1K99HL146948‐01A1
A Brain Tissue Metabolomic Signature Discloses Alzheimer’s Disease Post‐Mortem
Authors: Jasbi Paniz , Shi Xiaojian , Turner Cassidy , Jentarra Garilyn , Gu Haiwei
Published: April 2020

Alzheimer’s disease (AD) is the most common cause of dementia, accounting for an estimated 60 to 80% of cases, and is the sixth‐leading cause of death in the United States. A major challenge persists in the post‐mortem diagnosis of AD patients from those suffering mild cognitive impairment (MCI) and control patients with high pathology (HPC). Mass spectrometry (MS)‐based metabolomics has shown significant potential in disease diagnosis over the past two decades. In this study, we present a combination of gas chromatography‐MS (GC‐MS) for global metabolic profiling and analysis of long‐ and short‐chain fatty acids, in addition to liquid chromatography‐tandem MS (LC‐MS/MS) for the detection of targeted aqueous metabolites and lipids. Using this approach, 2084 metabolites and features were reliably detected and monitored in 48 tissue samples harvested from the superior frontal gyrus of male and female subjects post‐mortem. Samples were taken from four groups: 12 normal control (NC) patients, 12 subjects characterized as HPC, 12 with sub‐clinical MCI, and 12 diagnosed with AD. Multivariate significance testing informed the construction and cross‐validation (p < 0.05) of partial least squares‐discriminant analysis (PLS‐DA) models defined by a 9‐metabolite panel of potential diagnostic biomarkers. Receiver operating characteristic (ROC) analysis showed high predictive accuracy of the resulting PLS‐DA models for discrimination of NC (96.6%), HPC (91.7%), MCI (93.9%) and AD (95.1%). Pathway analysis revealed significant disturbances in lysine degradation (p = 0.007), fatty acid metabolism (p = 0.009), and the degradation of branched‐chain amino acids (p = 0.025). Over representation analysis of lipid signatures revealed significant alterations in glycerophospholipid (FDR q = 8.3E‐07) and choline metabolism (FDR q = 0.006). Network analysis showed significant enrichment in 11 enzymes, mostly of the mitochondria. The current results allow for improved clinical staging post‐mortem, expand basic knowledge of the metabolome related to AD pathogenesis, and reveal pathways that can be targeted therapeutically in future studies. This study also provides a promising basis for the development of larger multi‐site projects to validate these candidate markers in readily available biospecimens such as blood to enable the effective screening, rapid diagnosis, accurate surveillance, and therapeutic monitoring of AD across population groups.

Support or Funding Information

Support from the College of Health Solutions at Arizona State University and the National Institutes of Health is gratefully acknowledged.
Pharmacological or genetic depletion of senescent astrocytes prevents whole brain irradiation‐induced impairment of neurovascular coupling responses protecting cognitive function in mice
Authors: Yabluchanskiy Andriy , Tarantini Stefano , Fulop Gabor , Kiss Tamas , Balasubramanian Priya , DelFavero Jordan , Ahire Chetan , Csipo Tamas , Nyúl-Tóth Ádam , Lipecz Agnes , Sonntag William , Schwartzman Michal , Campisi Judith , Csiszar Anna , Ungvari Zoltan
Published: April 2020

Whole brain irradiation (WBI, also known as whole brain radiation therapy or WBRT) is a mainstream therapy for patients with identifiable brain metastases and as a prophylaxis for microscopic malignancies. WBI accelerates brain aging, causing progressive cognitive dysfunction in ~50% of surviving patients, thus compromising quality of life. The mechanisms responsible for this WBI side effect remain obscure, and there are no effective treatments or prevention strategies. Here, we test the hypothesis that WBI induces astrocyte senescence, which contributes to impaired astrocytic neurovascular coupling (NVC) responses and the genesis of cognitive decline. To achieve this goal, we used transgenic p16‐3MR mice, which allows the detection and selective elimination of senescent cells. We subjected these mice to a clinically relevant protocol of fractionated WBI (5 Gy twice weekly for 4 weeks). WBI‐treated and control mice were tested for spatial memory performance (radial arm water maze), astrocyte‐dependent NVC responses (whisker‐stimulation‐induced increases in cerebral blood flow, assessed by laser speckle contrast imaging), NVC‐related gene expression, astrocytic release of eicosanoid gliotransmitters and the presence of senescent astrocytes (by flow cytometry, immunohistochemistry and gene expression profiling) at 6 months post‐irradiation. WBI induced senescence in astrocytes, which associated with NVC dysfunction and impaired performance on cognitive tasks. To establish a causal relationship between WBI‐induced senescence and NVC dysfunction, se‐nescent cells were depleted from WBI‐treated animals (at 3 months post‐WBI) by genetic (ganciclovir treatment) or pharmacological (treatment with the BCL‐2/BCL‐xL inhibitor ABT263/Navitoclax, a known senolytic drug) means. In WBI treated mice, both treatments effectively eliminated senescent astrocytes, rescued NVC responses and improved cognitive performance. Our findings suggest that the use of senolytic drugs can be a promising strategy for preventing the cognitive impairment associated with WBI.

Support or Funding Information

This work was supported by grants the Oklahoma Center for the Advancement of Science and Technology, the Oklahoma Shared Clinical and Translational Resources (OSCTR) program funded by the National Institute of General Medical Sciences (GM104938), the NIA‐supported Geroscience Training Program in Oklahoma (T32AG052363), the Oklahoma Nathan Shock Center (P30AG050911), the Cellular and Molecular GeroScience CoBRE (1P20GM125528, sub#5337), and the NIGMS supported Center of Biomedical Research Excellence (CoBRE).
Extracellular vesicles from tumor microenvironment transforms endothelial cell phenotype via downregulation of TRPV4 channels
Authors: Guarino Brianna Denise , Adapala Ravi Kumar , Kanugula Anantha K. , Dougherty Julie A. , Paruchuri Sailaja , Mahmood Khan A. , Thodeti Charles Kumar
Published: April 2020

Angiogenesis, the formation of new blood vessels from existing ones, is a normal physiological process. However, deregulation of angiogenesis can lead to pathological states such as cancer, that is characterized by hyper‐permeable and tortuous vessels. We have recently shown a significant decrease in functional expression of the mechanosensitive ion channel, transient potential receptor vanilloid 4 (TRPV4), in tumor endothelial cells (TEC). Further, pharmacological activation of TRPV4 induced normalization of tumor vasculature and improved cancer therapy. However, the molecular mechanisms by which TRPV4 is downregulated in TEC is not yet known. To determine this mechanism, we focused on extracellular vesicles (EVs) derived from tumor cells. We first collected conditioned media (TCM) from tumor cells with and without pre‐treatment of an exosome inhibitor, GW4869. We found that treatment of human normal endothelial cells (hNEC) with TCM transformed them into tumor‐endothelial like (hTEC) phenotype as revealed by expression of TEM8, VEGFR2 membrane translocation, and abnormal tube formation. However, TCM from exosome inhibitor‐treated cells, failed to induce endothelial transformation. Further, we found that EVs isolated from TCM induced hNEC transformation to hTEC. Mechanistically, we found that tumor derived EVs induced functional downregulation of TRPV4 in hNEC as assessed by calcium imaging. Taken together, our results suggest that tumor derived EVs transforms normal endothelial cells via downregulation of TRPV4 channels.

Support or Funding Information

National Institutes of Health R15CA202847 and R01HL119705
Functional and Structural Characterization of Acrodysostosis Mutations of PKA RIα Reveals Mechanism Causing resistant to cAMP activation
Authors: Del-rio Jason , Wu Leon , Lu Tsanwen , Aoto Phillip , Taylor Susan S.
Published: April 2020

Acrodysostosis is a type of disease related to bone development which usually happened on gene PRKAR1A(PKA RIα) or PDE4D(PDE4D). Mutations in the PRKAR1A gene are linked to Carney complex disease (CNC) and Acrodysostosis‐1 (ACRDYS1) which show contrasting phenotypes. CNC mutations show increased protein kinase A (PKA) activity, whereas ACRDYS1 mutations result in decreased PKA activity and cAMP resistant holoenzymes. Here we characterize three ACRDYS1 mutants: Y371C, R366X and G198R biochemically and structurally to understand the molecular basis of Acrodysostosis disease mutants of PKA. The activation assay indicated that these three mutations show resistant to the cAMP activation, and almost complete loss of cooperativity for cAMP activation with a shift in the Hill coefficient from 1.7 in wild‐type to 1.2 in the mutants. The crystal structure of Y371C mutant at 3.36 Å resolution shows how PKA utilizes cyclic nucleotide‐binding (CNB) domains as building blocks to create a unique isoform‐specific homodimer interface, which agrees well with previously published wild‐type RIα structure. This structure also demonstrates the important role of Y371 which is essential for cAMP binding. The residues after Y371 are disordered in the mutant, and only one cyclic nucleotide ligand can be seen at its two CNB‐B sites. The molecular dynamics simulations show increased dynamics in Y371C mutant compared to wild type protein which agrees to the functional and structural results. These data allow us to better understand the molecular mechanism of Acrodysostosis and it might help the therapeutical and drugs development.

Support or Funding Information

This work was supported by NIH grant GM34921 (to S.S.T.).