bluuebunny/crossref_metadata_2025 huggingface dataset:
Acetylation‐phosphorylation cross‐talk: A role for HDACs in the regulation of PKCdelta/theta phosphorylation
Authors: Habibian Justine S. , Bolino Matthew , Ferguson Bradley S.
Published: April 2020

Post‐translational modifications (PTMs) such as acetylation and phosphorylation have been implicated in contractile dysfunction and muscle wasting. Moreover, mounting evidence demonstrates that histone deacetylase (HDAC) inhibitors attenuate muscle wasting in animal models of disuse and muscular dystrophy. Historically, lysine acetylation was studied in the regulation of gene transcription, where for instance HDACs remove acetyl groups on lysine residues of histone proteins to suppress gene expression. However, recent evidence has shown that non‐histone proteins are also regulated by acetylation; functioning in protein stability, intracellular signaling, mitochondrial function and muscle contraction. Despite our current understanding for lysine acetylation and protein phosphorylation in muscle, how these PTMs cross‐talk to regulate muscle physiology remains unclear. In our attempt to understand acetylation‐phosphorylation cross‐talk, we examined the role for HDAC inhibitors on protein kinase C delta (PKCδ) and delta/theta (PKCδ/θ) phosphorylation. PKC is composed of multiple isoforms and its activation has been linked to muscle disease and dysfunction. Myotubes were treated with or without a pan‐HDAC inhibitor (trichostatin A; TSA) in the absence or presence of a pathological agonist (i.e. PMA). Phorbol 12‐myristate 13‐acetate (PMA) increased phosphorylation of PKCδ and PKCδ/θ. Interestingly, PMA‐induced PKCδ and PKCδ/θ phosphorylation was amplified in myotubes treated with the pan‐HDAC inhibitor TSA. Class selective HDAC inhibitors confirmed these findings and further demonstrated that inhibition of class I HDACs (HDAC1, 2, 3) was sufficient to increase PKCδ and PKCδ/θ phosphorylation. It should be noted that total PKCδ and PKCδ/θ protein expression increased with class I HDAC inhibition concomitant to the increase in protein phosphorylation. These data suggest that HDAC inhibition increases total PKC gene/protein expression that can then be phosphorylated. Finally, we report that the HDAC 1/2 selective inhibitor Romidepsin also increased total and phosphorylated PKCδ and PKCδ/θ in response to PMA. Genetic knockdown studies are currently underway to elucidate the specific HDAC that regulates PKC gene/protein expression, while chromatin immunoprecipitation (ChIP) is being used to determine HDAC binding to the PKC promoter. Lastly, elucidating a functional consequence for HDAC inhibitor‐mediated regulation of PKCδ and PKCδ/θ protein expression and phosphorylation in muscle could yield significant insight into muscle wasting and function.

Support or Funding Information

This work is supported by the USDA NIFA (Hatch‐NEV00727, Hatch‐NEV00767), the Dennis Meiss & Janet Ralston Fund for Nutri‐epigenetic Research, the National Institute for General Medical Sciences (NIGMS) of the NIH (P20 GM130459) and the National Heart, Lung, and Blood Institute of the NIH (R15 HL143496) and NSF EPSCOR Track II (OIA‐1826801) to B.S.F.
Essential Anatomy for Emergency Medicine in the Undergraduate Medical Curriculum
Authors: Harmon Derek , Martindale Jim , Marzban Hassan , Farias Anna , McNary Thomas , Harrell Kelley , Keim Sarah , Kirera Francis , Cotter Meghan , 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 Emergency Medicine 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 Emergency Medicine 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 33 Emergency Medicine clinical educators at 9 medical schools. All (100%) Emergency Medicine clinical educators considered each anatomical region important to their specialty. Further data analysis has identified the highest ranked anatomical topics in each body region for the Emergency Medicine clerkship/elective.

Discussion and Conclusion

This database provides detailed information regarding the most clinically relevant anatomical topics as identified by Emergency Medicine clinical educators. This information can aid in focusing preclinical learning to best prepare medical students for success in their undergraduate and graduate clinical experiences.
Assessing the Relationship Between Students’ Approaches to Learning, Visuospatial Abilities, and Performance in an Undergraduate Human Anatomy Course
Authors: McWatt Sean C.
Published: April 2020

Learning is difficult to quantify. Several metrics for successful student learning have been explored, the most popular of which is academic performance. However, academic performance does not always accurately represent nuanced outcomes such as meaningful learning and skill development, nor is it a successful predictor of long‐term knowledge retention. One metric that has demonstrated alignment with such qualitative learning outcomes is student approach to learning; a framework that evaluates the depth with which students interact with their learning environment and the course content. However, the student approach to learning framework has yet to be adequately investigated for its reliability as a metric for learning in human anatomy, specifically. One measure that has been shown to predict learning achievement in human anatomy is visuospatial ability. Individuals with high visuospatial abilities are typically faster and more successful at performing three‐dimensional anatomy tasks than those with low visuospatial abilities. It has been suggested that this may be due to differences in strategies used for learning by students with high (versus low) visuospatial abilities. Despite this, the degree of correlation between students’ approaches to learning and visuospatial abilities has yet to be investigated. The present study therefore aimed to examine the relationship between students’ approaches to learning, visuospatial abilities, and performance in anatomy to investigate the validity of the student approach to learning framework as an alternative metric to evaluate anatomy learning. Physical Therapy, Occupational Therapy, and Kinesiology students (n = 35) completed a Mental Rotations Test and Revised 2‐factor Study Process Questionnaire after the end of their first anatomy course. Responses and final course grades were examined using regression analyses to detect their degrees of correlation. We hypothesized that visuospatial ability would correlate positively with both anatomy performance and deep approach to learning scores and have a negative correlation with surface approach to learning scores. However, no significant correlations were found between visuospatial ability and deep or surface approach to learning scores at p ≤ 0.05. Anatomy performance was positively correlated with deep approach to learning scores (β = 0.427, p = 0.02) and negatively correlated with surface approach to learning scores (β = ‐0.704, p < 0.01) but had no significant correlation with visuospatial ability at p ≤ 0.05. The data suggest that visuospatial ability does not significantly influence students' approaches to learning; however, additional research on larger populations is required to explore these relationships further.
Essential Anatomy for Family Medicine in the Undergraduate Medical Curriculum
Authors: Keim Sarah , Royer Danielle , Farias Anna , Harrell Kelley , Carpenetti Tiffany , Downs Mary Beth , Lewis Steven , McNary Thomas , Topping Daniel , Kirera Francis , Mork Amy , Wineski Lawrence , Granite Guinevere , Lopez Lisa , Zumwalt Ann , Sanky Charles , Laitman Jeffrey , Reidenberg Joy S. , Davies David , Marzban Hassan , Cotter Meghan , Hisley Kenneth , 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 Family Medicine 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 Family Medicine 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 93 Family Medicine clinical educators at 19 medical schools. The percentage of Family Medicine clinical educators that considered each anatomical region important to their specialty were (highest‐to‐lowest): Back (99%), Upper Limb (99%), Lower Limb (99%), Head & Neck (98%), Thorax (96%), Abdomen (96%), and Pelvis & Perineum (95%). Further data analysis has identified the highest ranked anatomical topics in each body region for the Family Medicine clerkship/elective.

Discussion and Conclusion

This database provides detailed information regarding the most clinically relevant anatomical topics as identified by Family Medicine clinical educators. This information can aid in focusing preclinical learning to best prepare medical students for success in their undergraduate and graduate clinical experiences.
Pentalinonsterol exhibits the immunomodulatory action in macrophages through activation of Phospholipase A2
Authors: Varikuti Sanjay , Shelton Blake , Kotha Sainath R. , Gurney Travis , Gupta Guarav , Fuchs James R. , Kinghorn Dougles , Srivastava Nidhi , Satoskar Abhay R. , Parinandi Narasimham L.
Published: April 2020

Leishmaniasis, a protozoan parasitic disease, is caused by the infection with the parasite, Leishmania spp. Pentalinonsterol (cholest‐4,20,24‐trien‐3‐one) (PEN) is a natural product recently isolated from the roots of Pentalinon andrieuxii. Previous in vitro and in vivo studies have demonstrated that P. andrieuxii root extracts possess potent immunomodulatory properties, which markedly increase the activity of macrophages and dendritic cells. Given the beneficial characteristics of this root extract, the objective of this study was to elucidate the mechanism of action of the immunomodulatory action(s) of PEN in macrophages on membrane phospholipid metabolism towards the anti‐leishmanial action of PEN. Phospholipase A2 (PLA2), plays an important role in membrane phospholipid homeostasis, cell signaling and generation of the eicosanoid inflammatory bioactive lipids. Therefore, in this study, we investigated PEN‐mediated modulation of PLA2 activity in RAW 264.7 macrophages and the bone marrow‐derived primary macrophages (BMDMs) from mice in vitro and how the upstream PLA2 activation would modulate the downstream cytokine release in the macrophages. Our results demonstrated that, (1) PEN induced PLA2 activation (arachidonic acid release), in a dose‐ and time‐dependent manner that was regulated upstream by mitogen‐activated protein kinases (MAPKs): (2) the PEN induced activation of PLA2 was attenuated by the cPLA2‐specific pharmacological inhibitors; and (3) cPLA2‐specific pharmacological inhibitors attenuated the release of the inflammatory cytokines from macrophages. We also observed that PEN enhances the Leishmania‐induced cPLA2 activation in the RAW macrophages. Our study demonstrated that PEN exerted immunomodulatory actions through cPLA2 activation in macrophages, which is advantageous for vaccine development against Leishmaniasis.

Support or Funding Information

Supported by the U.S. Army/Department of Defense and Center for Clinical and Translational Science, Ohio State University.
Reshaping commensal gut microbiota in early life lowers blood pressure potentially via a succinate mediated mechanism
Authors: Chakraborty Saroj , Galla Sarah , Cheng Xi , Yeo Ji-Youn , Mell Blair , Chiu Nathaline , Wenceslau Camilla F. , Kumar Matam Vijay , Joe Bina
Published: April 2020

Pediatric hypertension is recognized as an emerging global health concern. While new guidelines are developed for facilitating clinical management, the reasons for the prevalence of hypertension in children remain unknown. Genetics and environmental factors do not fully account for the growing incidence of pediatric hypertension. Because stable bacterial flora in early life are linked with health outcomes later in life, we hypothesized that reshaping of gut microbiota in early developmental stages of life affects blood pressure (BP) of pediatric subjects. To test this hypothesis, we administered amoxicillin (50mg/day/kg body weight with drinking water), the most commonly prescribed pediatric antibiotic, to alter gut microbiota of young, genetically hypertensive Dahl S rats (study 1) and dams during gestation and lactation to reshape microbiota of offspring (study 2). Reshaping of microbiota (determined by 16s sequencing), with reductions in Firmicutes/Bacteriodetes ratio observed in amoxicillin treated young rats and in dams. Amoxicillin treated rats (8 weeks old) also had lower blood pressure (radiotelemetry: 24‐hour average Systolic Blood Pressure in mmHg (SBP): 155±2*) compared to the untreated rats (SBP: 166±3) (n=10–12/group). In the young rats treated with amoxicillin, the BP lowering effect persisted even after the antibiotics were discontinued (10 weeks old‐last 2 weeks without Amoxicillin) (amoxicillin SBP: 196±4 vs Control SBP: 231±10*). Similarly, offspring from the dams treated with amoxicillin also showed lower systolic blood pressure (SBP: 201±4*) compared to the control rats (SBP: 222±6) (9 weeks old and n=6–8/group). Remarkably, in all cases, a decrease in BP was associated with lowering of Veillonellaceae, which are succinate‐producing bacteria. Elevated plasma succinate is reported in hypertension. Accordingly, serum succinate was measured via choloremtric assay and found lower in animals treated with amoxicillin (410±33 mg/L*) compared with control (508±30 mg/L). Our results demonstrate a direct correlation between succinate‐producing gut microbiota and early development of hypertension, and indicate that reshaping gut microbiota, especially by depleting succinate‐producing microbiota early in life may have long‐term benefits for hypertension‐prone individuals. Data presented as Mean±SEM. Non‐parametric Mann‐Whitney test*p<0.05.

Support or Funding Information

National Institutes of Health (R01HL143082)
Effect of Aging on Vascular Smooth Muscle Myogenic Contractility in Soleus Muscle Feed Arteries
Authors: Shin Song Yi , Seawright John , Trache Andreea , Woodman Christopher
Published: April 2020

Objective

We tested the hypothesis that vascular smooth muscle (VSM) myogenic constrictor responses decline with age in soleus muscle feed arteries (SFA). In addition, we asked whether pre‐treatment of SFA with a short duration (1 h) increase in intraluminal pressure, to mimic pressure associated with a bout of exercise, would have beneficial effects on myogenic responses in SFA.

Methods

SFA from young (4 mo) and old (24 mo) Fischer 344 rats were cannulated with glass micropipettes and endothelial cells were removed (denuded) by passing 5 ml of air through the lumen of the vessel. SFA were then pressurized to 90 cmH2O (p90) or 130 cmH2O (p130) for 1 h. At the end of the 1 h treatment, intraluminal pressure in all arteries was set to 90 cmH2O for examination of VSM myogenic constrictor function. After stable tone was achieved, myogenic contractile responses were assessed using step‐increases in intraluminal pressure from 90 cmH2O to 135 cmH2O in 15 cmH2O increments followed by step‐decreases down to 45 cmH2O.

Results

Myogenic responses of denuded old SFA (p90) was not different from denuded young SFA (p90). Pre‐treatment of SFA with increased pressure for 1 h improved myogenic constriction in young SFA (p130) but not in old (p130).

Conclusion

VSM myogenic responses were not altered with age in denuded SFA. Pressure treatment contributed to enhanced myogenic constrictor responses in young SFA.

Support or Funding Information

NIH grant R03AG064551, Sydney and J.L. Huffines Institute of Sports Medicine and Human Performance, and CEHD Research Fellowship, Texas A&M University
Reprogramming Cardiac Cells Into Cardiac Purkinje‐like Cells For The Treatment Of Heart Failure
Authors: Prodan Nicole , Raghunathan Suchi , Iyer Dinakar , Schwartz Robert J. , McConnell Bradley K.
Published: April 2020

Heart failure (HF) is a chronic condition where the heart loses its ability to efficiently pump blood to other organs in the body. Currently, there is no cure for HF, other than a heart transplant, which may not be available for most patients. Other treatments available for HF focus on reducing the symptoms, but do not properly address the loss of cardiac tissue and scar tissue formation commonly observed in this condition. We believe that a novel therapeutic approach for HF can be achieved through direct administration of cardiac electrical conducting cells to failing hearts. This is because cardiac conducting cells, such as Purkinje cells, can intrinsically propagate electrical signals in synchronicity with other cardiac cells, allowing the heart to contract and pump blood. Thus, direct injection of cardiac Purkinje cells into failing hearts may overcome mechanical issues from scar tissue, as well as restore some of the cardiac tissue that has been lost in HF. To create Purkinje‐like cells, a reporter gene containing a Connexin 30.2 promoter and mCherry fluorescent tag was first introduced into the cardiac cells. This was followed by 5 days treatment with specific small molecules. The reprogrammed cardiac Purkinje‐like cells are then being characterized by transcriptome, epigenetic state, and functional analyses through FACS sorting, immunostaining, qPCR analysis, RNA sequencing, ChIP‐seq and Patch clamp. Successfully reprogrammed cardiac cells are expected to exhibit Purkinje‐like properties such as genetic make‐up and functionality. Upon creating Purkinje‐like cells, the next step will be to test the effectiveness of the Purkinje‐like cells in restoring the electrical conduction, as well as improving cardiac function, in small animal models of HF.

Support or Funding Information

Robert J. Kleberg, Jr. and Helen C. Kleberg Foundation

National Heart, Lung, and Blood Institute (NHLBI) of the NIH (R15HL141963)
A Team‐Based Ultrasound Examination: Assessing Ultrasound Knowledge and Non‐Traditional Discipline‐Independent Skills During Practical Examination in the First‐Year Medical Anatomy Course
Authors: Andersen Kylie Jean , Rockey Nathan Giffen , Thomas Alexa Leigh , Linder Katharine Elizabeth , Oyemade Kafayat Adeola Enitan , St. Jeor Jeffery Dale , Bhagra Anjali , Lachman Nirusha , Pawlina Wojciech
Published: April 2020

In recent years, the use of point of care ultrasound (POCUS) in clinical practice has increased substantially
(1). Many medical schools have introduced ultrasound skills training into their curriculum which starts early in anatomy courses
(2). Few, if any, of these programs assess students’ ultrasound knowledge and skills in a simulated clinical team environment. Anatomy Ultrasound Objective Structural Practical Examination (OSPE) implemented as a part of practical examination provides such an opportunity to asses both traditional and non‐traditional discipline‐independent skills, competencies and attributes. As part of the seven‐week anatomy course at Mayo Clinic Alix School of Medicine, first‐year medical students attended four ultrasound sessions. Each session included a short didactic module followed by hands‐on practice guided by near‐peer teachers. For the final examination, four‐member student teams were required to work collaboratively to perform four different scans during which students interacted with multiple standardized patients and rotated responsibility for obtaining an image, orienting the image, and identifying assigned structures. Students were graded using a rubric and received a team‐score based on tasks associated with individual roles of team members. In addition to ultrasound skills, the nature of their professional interactions with patient, communication skills, and situational awareness were assessed. The average team score was 96%. The students completed a survey at the conclusion of the anatomy course in which they rated the OSPE experience very positively. Team‐based OSPE examination was a clinically relevant and effective mechanism to simultaneously evaluate anatomic knowledge, clinical skills, and non‐traditional discipline‐independent skills that were emphasized during the anatomy course.

Support or Funding Information
Agonist‐Dependent Modulation by the Long‐Acting Mu Opioid Receptor Antagonist, Methocinnamox (MCAM)
Authors: Smith Hudson R. , Zamora Joshua C. , Chavera Teresa S. , Jennings Elaine M. , Winger Gail D. , Woods James H. , Clarke William P. , Berg Kelly A.
Published: April 2020

Opioid overdose is a leading cause of death in the US. Naloxone (NLX), a mu opioid receptor (MOR) competitive antagonist, is the only treatment currently available for reversing opioid overdose. Unfortunately, NLX’s effects are limited by a short duration of action and ease of surmountability by opioid agonists. Methocinnamox (MCAM) is a novel MOR antagonist that has a long duration of action in vivo and holds promise as a better treatment for overdose and perhaps opioid use disorder. In this study, we compared the antagonist properties of MCAM to that of NLX and beta‐funaltrexamine (β‐FNA; an irreversible MOR antagonist). In HEK cells that express human MOR, DAMGO inhibited forskolin‐stimulated cAMP levels in a concentration dependent manner with an EC50 of 10 nM and a maximal inhibition of 40%. Pretreatment (15 min or 2 hrs) with NLX (100 nM, 10 x Ki) shifted the DAMGO concentration response curve (CRC) to the right, 100‐fold, in a fully surmountable manner that was independent of pretreatment time and fully reversed following NLX washout. Pretreatment with β‐FNA (10 nM) reduced the DAMGO maximal response in a time‐dependent, non‐surmountable and irreversible manner with no effect on the potency of DAMGO. By contrast to both β‐FNA and NLX, pretreatment with MCAM (10 nM) reduced the maximal response in a time‐dependent, non‐surmountable, non‐washable, and irreversible manner
and shifted the DAMGO CRC to the right 1000‐fold. We hypothesized that this rightward shift in the DAMGO CRC may be due to allosteric properties of MCAM at MOR. A hallmark of allosterism is ligand dependence, so we next tested effects of MCAM, β‐FNA and NLX on a different mu opioid agonist, fentanyl. As expected, pretreatment with β‐FNA reduced the maximal response to fentanyl in a non‐surmountable manner without affecting potency, whereas NLX pretreatment shifted the CRC of fentanyl to the right (decreased the potency) in a manner that was fully surmountable and independent of pretreatment time. By contrast, pretreatment with MCAM reduced the maximal response to fentanyl in a non‐surmountable manner
and shifted the CRC to the
left (increased potency). Altogether, these data suggest that MCAM may be an irreversible orthosteric antagonist and an allosteric modulator at MOR.

Support or Funding Information

Supported by NIH/NIDA RO1 grant DA048214
Prenatal Exposure of Mice to Beta‐methyl‐amino‐L‐alanine (BMAA) as a Model for Amyotrophic Lateral Sclerosis‐Parkinson’s Dementia Complex (ALS‐PDC).
Authors: Muthian Gladson , Charlton Clivel
Published: April 2020

Amyotrophic Lateral Sclerosis‐Parkinson’s Dementia Complex (ALS‐PDC) is a severe neurological disorder, caused by degeneration of upper motor
glutamine (UMG), nigrostriatal
dopamine (NSDA) and basal forebrain
acetylcholine (BFbA) neurons, and locally called Lytico‐bodig in Guam. The incidence of ALS‐PDC has decline, but the cause is still unknown. Several studies indicate that beta‐methylamino‐L‐alanine (BMAA), a non‐protein amino acid, consumed in food prepared from the cycad plant, is the cause of ALS‐PDC. The consumption of BMAA has been reduced, as well as the incidence of ALS‐PDC, and these implicate BMAA as a likely cause for ALS‐PDC, Studies, however, had produced no good model of ALS‐PDC. Since UMG, NSDA and MFbA neurons are impaired in ALS‐PDC, we targeted these neuronal groups in the fetus, by exposing C57BL/6J pregnant mice to BMAA during gestation day 8–18, the birth or neurogenesis of these neurons in the fetus. The goal was to produce impaired neurons that will succumb/die early during aging. The outcomes showed post‐natal changes in weight, behaviors, muscle functions as well as specific neurotransmitters and other neurochemicals. BMAA administered at 10, 50 and 100 mg/kg to the pregnant dams caused dose‐dependent reduction in the weight of two weeks pups by 12.5, 20.0 and 27.5% of the control. Body weight, however, recovered at 4–5 weeks post‐weaning. Prenatal BMAA also reduced open field motor activity: e.g. the total distance travelled was reduced by 8.8, 11.6 and 19% by the respective doses of BMAA. On the Rota‐Rod, the latency to fall was dramatically reduced to 71.4, 17.4 and 8.7% by the 10, 50 and 100 mg/kg of prenatal BMAA, as compared to control. Likewise, the no of revolution per min that the mice endured were reduced from the control value of 100%, to 67.8%, 25.4% and 16.9% for the 10, 50 and 100 mg/kg of BMAA. The data show that BMAA has effects on motor performance of offspring exposed prenatally. The continuation of the study will reveal underlying neurochemical changes that may be related to ALS‐PDC. The indication is that ALSPDC may have a fetal basis in which early subtle neuronal impairments occurred and symptoms precipitated later in life when UMN, NSDA and MFbA are impaired during aging.

Support or Funding Information

NIH
Gnotobiotic rats reveal an obligatory role of microbiota in blood pressure
Authors: Chakraborty Saroj , Cheng Xi , McCarthy Cameron G. , Mell Blair , Yeo Ji-Youn , Golonka Rachel , Yeoh Beng San , Mandal Juthika , Yang Tao , Saha Piu , Wenceslau Camilla F. , Kumar Matam Vijay , Joe Bina
Published: April 2020

Blood pressure is a cardinal vital sign that informs clinicians for both acute and long‐term clinical care. Elevated blood pressure or hypertension is the single largest risk factor for cardiovascular diseases which are the leading cause of human deaths. Current clinical management of blood pressure is focused on restoring homeostasis of the host alone, which targets an incomplete holobiont i.e., without accounting for commensal gut microbiota. Recent evidence from the CARDIA study in humans and multiple studies using animal models suggest that development of hypertension in the host is associated with alterations in microbiotal communities. Whether microbiota is a required component of the holobiont to maintain cardiovascular health is unknown. Here we examined whether microbiota is necessary for blood pressure and vascular homeostasis by functional evaluation of the gut homeostasis, hemodynamic, and vascular function of gnotobiotic rats reconstituted with microbiota to represent the complete holobiont. Gnotobiotic rats were used to represent incomplete holobionts. To reconstitute complete holobionts, gnotobiotic rats were co‐housed with conventionally‐raised rats. Acquisition of microbiota was evaluated through monitoring of gross ceca and fecal samples by metagenomic 16S sequencing. BP was recorded and vascular, renal, hepatic, cardiac and gut features were assessed using histology and ex vivo myography. Markers of innate immune effectors (Immune cell population, level of Lcn2, Gut permeability) were used to examine the nature and extent of host immune cell processes concomitantly occurring along with observations of host hemodynamics. Compared to the reconstituted holobiont represented by the animals exposed to microbiota, the incomplete‐holobiont represented by gnotobiotic rats, had significantly lower BP (SBP of germ free:109±8 mmHg, SBP of conventionalized:138±10mmHg*) and vascular contractility responses to phenylephrine (Emax (mN): germ‐free: 6.9±1.3, GFC: 11.7±0.7*). Acute exposure of the host to microbiota reconstituted gut microbiotal communities, significantly boosted their gut epithelial cell proliferation, innate immune function and restored vascular contractility. These data indicate that in addition to the dependency of the host on microbiota for essential bodily functions such as digestion of plant‐derived complex carbohydrates, the host is also dependent on microbiota for maintaining blood pressure and vascular function

Support or Funding Information

National Institutes of Health (R01HL143082)
Amygdalar Plasticity in Airway Disease
Authors: Kuan Shin-Ping , Atanasova Kalina , Liao Yan-Shin , Guevara Maria Valentina , Pompillus Marjory , Collins Emily , Moore Jonai , Febo Marcelo , Reznikov Leah R.
Published: April 2020

Asthma is one of the most common immunological diseases among humans. Chronic airway inflammation drives asthma pathophysiology. While much effort has focused on understanding the airway intrinsic effects of inflammation, chronic inflammation also activates stress‐responsive brain regions, including the amygdala. This is important because greater amygdala activity exaggerates inflammatory responses, and greater amygdala activation in asthmatic patients correlates with reduced bronchodilator responses. Within the amygdala, pyramidal neurons of the basolateral amygdala (BLA) encode aversive stimuli. These neurons are characterized by spiny dendrites, are excitatory (e.g., glutamatergic), and exhibit unique cellular and structural plasticity in response to aversive stimuli that is mechanistically linked to BLA function. Thus, pyramidal neurons might be key targets of inflammation that further drive asthma pathology. Here we test the central hypothesis that pyramidal neurons of the BLA encode airway inflammation and exacerbate asthma pathophysiology. Our preliminary data suggest that an acute asthma exacerbation increases activity of the murine BLA, as evidenced by c‐fos expression and manganese‐enhanced MRI. Further, we show that global pharmacological disruption of glutamatergic signaling mitigates airway disease features, including airway hyperreactivity and mucus hypersecretion. Finally, we show that airway inflammation increases dendritic spine density in pyramidal neurons of the BLA. These results identify novel therapeutic targets for asthma, and possibly other airway diseases.
Diverse Interactions of Adeno‐Associated Viruses (AAV) with Cell Receptor, AAVR – Binding Studies Supported by Cryo‐Electron Microscopy at 2.5 Å Resolution, Relevant to Gene Therapy
Authors: Chapman Michael Stewart , Silveria Mark , Meyer Nancy Lynn , Davulcu Omar Lynn , Xie Qing , Large Ed , Zane Grant , Neff Rachel , Hu Guiqing , Stagg Scott S.
Published: April 2020

Adeno‐associated virus (AAV) is a small human single‐stranded parvovirus. Interest centers upon the use of recombinant forms (rAAV) as delivery vectors in gene therapy, the first of which have been gaining FDA approval. An understanding of the molecular interactions on cell entry is a foundation for the design of vectors with greater tissue specificity. In 2016, we revisited the identity of cell receptors, with a genome‐wide deep‐sequencing of haploid human cells selected from gene‐trap mutant library. We enriched the population of AAV‐resistant cells, thorough fluorescence activated cell‐sorting, using an rAAV encoding mCherry, allowing us to identify host‐factors needed for AAV transduction. Foremost was an uncharacterized membrane protein, AAVR, that is needed for trafficking from the cell surface to the perinuclear trans Golgi network. After identifying the extracellular domains of AAVR that interact with AAV, we have expressed soluble domain fragments and have recently obtained structures of complexes at about 2.5 Å by cryo‐electron microscopy (EM). The highest resolution structures have come from complexes with just the two N‐terminal PKD domains, allowing us to minimize conflicts between receptors bound at adjacent sites on the virus surface related by symmetry. However, the high resolution structures are fully consistent with cryo‐electron tomography, where we see (at about 3nm resolution) not only the domains bound directly, but those linked by flexible hinges as they adopt several configurations. Thus, this is an illustration of a holistic understanding of a flexible complex coming from different constructs amenable to studies at different resolutions. AAV serotype 2 (AAV‐2) interacts primarily with AAVR’s polycystic kidney disease (PKD) domain 2 at a relatively conserved part of the viral surface which is also targeted by some of AAV’s most neutralizing monoclonal antibodies. Intriguingly, binding studies, mutation, and now EM structure, show that AAV‐5 interacts more exclusively with PKD domain 1 at a non‐overlapping site on the virus. In addition to providing a lead in understanding the differential cell specificities of different AAVs, these unexpected results pose interesting questions regarding the evolution of virus‐host interactions, specifically in the emergence of distinct sets of molecular interactions among related viruses that still target the same receptor.

Support or Funding Information

Funded by R35 GM122564 (MSC).
New Chemical Probes for Detection of Molecular Markers of Multiple Sclerosis
Authors: Dacon Nicholas , Kadnikov Dimitry V.
Published: April 2020

Multiple sclerosis is a neurodegenerative disease of central nervous system characterized by an autoimmune response against the myelin sheath. The causes and mechanism of the development of the pathology are still not fully understood, but one of the main molecular markers of the disease development appear to be an extensive citrullination of a myelin basic protein. Citrullination is a post‐translational modification, specifically a deimination, of a side chain of an amino acid arginine, which results in significant changes in protein structure. Therefore, reliable detection of this modification would be indispensable for the study and diagnosis of the disease. However, detection of this post‐translational modification by mass‐spectrometry is difficult due to a small change in mass of the amino acid and as a result, small molecule chemical probes emerged as a tool of choice for citrulline detection. While several probes have been reported, the field is still in its infancy and novel more potent and more efficacious probes are required. Our group has been engaged in the development of modular chemical probes for detection of citrulline. In such probes a reactive head that labels citrulline and a detection tag that allows visualization of labeled citrulline are synthesized and administered separately. Here we present design and synthesis of the chemical probes using 1,3‐dicarbonyl molecules as the reactive heads for the labeling of citrulline. To demonstrate the utility of these probes their reactivity with model compounds, such as N‐ethylurea , N,N‐diethylcitrullinamide, and a citrulline‐containing peptide have also been investigated and the results will be discussed.
A cadaveric study of the aortic arch branching patterns
Authors: Solis Laura , Bittles Leah , Rahimi Omid B. , Johnson Linda Y. , Sakaguchi Alan Y. , Occhialini Annette Y. , Kar Rekha
Published: April 2020

This study was conducted to investigate the variations in the aortic arch branching patterns in the cadavers donated to the University of Texas Health at San Antonio, San Antonio, Texas. Branching patterns of vessels from the aortic arch in 56 cadavers (31 males and 25 females) were recorded after dissection of the mediastinum by the medical students. A previous meta‐analysis of variations in the adult aortic arch branching has grouped the aortic arch variants into seven different types. Our survey of cadaveric specimens revealed three of these types. The normal variant (Type 1) where the aortic arch gives rise to the brachiocephalic trunk (BT), the left common carotid (LCC) and the left subclavian (LS) arteries occurred in 69.6% (21 male and 18 female, 95% confidence interval: 56.6% to 80.2%) of cadavers. The bovine arch (BA) variant (type 2) of the aortic arch is a known congenital anomaly with both clinical and surgical significance. It is described as the common origin of the left common carotid artery (LCC) and the brachiocephalic trunk (BT) from the aortic arch. BA was present in 25% of cadavers (95% confidence interval: 15.4% to 37.8%), distributed evenly among male and female cadavers (7 male and 7 female). Type 3 variant, where the left vertebral artery (LV) was branching directly from the arch between the LCC and the left subclavian artery (LS) was noted in 5.3% (95% confidence interval: 1.3% to 15.2%) of the cadavers. Interestingly, all the cadavers found to have the type 3 variant were male. Knowledge of variations in the branching patterns of vessels from the aortic arch is essential to surgical planning to ensure optimal patient outcomes.
Teratogenic effects of 1.5% EtOH in Cranial Blood Vessel development and Neurogenesis in Zebrafish larvae at 72hpf
Authors: Lee Hyuna , Huang Jocelyn , Song HyunJi , Son Dana , Atkinson Lindsay , Pia Thaddeo
Published: April 2020

Fetal alcohol spectrum disorder, which is observed in children who have been exposed to alcohol in utero, is a leading cause of neurodevelopmental disability. Studies in rodents have shown that prenatal ethanol exposure results in decreased blood flow from the umbilical artery to the fetal brain, compromising neuron formation and angiogenesis in the maturing brain. Ethanol (EtOH) has shown to disrupt coronary artery formation in the embryonic zebrafish, but the effects of ethanol on the developing cerebral blood vessels and neurogenesis in zebrafish larvae remains poorly understood. We exposed zebrafish larvae to 1.5% ethanol at 4 hours post fertilization (hpf) for 24 hours and imaged the brain at 72 hpf. We used Tg(fli1:EGFP) to image cerebral blood vessels and Tg(ETvmat2:GFP) to visualize monoaminergic neurons using confocal microscopy. Morphological data collected at 72hpf depicted significantly higher rates of craniofacial deformities, pericardial edema, and severe axial malformations in 1.5% EtOH‐treated embryos in comparison to control. Our preliminary results suggest that EtOH at 1.5% leads to reduced number of cranial vessels and altered vessel branching, and decreased density of telencephalic and hindbrain neurons. These findings lead to questions regarding molecular and vascular disparities in affected regions of the developing brain.

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I am currently seeking for funds (nonmember student registration fee) for our undergraduate students to attend and present this poster at the conference.
Production and Purification of N‐Hydroxy Succinyl Cadaverine (HSC) By Synthetic and Biosynthetic Methods
Authors: Ruhl Natalie , Hoffmann Katherine
Published: April 2020

Optimum conditions for the production and purification of N‐Hydroxy Succinyl Cadaverine, HSC, were investigated. HSC is not commercially available and is essential for research with an enzyme known as DesD. This enzyme will allow HSC to be trimerized and macrocyclized into a siderophore. The siderophore is capable of absorbing iron in the bloodstream that antibiotic‐resistant bacteria use as a life‐sustaining nutrient. Small‐scale organic synthesis was involved in the process of producing the precursor to HSC, known as SC. SC, succinyl cadaverine, is non‐hydroxylated HSC. A deprotection of Boc‐protected succinyl cadaverine was performed. Regular phase thin‐layer chromatography (TLC) was used to check reaction progress while reverse phase TLC was used to determine solvent conditions for purification of SC. Previous attempts to create SC yielded orange‐red puddles of oil. Reaction conditions were changed to theoretically yield white crystals, which indicates purity. After decreasing the equivalencies of TFA, changing the solvent of the reaction, and increasing the run time, white crystals were achieved. 21.5 milligrams of SC were produced in total. The appearance of the crystals increased confidence that the product was fairly pure. Regardless, purification was necessary. The solvent condition for purification of SC was determined to be 100% dichloromethane. This solvent condition yielded a Rf of ~0.5 on a reverse‐phase TLC plate. With this year being the fifth year of research on this project, the organic synthesis of HSC has proven to be very difficult and time‐consuming. New approaches have been considered to make the mechanism more efficient. A biochemical pathway of synthesis will now be investigated. High pressure liquid chromatography will be used to explore the separation conditions of materials between steps. The trimerized and macrocyclized version of HSC, known as DFO‐E, is $400/mg if purchased commercially, and HSC is not available at all. If DesD, a potential new antibiotic drug target, is to be fully investigated, efficient, and cost‐effective, means of creating HSC will be required.

Support or Funding Information

This research was supported in part by the National Science Foundation (NSF‐RUI grant 1716986 to K.M.H.), and by the John Stauffer Charitable Trust.
Ensemble Dynamics of Stellate Ganglion Neurons Reveal Differential Transduction of Cardiac and Pulmonary Inputs ‐ SPARC
Authors: Sudarshan Kostubh B. , Hori Yuichi , Swid M. Amer , Karavos Alexander B. , Wooten Christian , Mirhanian Gohar , Narinyan William , Armour J. Andrew , Kember Guy C. , Ajijola Olujimi A.
Published: April 2020

Background

The sympathetic nervous system exerts closed‐loop hierarchical control of cardiac function partially through peripheral sympathetic ganglia (PSGs). To what extent PSGs possess network processing capabilities useful for local cardiopulmonary integration outside the central nervous system is unknown.

Methods

To understand whether neurons within PSGs process cardiopulmonary inputs, and to gain insights into the modes and properties of such transduction, we performed extracellular recordings (via a linear 16‐electrode array) to determine intrinsic activity of left stellate ganglion (LSG) neurons in chloralose‐anesthetized pigs (n=8). Continuous respiratory and left ventricular pressures (RP and LVP) were recorded along with the ECG. Neuronal activity was examined during resting states and under various cardiopulmonary stressors. Linkages between neural activity and LVP and RP were examined at the local neural ensemble and global population levels such that ensemble behavior could be examined over short time scales (minutes) versus evolution of population level processing over hours, using a sliding window.

Results

Neural ensembles show a wide range of regional behaviors with activity that may cease or commence during the cardiorespiratory cycle such that no neuron is either purely cardiac or pulmonary, rather neurons exhibit cardiopulmonary integration. Global level processing was examined, over a sliding window, for: (i) the presence or absence of cardiac and respiratory periodicities, and (ii) the evolution of the degree to which greater or lesser ‘attention’ is globally paid to LVP and RP relative to random sampling. The results of (ii) show that the greatest attention is paid to diastole and near‐peak LVP. Regarding RP, little attention is paid to the respiratory pressure while the breathing rate from (i) is generally strongly evident. Interestingly, spiking density did not correlate with level of attention i.e. increased spiking levels does not imply that random sampling has ceased and attention has become focused on LVP or RP.

Conclusions

Activity of stellate ganglion neurons recorded in situ reveal complex local neuronal and global population properties that are dynamically phase‐ and rate‐locked with cardiac and pulmonary function respectively. The combination of (i) and (ii) are useful in finding consistency in neuronal attention across animals since this combination gives insight into the degree to which a population of neurons is processing information at the local and global levels within PSGs.

Support or Funding Information

NIH: DP2OD024323‐01 and OT2OD023848
Polarized Intracellular Endothelial Pannexin 3 Can Regulate Vascular Function
Authors: Wolpe Abigail , Ottolini Matteo G. , Alfaidi Mabruka , Johnstone Scott , Askew-Page Henry , Barr Kevin , Beier Frank , Orr A. Wayne , Redemann Stephanie , Penuela Silvia , Sonkusare Swapnil , Isakson Brant
Published: April 2020

Pannexin 3 (Panx3) expression is generally thought to be restricted to skin, cartilage and bone where it is routinely observed on intracellular compartments. However, multiple RNAseq studies have demonstrated consistent expression of Panx3 in endothelium throughout the vasculature. A physiological role for endothelial Panx3 has yet to be described. To assess the expression of Panx3 in intact endothelium, we first performed immunogold labelling on high pressure frozen third‐order mesenteric arteries. Panx3 preferentially localized to internal membranes in transverse sections of the endothelium. Next, we used multiple Panx3 antibodies raised against different topographical locations on third‐order mesenteric arteries prepared en face. We found that endothelial Panx3 was polarized downstream of blood flow, co‐localizing with a subset of calnexin‐positive perinuclear membranes, indicating association with the endoplasmic reticulum (ER). To test whether Panx3 was flow‐sensitive, primary cultures of human endothelial cells were seeded in parallel plate flow chambers and exposed to laminar shear stress (10 dynes/cm2 for 18 hours). Panx3 was polarized in the direction of flow similar to that seen in intact resistance arteries. To interrogate the physiological role of endothelial Panx3, we generated an inducible, cell‐specific Panx3 knockout mouse (Panx3fl/fl/Cdh5‐CreERT2+), which reduces both Panx3 mRNA and protein following tamoxifen injection. Panx3fl/fl/Cdh5‐CreERT2+ mice had normal blood cell parameters, heart function, body length, body weight, and fat content. Because we identified Panx3 in the ER perinuclear region, we began by assessing intracellular calcium on intact third‐order mesenteric arteries. Loss of endothelial Panx3 reduced intracellular calcium activity in endothelium under unstimulated conditions. Because calcium is vital to arterial dilation, we performed pressure myography. We found that resistance arteries from Panx3fl/fl/Cdh5‐CreERT2+ mice exhibit significantly enhanced vasoconstriction in response to phenylephrine, indicating impaired endothelial negative feedback on the smooth muscle cells. Due to the role of resistance arteries in blood pressure regulation, we hypothesized there may be a change in the blood pressure of these mice. We used radiotelemetry and found Panx3fl/fl/Cdh5‐CreERT2+ mice were hypertensive compared to tamoxifen‐injected littermate controls. The sum of this data suggests that endothelial Panx3 is polarized on the ER where it can regulate calcium activity in the endothelium, independent of ATP release, and may be important for vascular function.

Support or Funding Information

HL149221 (AGW) and HL120840 (BEI)