bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 InformationFunded by R35 GM122564 (MSC).
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.Support or Funding InformationI 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 InformationThis 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

BackgroundThe 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.MethodsTo 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.ResultsNeural 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.ConclusionsActivity 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 InformationNIH: 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 InformationHL149221 (AGW) and HL120840 (BEI)
Investigating Cellular Interactions Between the Rho GTPase TCL/RhoJ and Coronin Proteins
Authors: McRoss Jasmine M. , Grigoryants Angelina J. , Hamann Michael J. 
Published: April 2020

The Rho‐family GTPase TCL/RhoJ enhances pro‐angiogenic and pro‐metastatic cellular processes; however, contributing protein interactions are not well characterized. TCL is part of the Cdc42 subfamily of Rho GTPases but has a short, unique N‐terminal amino acid sequence that is important for GTP‐loading and plasma membrane localization. Additionally, we have evidence that TCL binds vesicular membranes through a protein/protein interaction when it is GDP‐loaded. We are evaluating whether Coronin proteins selectively interact with GDP‐loaded TCL, as Coronin proteins contain CRIB motifs that bind other Rho family GTPases when they are GDP‐loaded. To determine if the Coronins co‐localize with TCL at vesicular membranes, mCherry‐fusions of the seven Homo sapiens Coronin proteins will be transfected into HeLa cells along with YFP‐tagged TCL and mutants bearing constitutively active (Q79L) and dominant negative (T35N) mutations. These TCL mutations will mimic GTP/GDP bound forms of TCL and help identify nucleotide‐dependent interactions with Coronin proteins. Coimmunoprecipitation assays will also be performed to detect interactions between Flag tagged‐TCL and myc tagged‐Coronin proteins. These results will provide additional insights into binding partners that influence the role of TCL in pro‐angiogenic/metastatic processes.Support or Funding InformationThis work was supported by Bemidji State University Biology Department and the College of Business, Mathematics, and Science. Support was also provided through the Neilson Foundation, Bemidji, MN and Regenerative Medicine Minnesota (RMM‐2017‐EP‐04).
Methodology for Efficient and Biosecure Genetic Engineering of Biomedical Swine Models
Authors: Meudt Jennifer J. , Reichert Jamie L. , Lopez Ana Cecilia Escobar , Krentz Kathleen J. , Rubinstein Clifford Dustin , Lehman Brent P. , Schomberg Dominic T. , Monson Ricky L. , Willenburg Kilby , Frank Jennifer M. , Graff Keri D. , Shanmuganayagam Dhanansayan 
Published: April 2020

The genetic proximity of swine to human, the overwhelming anatomical, physiological and pathophysiological similarities, and the ability to mimic external factors in a practical manner make swine the ideal preclinical model for many human diseases. While the value of swine in biomedical research is well recognized, their use has been limited by the lack of tools to easily edit the swine genome to model human disease, and the lack of facilities, resources and expertise at most institutions for supporting large‐scale swine research. The recent implementation of new genetic editing tools such as the CRISPR/Cas9 platform make the creation of novel genetic swine models possible and practical. However, conventional approaches to genetic engineering of swine rely on ova obtained from commercial abattoirs, significantly compromising biosecurity, and often offering little control over the background genetic of the swine breeds upon which the genomic editing is performed. Here, we describe methods and workflow that we have developed that allow for efficient genetic engineering of swine, while maintaining control over background genetics and minimizing the biosecurity risk to the animals and the facility. The process centrally relies on the refinement of reproductive cycle synchronization and superovulation of swine and time‐precise coordination of the entire genetic engineering workflow.Support or Funding InformationThe research was supported by the Biomedical & Genomic Research Group Discretionary Fund (University of Wisconsin‐Madison), NF North Central, NF Network, and NF Team Foundation
Household Air Pollution Indicators and Epigenetic Changes in the Renin Angiotensin System Genes ACE and AGTR1
Authors: Swann Sellers , Beach Kayman , Ehmke Sarah , Rice Nancy A. 
Published: April 2020

Cardiovascular diseases (CVD) are the world’s leading cause of mortality and disproportionately affect individuals in low and middle income countries (LMICs). Hypertension (HTN) is a multifactorial disease that is the current leading cause of CVD. In this study, we investigated the epigenetic regulation of two key genes involved in regulation of blood pressure, angiotensin‐converting enzyme (ACE) and angiotensin II receptor type 1 (AGTR1), from a cohort of Kenyans known to have both a high prevalence of HTN and exposure to household air pollution (HAP). Genomic DNA, isolated from saliva samples, was used to quantify methylation of cytosine‐phosphate guanine (CpG) islands in the promoter regions of ACE or AGTR1 by pyrosequencing following bisulfite conversion. In preliminary samples (n=29) analyses indicate that when stratified by blood pressure, both ACE and AGTR1 are hypomethylated in hypertensive versus normotensive individuals (1.43 ± 0.316 vs. 1.524 ± 0.35 and 2.841 ± 1.35 vs. 2.104 ± 0.591, respectively). However, specific CpG3 dinucleotide in the AGTR1 promoter showed a significant 3% increase of methylation (5.652 ± 3.069 vs 2.638 ± 0.734; p=0.028) in hypertensive versus normotensive individuals. In this preliminary work, no significant associations were found between methylation and indicators of HAP, although a strong positive correlation exists between CpG2 and CpG4 methylation in AGTR1 (R=0.990; p<0.001). Our findings indicate epigenetic regulation of AGTR1 may have a role in modulating high blood pressure incidence in rural LMICs where HAP is epidemic.Support or Funding InformationWe acknowledge funding form the University of South Alabama to N.A.R.
Peer Assisted Learning Experience as an Extracurricular Activity during a Gross Anatomy course.
Authors: Pabon Daniela , Muñoz Nicolas Stozitzky , Rueda-Esteban Roberto Javier 
Published: April 2020

Multiple medical schools have modified classical teaching methods introducing new tools to improve learning processes
1. Amongst these, extra academic activities defined as out of the curriculum, of voluntary participation
2, and without grading, have been described as activities requiring auto discipline, teamwork, commitment and knowledge
3 as these are mostly carried out by an academic peer. These and other group activities in and out of campus should not be overlooked, as they also influence student performance in medical school. Additionally, peer assisted learning has many benefits, which include preparing students as future educators, improving their communication skills and reducing staff workload.The aim of this study is to describe the effect of a peer assisted learning based extracurricular activity on student performance during a Gross Anatomy course in an integrated curriculum medical school and the students’ perception on their own learning process.Quantitative and qualitative data acquisition tools were used to determine the existence or not of an association between the activity and students summative performance. Grades from two groups without the activities n=162 and two with these n=127 were considered with a total of 22 interventions. Additionally, quantitative data was triangulated with qualitative data obtained from a focal group with the student’s perception on their own learning process.No positive association (p > 0.05) between the grades of each group were found. However, as each exam is composed by a theoretical and a practical component, a decrease in the difference between theoretical and practical grades when peer assisted extracurricular activities were applied was evident. When qualitative data was analyzed, there was a general perception by students that they had less stress levels during the practical component after the activity.Peer‐assisted extracurricular activities show to be beneficial for both: those who attend and peers. Students consider this activity as helpful in reducing other factors such as stress and anxiety that may also be influencing their performance on the exam. However, not finding an association may occur because the students who are more academically able tend to seek out teaching and/or research opportunities and participated at these activities
2. This and other factors need to be addressed and evaluated in order to restructure this tool, so it can be improved in a way it can be implemented and fully analyzed in the future.Support or Funding InformationAnatomy Laboratory, Universidad de los Andes School of Medicine
Mitochondrial Fragmentation in Skeletal Muscle Derived Cells from an Old Male Donor May Relate to Decreased Oxygen Consumption Rates
Authors: Arevalo José Adan , Miller Marvin L. , Vazquez-Medina José Pablo , Brooks George A. 
Published: April 2020

With age, skeletal muscle mitochondria lose their ability to fuse and regenerate following stress and exercise leading to fragmentation. These phenomena can lead to reduced oxygen, muscle atrophy, reduced maximal O2 consumption, loss of metabolic flexibility and risk of developing age‐related diseases such as sarcopenia. Skeletal muscle cells derived from human donors can be used to investigate these physiological capacities in primary culture. Our objective is to investigate mitochondrial morphology and cellular maximal oxygen consumption rates (OCR) of skeletal muscle cells derived from healthy young and old male donors. Primary skeletal muscle cells derived from the Rectus abdominis muscle of healthy active eighteen and sixty‐nine year old men (SKM18M and SKM69M, respectively) were obtained from Cook MyoSite Inc. (Pittsburgh, PA). Cells were stained with MitoTracker Red (Cell Signaling; Danvers, MA) and mitochondrial morphology was observed using a Zeiss LSM 710 AxioObserver confocal inverted scanning microscope (Carl Zeiss; White Plains, NY). The mitochondrial network was analyzed using the Mitochondrial Network Analysis tool in ImageJ (MiNA, FIJI) to estimate mitochondrial footprint from a binarized image. Oxygen consumption rates were measured in intact cells using Seahorse Cell Mito Stress Tests on a XFp extracellular flux analyzer (Agilent Technologies; Santa Clara, CA). Primary cells derived from the young donor (SKM18M) had a larger mitochondrial footprint, longer branch length, and a greater number of network branches compared to the old donor (SKM69M) (Footprint: 34.65 ± 25.30 vs. 11.64 ± 9.53 μm2; Branch Length: 20.59 ± 7.23 vs. 12.10 ± 6.84 μm; Network: 17.25 ± 0.16 vs. 7.67 ± 4.97 counts). SKM18M also showed higher Basal and Maximal OCR compared to SKM69M (Basal: 38.78 ± 8.34 vs. 12.82 ± 2.07; Maximal: 60.09 ±10.84 vs. 20.52 ± 2.36 pmol/min/protein). We observed differences morphologically and metabolically between the primary skeletal muscle cells derived from young and old donors. These preliminary results give us an insight into human skeletal muscle‐derived cellular physiological capacity. Technology to observe human muscle mitochondrial fragmentation in vitro will help us elucidate the effects of aging, illness and injury on skeletal muscle mitochondrial fragmentation, dysfunction, and loss of metabolic flexibility.Support or Funding InformationFunded by the National Institute of Aging of the National Institute of Health; Grant Number R01‐AG059715.