bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for Progressive Shifts Towards α‐Myosin Isoform with Paradoxical Increases in Cardiac Hemodynamic Loads in Post‐Natal Healthy Rats
Authors: Geist Gail Elizabeth , Combs Ariana C. , Leinwand Leslie , del Rio Carlos L.
Published: April 2020

Introduction

The balance of fast (α) and slow (β) cardiac myosin heavy chain (MYHC) isoforms is known to change with both aging and disease. While pathological states with increased hemodynamic loads are often accompanied by shifts toward energy‐sparing β isoforms, the underlying mechanism(s) of these changes remain poorly understood. Neonatal rats are a promising model to study such cardiac MYHC remodeling, as they show a well‐documented transition (β to α) during post‐natal development. In these experiments, MYHC‐isoform switches and in vivo cardiac structure/hemodynamic changes were evaluated during post‐natal development.

Methods

Cardiac echocardiographic and systemic/left ventricular (LV) hemodynamics were evaluated in Sprague‐Dawley rats (n = 61) ranging from post‐natal day (PND) 3 to adulthood. Hemodynamics were obtained before/after challenges with sodium nitroprusside (SNP, 25 μg/kg IV) and/or phenylephrine (PE, 10 μg/kg IV) to study cardiovascular responsiveness. LV’s were also collected (n = 48) for myosin isoform analysis via SDS‐PAGE MYHC separating gels.

Results

As expected, post‐natal rats showed a progressive decline in β‐MYHC expression from PND3 (1.29 ± 0.05 α:β) to adulthood when α‐MYHC was dominant (12.78 ± 2.51 α:β). Concomitantly, postnatal rats showed progressive increases (P<0.05) in systemic/LV pressures (MAP: 46 ± 1 at <PND14 to 103 ± 2 mmHg, P<0.05), LV mass (0.16 ± 0.02g at <PND14 to 0.84 ± 017g) and diastolic dimensions (79 ± 6 at <PND14 to 288 ± 14 μL), with vastly preserved heart rates. Pressor responses to PE increased during maturation, while all age groups were sensitive to SNP‐mediated vaso‐relaxation. Ejection fraction was preserved during postnatal maturation with young pups showing lower rates of LV pressure development (dPdtmax 4052 ± 125 at <PND14 vs. 9478 ± 231 mmHg/s, P<0.05) consistent with the early expression of β‐MYHC.

Conclusions

Taken together, these data provide evidence of a maturing cardiovascular system in post‐natal rats showing progressive increases in systemic/ventricular pressures and cardiac dimensions, with paradoxical shifts towards α‐MYHC isoforms. In short, in rats, the cardiac β‐MYHC isoform consistently declines over maturation despite sustained increases in systemic load.
Characterization of New Optical Imaging Biomarkers of Peripheral Nerve Overstimulation
Authors: Tao Michael , Monroy Guillermo , Erfanzadeh Mohsen , Rafi Harmain , Vakoc Benjamin , Vasudevan Srikanth , Hammer Daniel
Published: April 2020

An animal study was designed to explore the potentially damaging effects of electrical overstimulation on peripheral nerves using a polarization‐sensitive optical coherence tomography (PS‐OCT) imaging and stimulation platform. Lewis rats (n = 15) were divided into 3 groups with 2 different levels of stimulation applied to the exposed sciatic nerve: Group 1 (sham control): 0.0 mA/0 Hz, Group 2: 3.4 mA/50 Hz, and Group 3: 6.8 mA/100 Hz. On day 1, the protocol contains Von Frey and walking track behavioral tests, followed by sciatic nerve exposure surgery (left leg). Electrical stimulation of the nerve was performed for one hour, with a one‐hour recovery period. PS‐OCT and optical coherence tomography angiography (OCT‐A) imaging were performed both prior to and during these periods. On day 7, this protocol was repeated without stimulation. OCT imaging was performed along a 5‐mm length of the sciatic nerve, including sites proximal and distal to injury. Both the stimulated and contralateral sciatic nerves were then resected and processed for histology.

Image analysis quantified the effects of electrical stimulation at the two time points and allows comparison between stimulation levels. In all cohort groups, PS‐OCT image features remained consistent on day 1. On day 7, we observed relatively few PS‐OCT or OCT‐A changes compared to day 1 in the sham control animals. Groups 2 and 3 showed a clear increase in OCT‐A perfusion in the stimulated region, while there is a drop in PS‐OCT signal in the same area. The PS‐OCT signal drop‐out appears more pronounced in Group 2 compared to Group 3. It is not yet clear if the PS‐OCT signal drop‐out is related to demyelination or from shadowing from hyperperfused tissue. Walking track video was analyzed frame‐by‐frame for animal foot spread to determine sciatic and tibial function indices. This data, along with Von Frey withdrawal forces, were analyzed with statistical comparison tests (ANOVA) and indicated no significant differences between the groups nor significant interactions when compared across stimulation group, pre‐ and post‐exposure, and ipsilateral‐contralateral leg. Histological samples were processed for histomorphometry and axonal damage proximal and distal to the injury, as well as for immunohistochemistry of axons, macrophages, and nuclei in the stimulated and unstimulated regions in all groups.

PS‐OCT and OCT‐A proffer real‐time observation of nerve changes with overstimulation that are undetectable by behavioral testing.

Support or Funding Information

NIH SPARC
Identifying Metabolic Pathways of c‐MET Tyrosine Kinase Inhibitor Tepotinib in Human and Mouse Liver Microsomes
Authors: Li Feng , MacKenzie Kevin R. , Nyshadham Pranavanand , Kerlec Kimberly A. , Matzuk Martin M.
Published: April 2020

Tepotinib (TPB) is a highly selective and potent c‐Met inhibitor that has shown promising activity in a phase I trial for non‐small cell lung cancer (NSCLC). Recent studies indicate that TPB can overcome resistance to epidermal growth factor receptor inhibitors driven by aberrant MET activation, and the Food and Drug Administration has granted a breakthrough therapy designation for tepotinib in patients with metastatic NSCLC harboring METex14 skipping alterations who have progressed on prior platinum‐based chemotherapy. The metabolism of TPB remains largely unknown, but is likely to play a critical role in both safety and efficacy: understanding the metabolism of TPB could improve safety and predict possible adverse effects and drug‐drug interactions. In the current study, we investigated the metabolism and bioactivation of TPB in human and mouse liver microsomes (HLM and MLM) using LC‐MS‐based metabolomic approaches. We identified over 20 TPB metabolites and adducts in liver microsomes. Monohydroxyl‐TPB (O+TPB) is the dominant metabolite in both HLM and MLM, and demethyl‐TPB is secondary to O+TPB. Using methoxyamine as a trapping agent, we infer that two aldehydes are generated in HLM; one of these was also observed in MLM. No adducts were observed in liver microsomes incubated with TPB in the presence of trapping agents glutathione or potassium cyanide. Using recombinant CYP450 isoenzymes, we showed that CYP3A4 and CYP3A5 are the primary contributors to the formation of both monohydroxyl‐TPB and the methoxyamine‐trapped TPB aldehydes. This study offers a comprehensive view of TPB metabolism and identifies metabolites that will be helpful to evaluate adverse effects of TPB and possible drug‐drug interactions in TPB‐treated hepatocytes and model organisms.

Support or Funding Information

This work was supported by Cancer Prevention & Research Institute of Texas (RP160805), the Eunice Kennedy Shriver National Institute of Child Health and Human Development (P01 HD087157‐01A1), Welch Foundation Grant (H‐Q‐0042), and Bill and Melinda Gates Foundation (INV‐001902) to Dr. Martin M. Matzuk and the National Institute of Diabetes and Digestive and Kidney (R01‐DK121970) to Dr. Feng Li.
Salivary Origins of Exogenous Cytokinins
Authors: Seng Stephannie , Connor Edward
Published: April 2020

Cytokinins are a class of phytohormones which play an important role in a variety of plant processes, such as cell growth and differentiation, formation of roots and shoots, and the reallocation of nutrients to form mobilizing sinks. Exogenous cytokinins found in plant‐manipulating insects are known to contribute to many effects on host plants. Perhaps the most apparent effect is that cytokinins, in the presence of auxin, leads to cell division and the proliferation of plant tissue; consequently, plant galls or tumors are formed. Cytokinins have also been implicated in the reallocation of sugars and nutrients for the oviposition site to act as a mobilizing sink, where the intention may be gall induction or sustenance for the organism. There are two known pathways for the biosynthesis of isoprenoid cytokinins. The first is the tRNA‐ipt pathway, in which an adenine residue is modified by the addition of an isoprenoid chain by the tRNA‐ipt gene. In plant plastids and bacteria, the methyl erythritol (MEP) pathway is the main source of the prenyl group, but in the cytosol of plants and insects, the prenyl group is synthesized via the mevalonate (MVA) pathway. Cytokinins are produced upon the degradation of the prenylated tRNA. To further investigate the role of cytokinins in gall induction by insects, I performed gene expression experiments using the gall‐inducing Eurosta solidaginis and its bacterial symbiont Wolbachia sp. I also performed targeted gene expression analyses in order to examine the expression of genes in the MEP, MVA, and tRNA‐ipt pathways. Up‐regulation of these pathways in the salivary gland implies that cytokinins are being synthesized in the salivary glands by either the insect and/or its bacterial symbiont. Along with the known widespread distribution of cytokinins in insects, these further imply that cytokinin production and secretion has been amplified and is used by the insects to manipulate their host plants. However, the genes involved in cytokinin production have also been found in other insect groups, so this plant manipulation may not just be exclusive to gall‐inducing insects, suggesting widespread hostplant manipulation among plant‐eating insects.

Support or Funding Information

NIH MBRS‐RISE: R25‐GM059298
Validation of Antibodies against Histone Modifications and Transcription Factors for ChIC/CUT&RUN Assays
Authors: Din Shabana , Saito Kan , Silverman Jennifer , MilliporeSigma Jeffrey Turner
Published: April 2020

DNA‐protein interactions in the nuclei control a wide variety of biological processes such as development, differentiation, and cellular response to environmental changes. Chromatin immunoprecipitation (ChIP) has been used to analyze association of proteins with genomic DNA sequences. Although there have been several improvements with the technology over the decades, fundamental challenges remain with ChIP, including high background that limits sensitivity, high amounts of cellular input, and artifacts resulting from cross‐linking and solubilization.

To overcome the challenges with ChIP, Cleavage Under Targets and Release Using Nuclease (CUT&RUN) was developed in Steven Henikoff’s lab by improving Chromatin Immune‐Cleavage (ChIC), which was originally developed in Ulrich Laemmli’s lab. In ChIC/CUT&RUN, binding sites of transcription factors in the genome were detected by targeting micrococcal nuclease conjugated with protein A (pA‐MN) through a specific antibody to detect genome‐wide transcription factor binding sites or histone modifications on native chromatin. Since the process doesn’t require cross‐linking and solubilization, and only the targeted fragments are released into solution and the majority of DNA is left behind, the results from ChIC/CUT&RUN assays have exceptionally low background levels compared with ChIP assays. As a result, ChIC/CUT&RUN provides high‐quality results using much smaller number of the cells (100 cells for a histone modification analysis and 1,000 cells for a transcription factor binding site analysis) than traditional ChIP assays.

Here we performed ChIC/CUT&RUN assays with various antibodies against popular histone modifications including H3K4me3 and H3K27me3, and transcription factors such as CTCF. The sequencing data was compared with ChIP‐seq data in the ENCODE database. The sequencing data of ChIC/CUT&RUN assays with each of the antibodies tested showed very similar patters to the ENCODE ChIP‐seq data with the same targets. The results not only confirmed the power of the ChIC/CUT&RUN technology but also validated the suitability and comparability of these antibodies for use in a variety of studies.
Can Classrooms Have an Impact on Student Stress?
Authors: Oyejide Kafayat , Hanna James , Daniel Kristy , Huertas Mar
Published: April 2020

Stress negatively impacts a person’s health, and stress in the school environment may negatively impact student performance. For the past five years, the graduation rate for undergraduate students has been falling. The main factors contributing to this decline may involve indirect or direct stress on the student. For instance, learning space and environment impact undergraduate students and their stress levels which impacts their graduation rates. We hypothesized that students in the SCALE UP (Students Centered Active Learning Environment for Undergraduate Programs) classroom have lower concentrations of salivary stress biomarkers (cortisol and α‐amylase) than students in traditional classrooms, and perform better academically than their counterparts. We screened students learning introductory biology in two different learning environments, a SCALE UP and a lecture hall, collected salivary samples twice a week, and asked the students to self rate their stress level from not stressed to very stressed. Saliva was collected at the beginning and end of each class period to measure a change in cortisol or α‐ amylase over the class period. We used an Enzyme‐Linked Immunosorbent Assay (ELISA) to quantify the concentration of salivary cortisol and a colorimetric method to determine the α‐ amylase. Students in the SCALE UP classroom had lower α‐amylase activity than those in a traditional lecture hall. Cortisol concentrations were not correlated with the classroom environment. The results of the α‐amylase assay aligned closer with the literature, indicating that salivary α‐amylase is better at identifying acute stressors, such as a classroom environment than cortisol. The setup of a SCALE UP class may have less of an impact on student stress than the pedagogy. The results of the study highlight the importance of pedagogy on student stress and the significance of salivary α‐amylase as an acute, noninvasive stress biomarker.

Support or Funding Information

This research was supported by the U.S. Department of Education HIS STEM program (841.031c), Award #P021C160036
The Need for a Morphogenetic Code
Authors: Neufeld Dan
Published: April 2020

We understand how regulation of gene expression is critical to development, growth, and health, but genes don’t control how cells build things. Cells behave. Cells have built every vestige of life that we see every day, but it is not known HOW precisely they do it. We understand very precisely how transcription and translation convert nucleic acids into peptides and proteins through execution of an elegant genetic code. Despite the fact that almost every cell contains information to code for an entire organism, no one has yet been able to turn that information into any recognizable structure. We need to understand why and how cells “behave” in certain ways to construct physical structure. Limb regeneration studies demonstrate that environments elicit certain cell behaviors that promote either wound healing or regeneration. We need to understand the morphogenetic code, hidden “in plain sight” in the extracellular milieu, that instructs cells to behave in defined patterns. We need a National Institute of Cellular Behavior and Morphogenesis.
Temperature Dependence of Hippocampal Short‐term Facilitation (STF) in Syrian Hamsters, a Hibernating Species
Authors: Horwitz Barbara , Horowitz John M.
Published: April 2020

Studies on non‐hibernating mammals have identified short‐term facilitation (STF) as a form of cellular neural plasticity that modulates information processing over a hippocampal neural network of CA3 and CA1 pyramidal neurons. The circuit has two branches: (1) a monosynaptic excitatory (E) feedforward pathway wherein CA3 axons directly excite CA1 neurons, and (2) a bi‐synaptic inhibitory (I) feed‐forward pathway. Pathway I includes a GABAergic inhibitory neuron such that CA3 axons excite GABAergic interneurons that in turn inhibit CA1 neurons. The present study evaluated the possibility that such a dual feedforward circuit supported STF in hibernating as well as in non‐hibernating Syrian hamsters (Mesocricetus auratus). We tested the hypotheses that: (1) at temperatures at (or above) 25° C, the hamster’s circuits support STF as in non‐hibernating species, and (2) as temperature declines from 25° to 20° (simulating brain temperatures encountered during entry into torpor), STF is attenuated. For this, we evaluated responses in 400μ hamster hippocampal slices, using tungsten stimulating electrodes placed to excite CA3 axons (Schaffer collaterals/commissural fibers) and a glass electrode positioned to record CA1 neuron evoked responses (fEPSPs). Paired‐pulse stimulation (with an interpulse interval of 30 msec and an interval between pairs of 60 sec) evoked a pair of responses – i.e., fEPSP1 followed fEPSP2. With bath temperature at (or above) 25° C, the initial slope of fEPSP2 was greater than that of fEPSP1. This finding supports the presence of a hamster facilitating circuit with at least an E feedforward pathway linking CA3 and CA1 neurons. To determine if the hamster circuit also has a bi‐synaptic inhibitory branch, paired‐pulse experiments were repeated in the presence and absence of picrotoxin, a non‐competitive GABA receptor antagonist. Picrotoxin altered fEPSP1 and fEPSP2 slopes (e.g., when GABAergic interneurons were blocked, fEPSP1 slope increased), indicating the hamster circuit also had an inhibitory feedforward branch. Additionally, paired‐pulse experiments with bath temperature lowered to 20° C resulted in a decreased ratio of fEPSP2/fEPSP1, marking a decline in STF. Nonetheless, at 20° C (and even at 15° C), single‐shock CA3 stimulation still evoked action potentials (population spikes) in CA1 neurons, showing that, despite a loss in facilitation, the circuit continued to relay signals from CA3 to CA1 neurons. Taken together, our data are consistent with the proposal that awake and behaving hamsters have a dual feed‐forward neural circuit supporting STF, as in non‐hibernating mammalian species, but when the hamsters are hibernating, STF is muted.
Gene Expression Patterns After Injury In Regenerating Young and Old Skeletal Muscle
Authors: Wang Fenyu , Hill Jonathon T. , Thomson David
Published: April 2020

Healthy skeletal muscle repair occurs through proper regulation of gene expression in many cell types within the regenerating muscle niche, including skeletal muscle, macrophages, fibroblasts, etc. However, the ability to repair muscles is compromised in old muscle, and the mechanisms for this are not fully understood. To further investigate factors leading to this age‐related defect in repair and regeneration, we conducted RNA sequencing on tibialis anterior muscle tissue from young (Y; 5 mo.) and old (O; 24 mo.) mice basally and at day 3, day 7, and day 10 after cardiotoxin‐induced injury. Principle component analysis demonstrated a general delay in global gene expression changes over the 10 day regeneration period in O muscles. Gene Set Enrichment Analysis (GSEA) of Hallmark Pathways showed that in Y muscles, inflammation and immune‐related pathways were upregulated at Day 3 followed by upregulated myogenesis‐related expression on days 7–10. In contrast, O muscles upregulated hypoxia‐ and metabolism‐related pathways at day 3, and showed continued upregulation of inflammation‐ and apoptosis‐ related gene expression through days 7–10, suggesting failure to resolve the inflammatory and cell stress response during the later regenerative period. This was associate with delayed Pax7 expression in the O muscles. As expected, upregulated expression of Wnt/beta‐catenin signaling pathway components, which are heavily involved in myogenesis, was dysregulated in old regenerating muscles. Our analysis indicates that disrupted gene regulation patterns post injury are a pivotal factor in the defective regenerative capacity of old muscles.

Support or Funding Information

This research was supported by a BYU Gerontology Award.
Collagen and Elastin Disarray Mechanisms Mediating Aortic Stiffness in Aging Pre‐Menopausal Female Monkeys.
Authors: Babici Denis , Kudej Raymond , McNulty Tara , Zhang Jie , Oydanich Marko , Berkman Tolga , Nishimura Koichi , Vatner Dorothy E. , Vatner Stephen F.
Published: April 2020

Whereas collagen and elastin content have been widely studied as mechanisms for mediating increased aortic stiffness with aging, collagen and elastin disarray has been less well studied, not only in all aging models, but most particularly in aging pre‐menopausal females. The current investigation compared collagen and elastin disarray in the aorta in aging, 24±1 years old, premenopausal female monkeys (M. Fascicularis and M. Mulatta) with disarray in young female monkeys (6 ± 1 years old). The non‐human primate model is unique for these studies, since most other aging animal models do not go through menopause. Collagen/elastin ratio and disarray were increased in the old, pre‐menopausal females compared to the young females in the thoracic aorta, but even more in the abdominal aorta, and the most in the iliac artery. For example, compared to values in the thoracic aorta of young females, the collagen/elastin ratio increased in the thoracic aorta of old female monkeys by 169%, and by 397% in the iliac artery of the old females. Compared to values in the thoracic aorta of young females, the disarray of elastin fibers increased by 106% in the thoracic aorta of the old female monkeys and by 350% in the iliac artery of the old female monkeys. Compared to values in the thoracic aorta of young females, the disarray of collagen fibers increased by 148% in the thoracic aorta of the old females and by 378% in the iliac artery of the old female monkeys. Compared to values in the thoracic aorta of young females, the number of breaks in elastin fibers increased by 158% in the thoracic aorta of the old females and by 883% in the iliac artery of the old female monkeys. Compared to values in the thoracic aorta of young females, the number of breaks in collagen fibers increased by 105% in the thoracic aorta of the old females and by 390% in the iliac artery of the old female monkeys. Thus, old pre‐menopausal female monkeys exhibit enhanced mechanisms of vascular stiffness. These mechanisms were increased in the thoracic aorta, but significantly more increased in the iliac artery.

Support or Funding Information

Study supported by National Institutes of Health grants R21 AG053514; R01 HL119464; R01 HL137405; R01 HL102472; R01 HL106511; R01 HL130848; R01 HL137368; UL1TR003017;
Effects of Western Diet on Histological Characteristics of Muscle in Leptin‐Resistant Pigs
Authors: Yankaway Raizel B. , Spooner Heather C. , Hernandez Gabriella V. , Smith Victoria A. , Burrin Douglas G. , Maj Magdalena , Manjarin Rodrigo , Blank Jason M.
Published: April 2020

Leptin‐resistant pigs offer a promising model for the study of non‐alcoholic fatty liver disease (NAFLD), particularly in pediatric populations. Muscle is a large and metabolically active tissue that may play an important role in the storage and metabolism of lipid during the development of NAFLD.

In this study, 10‐day old piglets were fed either a control diet (CON) (n=8) or a Western diet containing high levels of fructose, saturated fat and cholesterol (HFF) (n=12) every 6 hours for 70 days. Half of the pigs in each diet group were supplemented with a blend of probiotics containing Pediococcus, Lactobacillus and Bacillus. Longissimus dorsi muscle was collected on day 70 for immunohistochemistry and triacylglyceride (TG) quantification. Tissue sections were stained for lipid content with Oil Red O and muscle cells were fiber‐typed based on myosin heavy chain isoforms.

Muscle tissue from pigs fed HFF diets had higher total TG content, but a lower number of Oil Red O positive cells and a lower lipid staining intensity of these cells. HFF diet also caused a reduction in the fraction of type I (slow‐twitch) muscle fibers.

In summary, this staining pattern suggests that the muscle from HFF fed animals may have an increased accumulation of lipid in extramyocellular depots rather than within muscle cells and a decreased capacity to oxidize lipids. Such shifts in muscle metabolic characteristics may contribute to the progression of metabolic disease.

Support or Funding Information

The project was funded by ARI#58873, STRIDE#35466, and the William and Linda Frost Fund.
CRISPR Caspase 9 strategies to explore parkin loss‐of‐function Drosophila development
Authors: Houlihan Katie
Published: April 2020

Parkinson’s disease (PD) is a neurodegenerative disease in which dopaminergic (DA) neurons selectively degenerate within the substantia nigra pars compacta. Parkin loss‐of‐function Drosophila melanogaster, a model for familial PD, have neurodegeneration in structures homologous to those of the human substantia nigra. Mitochondrial dysfunction and reactive oxygen species (ROS) are implicated in PD‐associated neurodegeneration and in the parkin‐null fly phenotype. In healthy cells, ROS are involved in signaling and are regulated by antioxidant activity. Glutathione (GSH) is a major antioxidant that combats the effects of excess damaging ROS (oxidative stress) and may be dysregulated in parkin mutant Drosophila. Preliminary data from our lab has shown that parkin loss‐of‐function Drosophila (park25/25; Greene et al., 2003) have elevated hydrogen peroxide levels that are sustained as the fly ages, a transient increase in glutathione activity, and increased levels of protein oxidation. Whether oxidative stress persists because of low GSH levels, dysfunction of GSH, or excessive ROS is unclear. Our study aims to address the possibility that low levels of GSH contribute to the parkin loss‐of‐function fly phenotype. To this end, we utilized a modified CRISPR/Cas9 system to overexpress cystathionine beta synthase (CBS), a key, rate‐limiting‐limiting enzyme involved in the production of GSH. To overexpress CBS, we have used a modified Cas9 construct in which the enzymatic function of Cas9 has been deactivated (“dead Cas9” or “dCas9”). Transcriptional activators VPR have been fused to dCas9, resulting in overexpression of CBS. Since parkin–null Drosophila have developmental deficits, we have measured pupariation and eclosion efficiencies in flies overexpressing CBS to shed light on the potential use of this intervention for future studies. Parent flies are placed in a vial and allowed to lay embryos. First and second instar larva are collected and transferred to a secondary vial. The larva are then monitored, and the number of larva that pupate are recorded (pupariation efficiency). The number of flies that eclose (emerge from the pupa case) are also recorded and compared to the pupa count (eclosion efficiency). Additionally, the distance between pupa and the food level is measured to indicate larval mobility. Preliminary data indicate the overexpression of CBS increases pupariation efficiency and decreases pupariation times in the heterozygous parkin mutants. Parkin‐null larva CBS overexpression also appear to have increased climbing. This initial data implicates overexpression of CBS in neuroprotection of the park loss‐of‐function model. Additional experiments on neuron degeneration, mitochondrial functionality, and antioxidant activity can provide further insight into why parkin‐null dopamine neurons are susceptible to degradation. By incorporating Cas9 and dCas9 into our parkin‐null flies, we can modify expression of hundreds of genes in order to elucidate the pathways implicated in oxidative stress in this model.
Hypoxia‐Induced Syndecan 1 Shedding in Placental Trophoblasts is Associated with Increased Peroxide
Authors: Mahmoudi Elena , Cates Courtney , Chapman Heather , George Eric Matthew
Published: April 2020

Preeclampsia is one of the most common obstetrical disorders, affecting ~5% of pregnancies. It is one of the leading causes of maternal and fetal morbidity and mortality, as well as premature birth. While the origins of preeclampsia are obscure, it is believed that many cases are due to placental insufficiency and resulting ischemia/hypoxia. While many of the factors released by the ischemic placenta are known, the full scope of the molecular effects of ischemia on the placenta are not known. We have recently demonstrated that hypoxia causes shedding of the placental extra cellular matrix in vitro and in vivo, particularly the glycoprotein syndecan 1. It has also been reported that vascular ECM shedding can be affected by reactive oxygen species. Therefore, we wished to test the hypothesis that oxidative stress is the mediator of placental syndecan 1. We treated placental trophoblast derived cells (BeWo) for 24 hours with oxygen conditions mimicking healthy (8%) or ischemic (1%) placentas. To determine the effect of hypoxia on peroxide production, we used the peroxide indicator amplex red. In response to hypoxic conditions, amplex fluorescence was significantly increased (6728 ± 196 vs 8201 ± 501 AU, p<0.05), which was attenuated with the peroxide scavenger apocynin (4626 ± 100 AU, p>0.05). In response to hypoxia, SDC1 released from BeWos increased significantly (175 ± 10 vs 280 ± 13 pg/ml, p<0.05). There was no effect of the peroxide scavenger apocynin in normoxic conditions, but there was a significant reduction (185 ± 10 pg/ml, p<0.05) in hypoxic conditions. These data suggest that peroxide is a key mediator of hypoxia‐induced shedding of SDC‐1, and could play a key role in ECM shedding in placental ischemia in vivo. Future studies will examine the in vivo effects of peroxide scavenging on SDC shedding as well as its effects on other ECM components.

Support or Funding Information

This work was supported in part by NIH grants P01HL51971, P20GM104357, and R01HL137791
Role of Inositol Hexakisphosphate Kinase‐2 (IP6K2) in regulating mitochondrial brain functions
Authors: Nagpal Latika , Snyder Solomon H.
Published: April 2020

Inositol hexakisphosphate kinase (IP6K) isoform ‐ IP6K2, selectively controls cellular apoptosis. To explore the possible role of IP6K2 in degenerative brain damage, we elucidated its protein interactome in mouse brain to identify its functional partners. IP6K2‐immunoprecipitates from wild‐type mouse brain lysates were subjected to co‐immunoprecipitation followed by liquid chromatography mass spectrometric analysis. This revealed robust association of IP6K2 with creatine kinase‐B (CK‐B), an enzyme responsible for synthesis of phosphocreatine and ATP. Notably, both IP6K2 and CK‐B were highly expressed in the mouse cerebellum. IP6K2‐knockout mice (IP6K2‐KO) produced less phosphocreatine and ATP and generated higher levels of ROS and consequent protein oxidative damage in their cerebellum compared to wild‐type counterparts. As mitochondrial respiratory chains regulate energy metabolism/ATP production through glycolysis and TCA cycle coupled to oxidative phosphorylation and electron transfer, functional abrogation of any event that controls these processes can lead to abnormal electron transfer, increased ROS and consequent mitochondrial oxidative stress/damage and cell death as observed in the IP6K2‐KO mice. IP6K2‐KO mice also showed significant impairment of locomotive and cognitive functions. We thus propose to identify the precise metabolic events that are controlled by IP6K2‐CK‐B interaction and examine the possible causal role of oxidative stress and determine whether antioxidant treatment can restore normal brain functions. We plan to explore mitochondria‐targeted antioxidants like CoQ10/Mito Q along with derivatives of higher inositol phosphates (IP7‐product of IP6K2) in the IP6K2‐KO mice to reinforce the therapeutic potential of such anti‐oxidative treatment for the clinical management of diseases associated with mitochondrial dysfunction of the brain. Overall, our proposed study would help to establish the precise role of IP6K2‐CK‐B interaction in controlling cellular degeneration in the brain.
Vitamin D Impacts Nrf‐2 Signaling and Antioxidation Pathways that Modulate Cellular Aging
Authors: Sabir Zhela L. , Livingston Sarah , Whitfield G. Kerr , Haussler Mark R. , Jurutka Peter
Published: April 2020

Recent studies suggest that the biologically active form of vitamin D, 1,25‐dihydroxyvitamin D (1,25D), plays a role in potentially delaying the aging process, functions as an anti‐carcinogenic agent, and also prevents oxidative damage. 1,25D binding to the vitamin D receptor (VDR) promotes heterodimerization of VDR with the retinoid X receptor (RXR) to stimulate transcription of numerous vitamin D target genes. The VDR‐RXR complex controls transcriptional activity targeting various tissues such as the kidney and colon, regulates immune defenses, and controls cellular proliferation. The nuclear factor (erythroid‐derived 2)‐like 2 (Nrf‐ 2), is a transcriptional regulator that stimulates expression of genes encoding antioxidant enzymes via a DNA sequence referred to as an antioxidant‐responsive element (ARE). Nutraceuticals such as urolithin‐A have been proposed to play a role in this pathway. The current study was designed to investigate how vitamin D‐VDR signaling influences Nrf‐2 activity. Therefore, we utilized an ARE‐luciferase reporter plasmid to measure Nrf‐2 activity in human embryonic kidney cells (HEK‐293) in the presence of VDR. Our results indicate that VDR does not target genes with an ARE sequence directly. Instead, when cells were transfected with both Nrf‐2 and VDR, Nrf‐2 activity was significantly inhibited in a 1,25D‐ and VDR‐dependent manner. However, when cells were treated with urolithin‐A alone, or with vitamin D in conjunction with urolithin‐A, a nutraceutical that is hypothesized to cooperate with vitamin D, Nrf‐2 activity was upregulated. Additionally, real‐time quantitative polymerase chain reaction (qPCR) was employed to test the expression levels of GCLC and HMOX1 (two Nrf‐2 target genes), in the absence and presence of 1,25D, to assess if the mRNA expression results were consistent with the luciferase reporter assays. Thus, the current study reveals that VDR likely targets Nrf‐2 genes indirectly by influencing the activity of the Nrf‐2 transfactor. Collectively, our results demonstrate, for the first time, that VDR either binds directly to the Nrf‐2 protein to inhibit its activity, or 1,25D‐VDR represses Nrf‐2 gene expression resulting in a diminution of Nrf‐2 protein production. Modulation of Nrf‐2 activity by the vitamin D pathway appears to have a regulatory role in anti‐oxidation and cellular aging pathways.

Support or Funding Information

National Institutes of Health; grant numbers: DK033351 and CA140285.
Selective Breeding in Domestic Dogs: How Selecting for a Short Face Impacted Canine Neuroanatomy
Authors: Selba Molly , Bryson Emily , Rosenberg Ciele , DeLeon Valerie
Published: April 2020

Domestic dogs (Canis lupus familiaris) exhibit a wide range of cranial morphologies. This diversity in skull shape is largely the result of modern selective breeding practices. This project investigates the impact of selection for facial reduction on canine neuroanatomy. Previous research has indicated that the breeding for reduced faces in dogs has led to global impacts in the shape of their skull and mandible. This previous research has largely failed to consider, however, the impact of these breeding practices on the soft neuroanatomical tissue. We generated endocasts from an existing set of clinically‐obtained CT scans. This sample consists of a variety of dogs with various cranial morphologies representing four known breeds as well as a comparative sample of dogs of unknown breed. We recorded three‐dimensional coordinate data for 31 landmarks representing various gyri, sulci, and other neuroanatomical landmarks. Placing landmarks at these locations allowed us to analyze differences in shape of the endocasts. Using geometric morphometric analysis, we found that the endocast shape variance in this sample is correlated with cephalic index and as a result, it is clear that selection for facial reduction has caused a perceivable effect on canine neuroanatomy. Furthermore, we found that the majority of the shape variance in the sample was associated with olfactory anatomy. Despite this fact, when the olfactory anatomy‐associated landmarks were excluded from the sample, the rest of the brain morphology was also correlated with cephalic index. The results of this project indicate that modern breeding practices, specifically the selection for dogs with squished faces, have significantly impacted canine neuroanatomy.
A Hybrid Approach to Learning Endocrine Physiology: Investigating the Efficacy of Using “MicroVids” to improve student learning
Authors: Pierre-Louis Gandhy , Brainard Robert
Published: April 2020

Introduction

With advances in technology, many lectures are now recorded and uploaded online for re‐viewing by students. However, lectures are long ranging from 50–120 minutes and students need to devote a large amount of time in order to review lecture, which can deter students from viewing recorded lectures. Therefore, we proposed creating short videos (MicroVids) reviewing only one specific concept from lecture and embedding the MicroVid into the printed notes via QR code, so that as students review the printed notes, they have a quick and easily accessible option for viewing professor‐explained concepts without searching the entire recorded lecture for the desired material.

Objectives

To investigate the efficacy of utilizing MicroVids as a tool to improve student learning of the endocrine system.

Methods

A series of 8 MicroVids were created reviewing concepts in the endocrine section of Anatomy and Physiology 1. The MicroVids were uploaded to YouTube and the URLs for each video was linked to a QR code that students could either click on to access the MicroVid (electronic notes) or scan with a QR code reader while reviewing their notes. Students (n=18, experimental group) taking an AP1 class during the Fall 2019 semester were given 8 MicroVids as supplemental material. After one week of access to each group’s MicroVids, students were given a survey to gather student feedback. Exam results from endocrine questions on their summative assessment was compared a control group of students taking AP1 the same semester as those that received Microvids and also compared to students who took AP1 Fall 2018.

Outcome

This approach garnered an overwhelming positive feedback. Two major themes from surveyed students were: 1) the MicroVids were easily accessible and 2) they helped students capture information they missed from their lecture notes. In the class that received the Microvids, 94% of the students watched the videos and 61% of students reported that it helped them succeed in their summative assessment. Additionally, while there was no statistically significant difference in exam grades between the class of students who watched the MicroVids versus the students of the control group, there was a modest improvement on the grasp of the endocrine system between poor test performers in the experimental group as compared to the poor test performers in the control group (an average 2.8 percentage points difference). While modest, the observation that poor test performers in the experimental group outperformed their control counterparts on their assessment is consistent with other reports that demonstrated similar positive learning outcomes when an active learning strategy was implemented in the classroom.
Hybrid Genome Assembly of a Regenerative Chordate
Authors: Sumner Jack Thomas , Wax Sarah , Andrasz Cassidy , Anderson Paul , Keeling Elena , Davidson Jean
Published: April 2020

Modern genome sequencing technologies open new avenues for research on biological processes in non‐model organisms. Colonial ascidians are a diverse group of invertebrate chordates capable of whole body regeneration, an unusual trait that is absent from vertebrates. Although genomic data are available for a few related species, there exist no such data for the highly regenerative species Botrylloides violaceus. Using a de novo hybrid assembly approach, we present the first draft of the B. violaceus genome sequence. By integrating short read and long read sequencing technologies, genome assembly becomes cost‐effective and easily accessible. Comparative genomic analysis between B. violaceus and other chordate genomes reveals insights into the evolution of gene families implicated in regeneration and development.

Support or Funding Information

Warren J. Baker and Robert D. Koob Endowments, California Polytechnic State University; The Frost Fund, College of Science and Mathematics, California Polytechnic State University
Early Post‐Natal Maternal Effects on Voluntary Exercise, Maximal Aerobic Capacity, and Associated Traits in Mice
Authors: Cadney Marcell D. , Schwartz Nicolas , McNamara Monica , Schmill Margaret , Castro Alberto , Hillis David , Garland Theodore
Published: April 2020

During the suckling period, many mammals progress through numerous developmental milestones, including critical periods of the central nervous system, musculoskeletal system, and cardiovascular system – each an important element of physical activity and exercise capacity. In a novel experimental mouse model, four replicate lines were selectively bred for ~90 generations for high voluntary wheel‐running behavior (high‐runner; HR) while four non‐selected Control (C) lines were also maintained. As a result of selection, HR mice run approximately 3‐fold the distances of C mice on a daily basis. Various other physiological and behavioral differences have also evolved in the HR lines, including higher maximal oxygen consumption (VO2max). The aim of the present study was to assess the role of the maternal environment during the birth‐weaning period in the expression of the many differences between HR and C lines using a cross‐fostering paradigm. Cross‐fostering HR mice to C dams (and vice versa) at birth and until weaning allowed us to determine the relative contribution of genetic and early environmental factors in shaping the HR phenotype. Mice were weaned three weeks after birth, housed in same‐sex cages of four, and adult testing began at approximately six weeks of age with measurement of VO2max, daily wheel running over a six‐day period, food consumption, home‐cage activity, body fat composition, and organ masses following dissection. As expected, HR mice ran approximately ~3‐fold more revolutions/day on wheels than their C counterparts. Preliminary analyses show that rearing by an HR dam has a negative effect on adult wheel running in females. This result suggests that the maternal environment provided by HR dams during the suckling period may play a role in the selection limit observed in HR lines.

Support or Funding Information

NSF GRFP
The BS11 prevents the atherosclerosis via liver X receptorα‐Mediated cholesterol metabolism in western diet‐induced obesity model
Authors: Park Seonghwan
Published: April 2020

In traditional Korean medicine (TKM), blood stasis syndrome (BSS) is defined as a condition that is stagnant due to poor circulation of blood in the body. Recent studies have revealed that blood stasis is related to metabolic diseases such as obesity, atherosclerosis, and type2 diabetes. The BS11 is a typical prescription for treating blood stasis in TKM, but the few studies reported the effect of BS11 on metabolic diseases. In this study, we administered BS11 (300 mg/kg/day and 600 mg/kg/day) to western diet (WD)‐induced obesity mice. Also, we applied liquid chromatography mass spectrometry (LC/MS)‐based lipidomics, and metabolomics analysis. In our results, WD‐induced obese mice weighted more than normal diet mice, especially at 13 weeks. On the other hand, BS11 groups showed no significant change compared to WD group. In histopathological experiments, it is considered that BS11 has favorable inhibitory effects on the WD‐induced organ damages. In both concentrations of BS11 groups, aortic wall thickening, lipid deposition with MCP‐1 and expressions of ICAM‐1 and VCAM‐1 were reduced. Hepatic steatosis, adipocyte hypertrophy and inflammatory cell infiltration were improved by BS11 treatment. In particular, BS11 reduced cholesterol levels in WB mice, one of the major causes of atherosclerosis. Moreover, we confirmed the unique lipid profile after BS11 treatment. Thus, we investigated the hepatic genes related cholesterol metabolism. BS11 treatment significantly altered liver X receptor alpha (LXRα) regulated cholesterol 7 alpha‐hydroxylase (CYP7A1) and ATP‐binding cassette sub‐family G member 8 (ABCG8), indicating alteration of cholesterol excretion by BS11 treatment. In conclusion, it is expected that appropriate treatment of BS11 will be provided a novel therapeutic candidate for blood stasis syndrome through improve cholesterol metabolism.
“(−)‐Epicatechin reduces inflammation biomarkers in plasma and ameliorates cardiac fibrosis in a female rat model of pre‐HFpEF”
Authors: Bustamante-Pozo Moises Muratt , Garate-Carrillo Alejandra Alejandra , VILLARREAL FRANCISCO , Ito Bruce Robert , CEBALLOS GUILLERMO , Ramirez-Sanchez Israel
Published: April 2020

The consumption of polyphenolic compounds, specifically flavonoids, have been associated with beneficial impacts on cardiovascular health. Flavonoids are found in plants and fruits such as cacao, berries, grapes and tea leaves. Cacao seeds contain high levels of the flavanol (−)‐ epicatechin. (−)‐Epicatechin (Epi) is therefore present in products such as dark chocolate or cocoa powder and has proven to have beneficial effects on the cardiovascular system. In the heart, it can reduce infarct size and apparently, myocardial fibrosis. Thus, we aimed to examine the potential of Epi to reduce myocardial fibrosis and improving passive and active left ventricular function and structure in an aging female rat model of pre‐HFpEF characterized by low estrogen levels, excess weight and hypertension. Female, 18‐month‐old, Fischer F344 rats were allocated into an ovariectomy + 10% fructose (OVF), and ovariectomy + 10% fructose + Epi (OVFE) (n=9–10/group). Epi treatment (1 mg/kg/day) was provided by gavage during a 30‐day period (from 21 to 22 months of age) while control animals received water. Left ventricular (LV) structure/function was assessed monthly by echocardiography. At 22 months of age, animals were anesthetized, and LV function was evaluated using a Millar conductance catheter. Histological measures of chamber morphometry, inflammation and collagen density (fibrosis) were also obtained. With treatment, no major structural and functional modifications were detected by echocardiography or by conductance‐catheter based hemodynamics. Passive left ventricular pressure volume curves were significantly right shifted with Epi while circumferential epicardial strains were reduced. The histological analysis of collagen quantification using Sirius red indicated that Epi treatment significantly reduced overall levels of myocardial fibrosis. Epi also significantly reduced plasma levels of pro‐fibrotic and pro‐inflammatory cytokines. In conclusion, Epi appears capable of reducing cardiac fibrosis in an animal model of pre‐HFpEF which correlate with reductions in cytokine plasma levels. However, reductions in tissue fibrosis did not translate into major modifications in passive or active chamber structure and function. This model may serve to understand the role that estrogen depletion and weight gain may have in the development of HFpEF.

Support or Funding Information

Funding was provided by the Department of Defense PR150090 and National Institutes of Health DK98717, AG47326 to F.V. and Consejo Nacional de Ciencia y Tecnologia, Mexico to G. C.