bluuebunny/crossref_metadata_2025 huggingface dataset:
Investigating the Effects of Histone H3.3 Point Mutations in Neural Crest Cells and Craniofacial Development
Authors: Nzirorera Nadine , Jabado Nada , Majewska Loydie
Published: April 2020

Craniofacial development is an intricate process that requires the meticulous and coordinated expression of many genes. Histone proteins which are fundamental to both the structural integrity and organization of DNA into nucleosomes and consequently, chromatin play an important role in coordinated gene expression. Furthermore, the modification of histone tails is now known to be an essential mechanism for coordinated expression of a large number of genes. Recently, a point mutation in the gene h3f3a which encodes the histone variant Histone H3.3, was identified in a forward genetic screen in zebrafish. H3.3A was found to be important for neural crest cell induction and generation of cranial neural crest cell derived head bones. The authors proposed that incorporation of histone H3.3 was required for expression of genes important for cranial neural crest cell specification, whereas remodelling of this histone tail may be important for later stages in neural crest cell differentiation. However, it remains to be confirmed if disrupted modification of histone tails is also important in neural crest cells. We postulate that modification of the H3.3 tail is required for coordinated expression of genes important for neural crest migration and/or differentiation. To test this hypothesis, we used CRISPR/Cas9 to generate a mouse line with a conditional missense mutation in H3f3a. The mutation generated was previously shown to result in an abnormal increase in methylation of K27 and a decrease in methylation of K36 of the histone H3.3 tail. In this poster, we will present preliminary data using the Wnt‐1 Cre2 mouse line to drive expression of this dominant negative mutation in neural crest cells. Alcian Blue Staining will be used to analyze cartilage in E14.5 embryos and alizarin red will be used to stain bones formed in heads of E17.5 embryos. Bones will be analyzed in control and mutant embryos. Our result will enable us to uncover if K27 or K36 methylation is important in neural crest cell formation, migration and/or differentiation. This work will begin to unravel the role of epigenetics in neural crest cell formation.

Support or Funding Information
Changes in Motor Function in Response to Living in an Extreme Underwater Saturation Environment under High Stress and Increased Workload
Authors: Ari Csilla , D’Agostino Dominic Paul , Bharwani Sahil , Rehsi Anand , Moss Sara , Schmer-Galunder Sonja , Fiore Stephen
Published: April 2020

Understanding changes in individual motor function and how it relates to workload, personality types, and shared knowledge is vital to the success of team‐based missions. Such changes may present a challenge in long duration spaceflight and other missions in extreme environments. Therefore, we studied grip strength, dexterity, stress level, task load and the personality types of participants living in an extreme environment during the NASA Extreme Environment Mission Operation (NEEMO) 23 in June 2019. The Aquarius Reef Base is home to NEEMO missions and is located at 20 m (66 ft) with 2.6 atm (air) pressure inside the habitat, where CO2 levels are often elevated similar to the International Space Station.

Motor strength was assessed using a grip dynanometer and dexterity was measured using a pegboard. The tests were performed by 6 subjects (2 males, 4 females) 1 day pre‐mission, during the mission (grip test twice) and 1 day post‐mission. Dexterity test was also performed at the beginning and at end of Extravehicular Activities (EVAs). NASA TLX tests measuring mental, physical, temporal effort, performance, general effort and frustration, were assessed immediately after returning from EVAs. To monitor changes in stress level, Short Stress State Questionnaire (SSSQ) and Generalized Anxiety Disorder (GAD7) scores were collected. Measures of social cognition, including Reading the Mind in Eyes and Big 5 personality tests, were performed once before the mission.

There was a trend of decreased grip strength at the end of mission and post‐mission, compared to baseline and beginning of mission. The dexterity scores at the beginning of EVAs were significantly higher, compared to before and during mission. This suggests that tasks with fine motor function can be affected by context such as in‐water performance. At the end of the EVAs, the dexterity score was significantly higher compared to before mission and during mission as well. There was a trend of worse performance on the dexterity test at the end of EVA, compared to the beginning of EVA. A trend of increasing GAD7 scores at half time of the mission and post‐mission could be observed. There were several differences in NASA TLX scores between different subjects.

These results indicate that strength and dexterity was influenced during EVAs. This may have been due to both physical and psychological stress during the extreme underwater mission in a saturation environment. Further examination of these trends is recommended.

Support or Funding Information

Ketone Technologies LLC provided partial funding for the study. SIFT provided partial salary for CA.
The “Function vs. Disease Dilemma” in Contemporary Medicine: Physical Frailty & Sarcopenia as a Prototypic Condition of New‐Generation Geriatric Medicine
Authors: Tosato Matteo
Published: April 2020

Due to the worldwide demographic transition, healthcare systems are facing new demands. Health services—with their approach of mostly single acute conditions—are indeed confronted with an expanding older population characterized by specific medical needs related to multimorbidity and functional impairment. The European research project “Sarcopenia and Physical fRailty IN older people: multi‐componenT Treatment strategies” (SPRINTT) project is specifically designed to overcome existing barriers for efficient public health interventions against frailty, and promote the implementation of successful aging strategies across Europe. The just‐completed SPRINTT randomized clinical trial (RCT) compared the efficacy of a multicomponent intervention (based on long‐term structured physical activity, nutritional counselling, and an information and communication technology intervention) versus a Healthy Aging Lifestyle Education program for preventing incident mobility disability in community‐dwelling older persons with physical frailty and sarcopenia. For the RCT, we enrolled 1,517 community‐dwellers, aged 70 years and older (750 per treatment arm), non‐disabled older persons exposed to increased vulnerability to stressors. The identification of such population relied on three key elements: low muscle mass measured by DXA (identified using FNIH cutpoints); clinical signs of physical frailty (i.e., weakness, slow walking speed, and poor balance) assessed using SPPB; absence of major mobility disability (able to walk 400 meters within 15 minutes at baseline. The primary outcome was the incidence of mobility disability (i.e., incident inability to walk 400 meters). Secondary outcomes included, among others, changes in physical performance and function; ability of selected biomarkers to predict the rate of change in muscle mass; incidence of falls. The SPRINTT RCT was conducted in 15 study sites located in nine European countries. The mean age of our sample was 79 years (SD ± 5.8), 72% were women, BMI was 28.6 kg/m2 (± 5.9), with 37% of the population showing BMI >30 kg/m2, Handgrip strength was 20.1 kg (SD 8.2), SPPB score was 6.71 (SD 1.4), aLM in men was 21.0 kg (SD 4.06) and 15.0 kg in women (SD 1.98), the aLM/BMI was 0.71 (SD 0.06) in men and 0.51 (SD 0.07) in women. Data analysis is ongoing and final results may be available at the date of the EB meeting.

Support or Funding Information

Innovative Medicines Initiative – Joint Undertaking (IMI‐JU 115621)
Mutation of Class‐Distinctive Residues in Gα13 to Identify Determinants of RH‐RhoGEF Binding Selectivity
Authors: O’Shea Corin R. , Tagliatela Alicia C. , Meigs Thomas E.
Published: April 2020

Gα13 belongs to the G12/13 subfamily of heterotrimeric guanine nucleotide‐binding proteins that play a signaling role in cell growth and tumorigenic pathways, cytoskeletal rearrangements, and metastatic invasion. A structural bioinformatics analysis [Temple et al., 2010, PLOS Comput. Biol., 6(10): e1000962] identified a set of “class‐distinctive” residues in Gα13 which correspond to a different residue conserved in the other G protein subfamilies: Gs, Gi, and Gq. Using this information, we created a panel of Gα13 point mutants that replace each class‐distinctive residue with its putative ancestral form. In order to distinguish our mutant constructs from native Gα13 in cultured human embryonic kidney cells, we introduced a myc epitope tag to all Gα13 mutants, positioned within the αB–αC loop of the helical domain. Installation of this epitope tag was non‐disruptive to SRF signaling by Gα13, and allowed us to differentiate its recombinant and native forms in protein‐protein interaction experiments. While characterization of these Gα13 mutants is ongoing, we have identified a class‐distinctive Phe at position 234 for which ancestral substitution appears to cause selective uncoupling of protein binding within the Gα13‐responsive, RGS‐homology (RH) RhoGEFs. These findings shed light on the mechanism of Gα13 interaction with the individual RH‐RhoGEFs‐p115RhoGEF, PDZ‐RhoGEF, and leukemia‐associated RhoGEF‐ and further suggest that class‐distinctive Gα13 mutants may reveal binding determinants for additional effector proteins.

Support or Funding Information

We acknowledge support from the UNC Lineberger Comprehensive Cancer Center (University Cancer Research Fund), and the North Carolina GlaxoSmithKline Foundation.
Autophagic Burden on Calcium Signaling in Sympathetic Activation
Authors: Hayden Matthew R. , Wauson Eric , Tran Quang-Kim
Published: April 2020

Signaling via calcium plays essential roles in all aspects of cell functions. Macroautophagy is a catabolic process that regulates important aspects of cell metabolism. While many studies have indicated that calcium is involved at various steps in the regulation of autophagic flux, the impact of autophagic flux on calcium signaling is unknown. We conceptually hypothesized that an “autophagic burden” exists in which enhanced requirement of the cell to process autophagic cargos would exert negative impact on calcium signaling. To test this hypothesis, we utilized an “non‐stimulation” approach to increase the “autophagic burden” by heterologously expressing in cells the wildtype microtubule‐associated proteins 1A/1B light chain 3B (LC3) or a mutant version in which the C‐terminal glycine responsible for ATG4‐mediated cleavage for LC3 lipidation has been removed (LC3DG) and examined the impact on adrenergic receptor‐mediated calcium signaling. HEK293 cells produced oscillatory intracellular calcium signals in response to agonists of b adrenoceptors (isoproterenol), α1A adrenoceptor (A61603), or a combination thereof (10 mM norepinephrine, (NE)). Cells expressing a fusion between the RFP mKate2 and wildtype LC3 (mKate2‐LC3) responded to 10 mM NE with substantial reductions in the number of intracellular calcium oscillations in the physiological ranges of 300 – 600 nM, 600 – 900 nM, or 900 – 1200 nM, as compared to the non‐expressing cells examined in the same microscopic fields, recognized by absence of mKate2 fluorescence. In contrast, cells expressing the mKate2‐LC3DG produced the same number of oscillatory calcium signals compared to non‐expressing cells in the same microscopic fields across the same ranges of cytoplasmic calcium stimulated by NE. The data suggest that an “autophagic burden” exists that may dampen the effects of acute sympathetic stimulation significantly via reduction in adrenoceptor‐mediated calcium signals.

Support or Funding Information

IOER Grant 091707 to Q‐KT
Lysine 33 polyubiquitin chain formation by the HECT Ubiquitin E3 Ligase AREL1
Authors: Ladda Emily Rose , Spratt Donald
Published: April 2020

Apoptosis Resistant E3 Ligase 1 (AREL1) is a member of the Homologous to E6AP Carboxyl Terminus (HECT) E3 ubiquitin ligase family. All HECT E3 ubiquitin ligases contain a conserved HECT domain that facilitate the covalent attachment of ubiquitin onto their intracellular substrates. Ubiquitylation is involved in numerous cell signaling pathways that can result in proteasomal degradation, DNA damage repair, and cell trafficking, among others. The biological function of AREL1 is to inhibit intrinsic apoptosis by ubiquitylation of pro‐apoptotic inhibitor of apoptosis (IAP) antagonist proteins, SMAC, ARTS, and HtrA2, for proteasomal degradation. Interestingly, previous studies have found that AREL1 is the only HECT E3 ubiquitin ligase identified to date that is capable of building polyubiquitin chains via lysine 33 linkages, and the biological role of this linkage remains unclear. Using circular dichroism (CD), in vitro ubiquitin activity assays, disulfide complex formation, and multidimensional heteronuclear NMR spectroscopy, we are currently deciphering the unique molecular mechanism used by AREL1 to build the lysine 33 poly‐ubiquitin chains.

Support or Funding Information

NIH NIGMS Maurine H. Milburn Fund Clark University Carlson School of Biochemistry and Chemistry
Structural and Mechanistic Characterization of HERC2 E3 Ubiquitin Ligase C‐lobe with Implications in Cancer, Prader‐Willi Syndrome, and Eye Color
Authors: Rich Kayla , Schwaegerle Noah , Spratt Donald
Published: April 2020

HERC2 (HECT domain containing and RCC1‐like domain containing E3 ubiquitin ligase 2) is a HECT E3 ubiquitin protein ligase that is implicated in breast cancer, Prader‐Willi syndrome, and eye color. HECT E3 ubiquitin ligases covalently attach ubiquitin onto substrates to target proteins for proteasomal degradation, initiate DNA damage repair, control cell cycle and intracellular trafficking, and regulate transcription. HERC2 has proposed implications with DNA damage repair and proteasomal degradation. The HERC2 gene has been linked to the neurodevelopmental disorder Prader‐Willi syndrome and is part of the cis‐regulatory module for OCA2 (oculocutaneous albinism 2). To date, only a few substrates of HERC2 have been identified including BRCA1 (breast cancer 1). Using biochemical and biophysical techniques, such as circular dichroism and multidimensional heteronuclear NMR spectroscopy, the HERC2 C‐lobe structure was characterized. Our studies show that HERC2 is mainly α‐helical with a flexible acidic C‐terminal tail extension. We demonstrate that the C‐terminal tail extension is important for polyubiquitin chain formation through intramolecular interactions with ubiquitin. The truncation of the HERC2 C‐terminal tail decreases protein stability and function. These structural and mechanistic studies on the HERC2 C‐lobe are important for elucidating the cellular role of HERC2 along with its implications with disease.

Support or Funding Information

NIH‐NIGMS FundingAbbVie summer research grantClark University
Higher Susceptibility to Skeletal Muscle TA (Tibialis Anterior) Injury with Increased Inflammation in Aged Mice.
Authors: Tipparaju Srinivas M. , Virzi Jazmie M. , Puig Viviana M. , Brotto Marco
Published: April 2020

Background

Skeletal muscle contributes to a major portion of normal human body weight and plays a key role for physiological functions including growth, development and a host of metabolic regulating roles. Therefore, skeletal muscle injury is a major concern, particularly in pediatric and older populations.

Objective

To evaluate the differences in skeletal muscle injury, we utilized male Wild type C57BL6 young (5‐month old; n=4), and mature‐aged (12‐month old; n=4) aging mouse models.

Methods

We utilized Barium Chloride injection to induce inflammatory damage to the tibialis anterior muscle (TA), and monitored early skeletal muscle changes during 72 hours. Immunohistochemistry and morphometric analysis was conducted to identify changes between saline treated vs. barium chloride treated TA muscle.

Results

H&E staining from injured TA muscle identified an area of damage of ~52% in the young compared to a significantly larger area of damage of ~78% in mature mice. We also performed histomorphometric analyses to identify additional morphological changes. We found changes consistent with an increased cross sectional area post injury along with increased inflammation in the injured TA muscle.

Conclusions

The barium chloride TA muscle injury model allows for evaluation of TA muscle injury as early as 72‐hour post injection. Barium chloride injured muscles show significantly increased inflammation compared with saline injected muscle. The mature‐aged 12‐month old mice demonstrated greater injured area and enhanced inflammatory response, suggesting that even in mature mice, there is an increased susceptibility to muscle damage. These findings might have previously unforeseen consequences for older adults reaching middle age.

Support or Funding Information

NIH R01 DK119066 and William N. Saunders Endowment in geriatric pharmacotherapy to (SMT). MB was partially supported by NIH‐National Institutes of Aging PO1 AG039355 (M.B.), and R01AG056504 and R01 AG060341 (M.B.), and the George W. and Hazel M. Jay and Evanston Research Endowments (MB).
Effect of Physical Activity on Oxidative Stress and Endothelial‐Dependent Cutaneous Microvascular Function in Non‐Hispanic Blacks: A Pilot Study
Authors: Miller James , Turner Casey , Wong Brett
Published: April 2020

Cardiovascular disease is a dominant cause of morbidity and mortality in non‐Hispanic Blacks (NHB). This may be due, in part, to vascular endothelial dysfunction which causes reduced nitric oxide (NO) bioavailability and increased oxidative stress. Conversely, physical activity has been shown to attenuate and treat symptoms of cardiovascular disease, endothelial dysfunction, and oxidative stress. It remains unknown whether physical activity reduces oxidative stress and enhances endothelial‐dependent vascular function in NHB. The purpose of this study was to examine the extent to which physical fitness influences oxidative stress and nitric oxide‐dependent cutaneous vascular function in NHB. On the first visit, young, healthy NHB participants (n = 3 regularly active; n=2 inactive) completed a graded exercise test on a cycle ergometer to determine V̇O2max to confirm self‐reported physical activity status. On the second visit, participants were instrumented with 4 microdialysis fibers for infusion of 1) lactated Ringer’s (control), 2) 0.1 mM 1400W (iNOS inhibitor), 3) 0.1 mM tempol to inhibit oxidative stress, 4) and combined tempol + 1400W, respectively. Following infusion of experimental drugs, local skin temperature was increased via rapid local heating to 39°C at a rate of 0.1°C/sec. Once skin blood flow plateaued, 20 mM L‐NAME was infused to quantify NO‐dependent vasodilation. Following L‐NAME infusion, the local heaters were increased to 43°C and 54mM of sodium nitroprusside was infused to elicit maximal endothelium‐independent cutaneous vasodilation. Skin blood flow was measured via laser‐doppler flowmetry (LDF) throughout the local heating protocol. Cutaneous vascular conductance (CVC) was calculated as LDF divided by mean arterial pressure and normalized to maximum (%CVCmax). iNOS inhibition increased %NO‐dependent vasodilation in both active (67 ± 10% vs. 54 ± 7%) and inactive (69 ± 4% vs. 56 ± 10%) groups compared to control. Combined inhibition of iNOS and oxidative stress increased %NO‐dependent vasodilation in both active (72 ± 9%) and inactive (71 ± 13%) NHB compared to control but these were not different compared to inhibition of iNOS or oxidative stress alone. Interestingly, inhibition of oxidative stress significantly increased %NO‐dependent vasodilation in only the active group compared to control (74 ± 8%). Taken together these data indicate that inhibition of iNOS with 1400W increases endothelial‐dependent cutaneous vasodilation in active and inactive NHB while inhibition of oxidative stress increases endothelial‐dependent cutaneous vasodilation in active NHB.
Tumor Stroma Targeting Nanoparticles Pro‐drug Approach for Treating Pancreatic Ductal Adenocarcinoma
Authors: Alzhrani Rami , Sau Samaresh , Aboukameel Amro , Alsaab Hashem , Azmi Asfar , Iyer Arun
Published: April 2020

Pancreatic ductal adenocarcinoma (PDAC) is the third‐highest cause of cancer‐related death in the United States. The major challenges that limit the success of anti‐PDAC therapy are tumor heterogenicity and dense desmoplastic tumor stroma; that in turn promote drug resistance. Therefore, we developed nanoparticles (NP) pro‐drug that can overcome the stroma barrier and deliver the payload to the core of the tumor. We utilized nanoengineering technology to target dual CD44 and c‐Met biomarkers that have a crucial role in developing drug resistance in PDAC. Our western blot data analysis in several PDAC cell lines, such as PANC‐1, AsPc‐1, and BxPc3 revealed a high expression of CD44 and cMet. Thus, developing CD44/cMet dual‐targeted NP pro‐drug (C44MNPs) is expected to enhance drug delivery efficiency and efficacy. To test the tumor selectivity of C44MNPs on orthotopic PDAC model, near‐infrared (NIR) fluorescence dye was conjugated with C44MNPs, namely NIR‐C44MNPs. The biodistribution of NIR‐C44MNPs in the orthotopic model showed superior tumor accumulation with seven fold higher uptake than controls. Interestingly, NIR‐C44MNPs showed minimum liver accumulation, suggesting the ability of our NPs in overcoming liver toxicity (an observed problem with conventional NPs). Finally, C44MNPs was chemically conjugated with gemcitabine (GEM), namely C44MNPs ‐GEM pro‐drug, to improve the overall GEM therapeutic efficiency. In vitro data revealed that C44MNPs‐Gem pro‐drug has an excellent PDAC killing compared to free GEM. Moreover, C44MNPs‐GEM pro‐drug in combination with Everolimus showed extraordinary PDAC cell killing of D44M‐GEM with a high synergistic effect ( combination index [CI] value of 0.2–0.5). A significant tumor inhibition was achieved in an ASPC‐1 xenograft model by using combination therapy (C44MNPs‐GEM pro‐drug + EVR) compared with controls. Our current findings are highly encouraging and opens up a new avenue of using nanotherapeutics to overcome the current barriers of drug delivery for effective PDAC therapy.
Valproic Acid Upregulates The Expression of The Neurotrophin Receptor TrkC in Human Neuroblastoma Cells
Authors: Dedoni Simona , Marras Luisa , Olianas Maria C. , Ingianni Angela , Onali Pierluigi
Published: April 2020

Preclinical and clinical studies have shown that the Trk receptors of neurotrophic factors play an important role in regulating the biological properties and the clinical evolution of neuroblastoma, the most frequent extracranial solid tumor in childhood. We have recently reported that treatment of retinoic acid (RA)‐differentiated human neuroblastoma cells with valproic acid (VPA) and other histone deacetylase (HDAC) inhibitors downregulates the expression and signaling of the BDNF receptor TrkB, a marker of unfavorable and aggressive neuroblastoma. In the present study we report that VPA, at therapeutically relevant concentrations, upregulates the expression of the neurotrophin 3 receptor TrkC in undifferentiated and RA‐differentiated SH‐SY5Y neuroblastoma cells. The exposure to VPA markedly increased the TrkC mRNA and the levels of the full length and truncated forms of TrkC protein. This change was associated with enhanced expression of TrkC at the plasma membrane, as indicated by cell surface biotinylation experiments and immunofluorescence assays in non permeabilized cells. The upregulation of TrkC was time‐dependent, being evident after 24 h of drug addition, and was accompanied by the emergence of apoptotic cell death. VPA increased the levels of the transcription factor RUNX3, a positive regulator of the NTRK3 gene encoding TrkC, and inhibition of this response by siRNA treatment attenuated the upregulation of TrkC. Moreover, pharmacological blockade of either ERK1/2 or JNK, but not p38MAPK, signaling pathways reduced VPA‐induced TrkC expression. VPA was found to induce TrkC also in MYCN‐amplified BE(2)C, NB‐1 and Kelly neuroblastoma cells and its effect was mimicked by other HDAC inhibitors, such as romidepsin and entinostat. These data indicate that VPA upregulates TrkC by activating a transcriptional programme involving epigenetic changes and MAPK signaling. As the expression of TrkC has been associated with a favorable outcome in neuroblastoma, the upregulation of this neurotrophin receptor may constitute an important component of the antineuroblastoma activity of VPA.

Support or Funding Information

Sardegna Ricerche‐Progetto IBERNAT‐NBL P.O.R. Sardegna 2014–2020, CUP F21B17000730005.
The effects of probiotic supplementation and eating a high fat diet on sensitivity of rats to the behavioral effects of methamphetamine
Authors: Sandoval Oscar , Alfsen Amanda , Beltran Nina , Serafine Katherine
Published: April 2020

Eating a high fat diet can lead to obesity, insulin resistance, and can cause dysfunction to dopamine systems. For example, rats eating high fat laboratory chow are more sensitive than rats eating standard chow to the behavioral effects of drugs that act on dopamine systems (i.e., methamphetamine). Dietary supplementation with probiotics (including a combination of lactobacillus and bifidobacterium) has been shown to aid in digestion and decrease depressive‐like behavior among rats eating high fat chow. It is not known if probiotic supplementation can similarly decrease the effects of eating high fat chow on sensitivity of rats to the behavioral effects of dopaminergic drugs. To test the hypothesis that dietary supplementation with probiotics prevents high fat diet‐induced enhanced sensitivity to methamphetamine, rats eating standard chow (17% kcal from fat), high fat chow (60% kcal from fat), or eating standard or high fat chow with daily probiotic (1 billion CFU) supplementation were tested once weekly using cumulative doses of methamphetamine (0.1–3.2 mg/kg; i.p.) Dose‐response curves were analyzed using two‐way repeated measures ANOVAs using Tukey’s multiple post‐hoc comparisons tests where appropriate. After 5 weeks of testing, there were no significant differences in drug sensitivity to methamphetamine among rats eating different diets. Although the experiment is ongoing, previous literature suggests that sensitivity to methamphetamine will increase with continued testing (e.g., sensitization) and that rats eating high fat chow will be more sensitive to this effect than rats eating standard chow to drugs that act on dopamine systems.

Support or Funding Information

O.S. is supported by the National Institute of General Medical Sciences of the National Institutes of Health (NIH) under R25GM069621. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
Impact of Ampakine CX‐717 on Diaphragm EMG Activity and Breathing in Non‐anesthetized Freely Behaving Rats
Authors: Rana Sabhya , Sunshine Michael D. , Fuller David D.
Published: April 2020

Glutamatergic neurotransmission is partly mediated by ionotropic AMPA receptors, and these receptors play an important role in the neural control of breathing. Ampakines are positive allosteric modulators of AMPA receptors. These drugs can stimulate breathing during hypo‐ventilated states, such as following an opioid overdose. Our laboratory has recently shown that acute exposure to an ampakine, CX717 (15 mg/kg), can acutely stimulate neural drive to the diaphragm in anesthetized but otherwise healthy rats. The goal of the current work is to evaluate the impact of ampakine on diaphragm muscle activation in freely moving non‐anesthetized rats. Ventilation and metabolic rate (VCO2) were measured using whole body plethysmography and diaphragm EMG was assessed using intramuscular electrodes. Spinally intact rats (n=2) were given a single intravenous bolus of CX717 at four months after implantation of diaphragm EMG electrodes. Delivering CX717 at 15 mg/kg had no detectable impact on VCO2, inspiratory tidal volume or diaphragm EMG burst amplitude. In addition, at 30 minutes following IV ampakine, both animals showed a robust increase in diaphragm EMG activity, tidal volume, and the ratio of ventilation VCO2 (VE/VCO2) during an acute hypoxic challenge (5 min, 10.5% inspired O2). Qualitatively, we observed a possible sedative effect as shown of lack of movement and exploratory behavior following ampakine exposure. Our preliminary conclusion is that ampakine CX717 at a dose of 15 mg/kg has little impact on breathing in the otherwise healthy rat. Ongoing studies are evaluating impact of the vehicle (2‐hydroxypropyl‐beta‐cyclodextrin) versus ampakine using a cross‐over study design in spinally intact rats and in rats with spinal cord injury.

Support or Funding Information

5 R01 HL139708 02 (DDF)F31 HL145831‐01 (MDS)
Detection of mRNA Encoding Organic Anion Transporters in Zebrafish Eyes
Authors: Coles-Ruiz Melanie , McIntosh Roxanne , García Dana M.
Published: April 2020

The solute carrier 22 (slc22) family is a group of transmembrane proteins that specialize in the transport of organic ions. A sub‐category of slc22 consists of organic anion transporters (OAT) that transport cyclic nucleotides such as cyclic adenosine monophosphate (cAMP). In the process of light to dark adaptation of fish eyes, cAMP is hypothesized to be a regulatory signal between the photoreceptors and retinal pigment epithelium (RPE) that drives pigment granule aggregation in the latter. We hypothesize that cAMP is exported from the photoreceptors into the sub‐retinal space, and one of the slc22 OAT proteins imports cAMP into the RPE. To test this hypothesis, reverse transcription polymerase chain reaction (RT‐PCR) was used to test for the presence of mRNA encoding seven different OAT proteins (1, 2a, 2b, 2c, 2d, 2e and 3) in whole eyes from zebrafish. Messenger RNA encoding each of the OAT proteins was detected, corroborating results of earlier studies. Experiments are underway to test which of the OAT genes are expressed in isolated RPE. Identification of the OAT proteins in RPE is an important step in illuminating a novel signaling pathway.

Support or Funding Information

This project was supported by Texas State University in the form of an undergraduate research fellowship to MCR and funds from the Office of the Provost awarded to DMG in recognition of her service as Presidential Fellow.
Upregulation of G protein‐couple receptor 68 (GPCR68) in pulmonary artery smooth muscle cells from animals and humans with pulmonary hypertension
Authors: Rahimi Shamin , Babicheva Aleksandra , Rodriguez Marisela , Jain Pritesh , Rahimi Shayan , Yuan Jason X.-J.
Published: April 2020

Introduction

Pulmonary arterial hypertension (PAH) is a progressive and fatal disease with no cure. It is characterized by elevated pulmonary vascular resistance (PVR) and pulmonary arterial pressure (PAP) in part due to pulmonary vascular remodeling and sustained pulmonary vasoconstriction. The increased pulmonary arterial smooth muscle cell (PASMC) proliferation contributes to excessive pulmonary vascular remodeling while a rise in cytosolic free Ca2+concentration ([Ca2+]cyt) in PASMC is a major trigger for pulmonary vasoconstriction and a key stimulus for PASMC proliferation. G‐protein coupled receptors (GPCR), which are attractive pharmacological targets, are important regulators of pulmonary vascular tone and PASMC phenotype. Stimulation of GPCR leads to Ca2+ influx and increased [Ca2+]cytdue to store‐operated Ca2+ entry (SOCE) via inositol 1,4,5‐trisphosphate (IP3) production and receptor‐operated Ca2+entry (ROCE) via diacylglycerol (DAG) production. GPCR68 (or OGR1) is a proton‐sensing GPCR that responds to extracellular acidity and regulates a variety of cellular functions. As a Gq/11‐coupled receptor, GPR68 activation is known to lead to Ca2+ release from the intracellular stores in IP3‐dependent manner but the role of GPCR68 in the development of PAH is still unknown.

Methods

Lung, heart, brain, liver, and kidney tissues were isolated from control rats. Pulmonary hypertension model was induced by a single dose of monocrotaline (MCT) in rats. PASMC isolated from control or MCT rats and healthy subjects or patients with idiopathic pulmonary arterial hypertension (IPAH) were cultured in 5% fetal bovine serum (FBS) smooth muscle growth media and incubated in a humidified 5% CO2 atmosphere at 37°C. Total RNA was isolated from tissue or cells using TRIZOL method. mRNA expression of GPCR68 was quantified by RT‐PCR and qRT‐PCR. The data was analyzed using ImageJ and statistical analysis was performed using paired or unpaired Student’s t‐test.

Results

RT‐PCR analysis revealed that GPCR68 is expressed in brain, lung, heart, liver, and kidney in normal rat tissues (n=5). GPCR68 mRNA level was significantly higher in lung tissue and PASMC isolated from MCT‐treated rats compared with controls. Moreover, our data shows that GPCR68 is expressed in both human PASMC and pulmonary arterial endothelial cells. qRT‐PCR showed that GPCR68 was upregulated in PASMC isolated from IPAH patients compared with cells isolated from healthy subjects.

Conclusion

Our finding suggest that GPR68 is involved in the development and progression of PAH and may play an important role in Ca2+ signaling and enhanced [Ca2+]cyt in PASMC from animals and humans with PAH.

Support or Funding Information

This work was supported, in part, by grants from the National Heart, Lung, and Blood Institute of the National Institutes of Health (HL125208, HL135807, and HL126609).
Effects of Glyphosate on Locomotion and Anxiety‐like Behavior in Rats
Authors: Reyes-Sepúlveda Carlos Javier , Cáceres-Chacón Mauricio , Alvelo-Fernández Paola , Haddock-Martínez Hector , Ramos-Sánchez Raúl , Martínez-Guzmán Osmarie , Rivera-López Melissa , Sierra-Mercado Demetrio
Published: April 2020

Glyphosate is the active ingredient in several commercial herbicides. The development of glyphosate‐resistant seeds has led to an increase in the use of glyphosate in agriculture. This has resulted in the exposure of non‐agricultural lands and bodies of water to glyphosate. Though initially considered safe for mammals, recent studies in rats have shown that administration of glyphosate leads to alterations in locomotion and anxiety‐like behavior. These studies generally used unrealistically high concentrations of glyphosate. Therefore, the effects of a dose of glyphosate considered safe by the Environmental Protection Agency through drinking water remain unclear. To determine if these levels affect locomotion and anxiety‐like behavior, we provided rats with access ad libitum to glyphosate‐contaminated drinking water or filtered water for four weeks. Glyphosate‐contaminated water was prepared for a target dose of 1.75mg/kg of glyphosate daily. We hypothesize that intake of glyphosate‐contaminated drinking water will decrease locomotion and increase anxiety‐like behavior. Locomotion was assessed in an open field. We observed that glyphosate did not decrease the overall distance travelled (control: 30.67m, glyphosate: 23.70m; p=0.058) nor did it affect average speed (control: 0.102m/s, glyphosate: 0.079; p=0.058). Anxiety‐like behaviors were also measured in the open field. Here, glyphosate did not increase the amount of entries to the center (control: 25.5, glyphosate: 27.5; p=0.726) nor time spent in the center (control: 74.22s, glyphosate: 87.9; p=0.572). In conclusion, this dose and exposure time of glyphosate did not affect locomotion or anxiety‐like behavior. Future directions include performing immunohistochemistry to assess neuronal activity in brain regions involved in locomotion and anxiety‐like behaviors.

Support or Funding Information

Young Investigator grant (NARSAD) of the Brain & Behavior Research Foundation, PRCTRC Pilot, NIGMS COBRE and RCMI8G12MD00760 to DS‐M; NSF PRCEN fellowships to CJR‐S, MC‐C, HH‐M, RR‐S and MR‐L, UPR Med Sci Campus Chancellor’s Office and School of Medicine Deanship.
Dietary salt and endothelin A receptor activation regulates T cell responses in a tissue‐dependent manner
Authors: Molina Patrick , Pollock Jennifer
Published: April 2020

Numerous studies have investigated the role of high salt diet (HSD) by utilizing salt‐sensitive animal models or co‐administration with pro‐hypertensive insult. HSD is implicated in altering tissue‐dependent immunologic responses. Endothelin (ET‐1) is a major vasoconstrictor peptide synthesized in response to HSD. The ET‐1/endothelin A (ETA) receptor pathway has been implicated in regulating inflammatory responses, and to blunt the immunologic response with pro‐hypertensive conditions. However, the singular role of HSD on renal, colonic and splenic T cells is not fully understood. Therefore, we hypothesized that ETA receptor activation blunts T cell responses with HSD. Utilizing flow cytometry, we immunophenotyped renal, colonic, and splenic T cells in 12‐wk old male C57BL6/J mice provided normal salt diet (NSD, 0.4% NaCl food), ETA blockade (atrasentan, 10mg/kg/day) with NSD, HSD (4% NaCl food plus 1% NaCl drinking water), or co‐administration of atrasentan and HSD for 5 weeks. Kidneys, colons and spleens were collected for single cell suspensions. Isolated cells were stimulated followed by fluorescent antibody staining for flow cytometric analysis. Data are expressed as cell number means SEM (n=4/group) with statistical differences calculated by two‐way ANOVA with Tukey’s correction for multiple comparisons. HSD significantly blunted the total number of renal CD4+T cells mice compared to NSD as well as atrasentan treatment without further suppression of CD4+T cells under co‐administration of both HSD and atrasentan (NSD: 5217 619; Atrasentan: 1.8 0.15*; HSD: 2.4 0.32*; Atrasentan/HSD: 1.8 0.15*, x103, *p<0.001). Colonic CD4+T cells were similarly blunted under Atrasentan‐only and HSD‐only administration, without further reduction under co‐administration. However, splenic CD4+T cells were significantly expanded under chronic atrasentan administration (NSD: 2.66 1.45; Atrasentan: 7.63 0.61#; HSD: 6.63 0.69#; Atrasentan/HSD: 6.46 0.96#, x106, #p<0.05). We next examined IL‐17A production by CD4+T cells, which allows us to determine TH17 cells. Compared to NSD, HSD or atrasentan significantly blunted the total number of renal TH17 cells (NSD: 65 9; Atrasentan: 22 5*; HSD: 26 4*; Atrasentan/HSD: 20 3*, *p<0.001). Colonic TH17cells were similarly blunted. However, splenicTH17cells were seemingly unaffected under chronic atrasentan administration with similar results under HSD and combination of HSD/Atrasentan (NSD: 2.9 1.4; Atrasentan: 8.6 1.6; HSD: 7.6 2.8; Atrasentan/HSD: 5.5 1.6, x104, p>0.05). These findings suggest that HSD or ETA receptor activation may regulate T cell responsiveness in a tissue‐dependent manner.

Support or Funding Information

U01HL117684, P01HL69999, AHA SFRN 24450002, P01HL136267, 1 F31 HL151264‐01
Disturbed blood flow acutely increases endothelial microparticles and decreases flow mediated dilation in patients with heart failure with reduced ejection fraction
Authors: Sales Allan Kluser , Oliveira Thiago O. , Mendonca Gustavo S. M. , Fonseca Guilherme , Braga Pedro G. S. , Rocha Helena N. M. , Rocha Natalia G. , Lima Marta F. , Negrao Carlos E. , Nunes Alves Maria Janieire N.
Published: April 2020

Introduction

Disturbed blood flow, characterized by high retrograde and oscillatory shear rate (SR), is associated with a proatherogenic phenotype. The impact of disturbed blood flow in patients with heart failure with reduced ejection fraction (HFrEF) remains unknown. We tested the hypothesis that acute elevation to retrograde and oscillatory SR provoked by local circulatory occlusion would increase endothelial microparticles (EMPs) and decrease brachial artery flow‐mediated dilation (FMD) in patients with HFrEF.

Methods

Eighteen patients with HFrEF aged 55±2 years, with left ventricular ejection fraction (LVEF) 26±1%, and 14 healthy subjects aged 49±2 years with LVEF 65±1 randomly underwent experimental and control sessions. Brachial artery FMD (Doppler) was evaluated before and after 30 min of disturbed forearm blood flow provoked by pneumatic cuff (Hokanson) inflation to 75 mm Hg. Venous blood samples were collected at rest, after 15 and 30 min of disturbed blood flow to assess circulating EMP levels (CD42b−/CD31+;flow cytometry).

Results

At rest, FMD was lower in patients with HFrEF compared with healthy subjects (p<0.001), but blood flow patterns and EMPs had no differences (p>0.05). The cuff inflation provoked a greater retrograde SR both groups (p<0.0001). EMPs responses to disturbed blood flow significantly increased in patients with HFrEF (p=0.03). No changes in EMPs were found in healthy subjects (p>0.05). Disturbed blood flow decreased FMD both groups. No changes occurred in control condition.

Conclusion

Disturbed blood flow acutely decreases FMD and increases EMP levels in patients with HFrEF. These findings suggest that patients with HFrEF are vulnerable to blood flow disturbances.

Support or Funding Information

FAPESP Grants (2014/11671‐6 and
2017/25613‐6)
Impact of Premedical Anatomy Experience, its Perceived Benefit, and Study Effort on Student Performance in Medical Gross Anatomy
Authors: Goldenstein Allison J. , Howell Megan M. , Pazdernik Vanessa K. , Houser Jeremy J.
Published: April 2020

Predicting medical student success in Gross Anatomy could be beneficial for implementing early interventions for those who may struggle. In our earlier studies, previous anatomy experience did not clearly explain differences in performance in Gross Anatomy. These previous studies failed to assess student perception of previous courses and how this may affect study habits. This current study investigated the effect of study effort in conjunction with premedical anatomy experience and the student‐perceived benefit of that course on their success in the medical Gross Anatomy course.

Two surveys were administered to students. The first survey was distributed during orientation week prior to semester one inception. This survey covered the number, type, and level of anatomy courses taken prior to matriculation into medical school. Additionally, students were asked to rate the perceived benefit of the course for mastery of anatomical concepts and the potential benefit for success in medical Gross Anatomy. The second survey was administered during the first week of second semester, asking their average study efforts dedicated to lecture‐based and laboratory‐based examinations. These responses were matched with each student’s semester, lecture, and laboratory grades from the first semester of Gross Anatomy. Model selection methods determined final predictors for overall semester grade and grade on the first lecture exam. Tests for linear trends across increasing degrees of perceived benefit of their previous anatomy experience on their success in the Gross Anatomy course were performed.

The best predictors were lecture effort and the perceived benefit of their previous anatomy experience on their success in the Gross Anatomy course. Given the same amount of effort, the more beneficial the student believed their previous anatomy courses were toward future success in the medical Gross Anatomy course, the better the Gross Anatomy course grade. This was true for those who responded that the course was ‘slightly beneficial’ (mean=0.81, SE=0.009) up to ‘very beneficial’ (mean=0.86, SE=0.007; P<.001). Likewise, controlling for the overall semester grade, among students who responded that the course was at least ‘slightly beneficial’ up to ‘very beneficial’, the lower the average study effort given on lecture examination study (‘slightly beneficial’ mean effort=0.87; ‘very beneficial’ mean effort=0.82; P=.002). These results were similar for the first lecture examination grade.

Prior anatomy experience alone does not predict success in medical Gross Anatomy. However, a student who perceives more benefit from a previous anatomy course will generally put in less effort to attain a particular grade in Gross Anatomy. As this model of prediction is the same for the first lecture exam, early interventions may be implemented to minimize poor performance.
Biophysical Characterization of CowN from Gluconacetobacter diazotrophicus
Authors: Bretzing Kevin , Owens Cedric , Verde Christine Lo , Espinoza Alejandro
Published: April 2020

Gluconacetobacter diazotrophicus is a nitrogen fixing bacterium that associates with plants and plays a crucial part in providing fixed nitrogen to many crops such as sugar cane. The enzyme responsible for reducing atmospheric nitrogen to ammonia is nitrogenase. The presence of carbon monoxide gas will inhibit nitrogen fixation by nitrogenase. Many diazotrophs have a mechanism of protecting nitrogenase in vivo. In G. diazotrophicus, a protein called CowN protects nitrogenase from inhibition by carbon monoxide. Here, we present structural and biophysical characterization of CowN. Following heterologous expression of CowN in Escherichia coli and purification by affinity and size exclusion chromatography, CowN is found in two different oligomeric states; monomer and large soluble aggregate. Dynamic light scattering indicates the monomer has a radius of 1.9 nm, whereas the aggregate is larger than 5 nm. While the monomer protects nitrogenase from carbon monoxide inhibition, the aggregate does not. To determine what causes CowN aggregation and if it represents a physiologically relevant process, we investigated the mechanism of CowN aggregation. We discovered that high salt and protein concentration increase the propensity of CowN to aggregate. We further determined that under reducing conditions the monomeric state is preferred, suggesting that oxidation of CowN’s single cysteine residue plays a role in aggregation. Together, the data suggests that CowN may be most active under oxidizing conditions and, further, that aggregation might represent a mechanism to inactivate the enzyme at high concentration.

Support or Funding Information

This research was supported by the Chapman Center for Undergraduate Excellence, USDA‐NIFA (Grant no. 2015‐67012‐22895) and the National Science Foundation (Grant no. 1905399).
The Effect of Gas6‐Axl Double Knockout on Satellite Cell Proliferation and Skeletal Muscle Regeneration After Injury
Authors: Mervis Matthew J. , Matsumura Marc S. , Olsen Zachary E. , Hirschi-Budge Kelsey M. , Reynolds Paul R. , Arroyo Juan A. , Thomson David
Published: April 2020

Growth arrest‐specific 6 (Gas6) is an extracellular signaling molecule that is known to play a role in cell proliferation, inflammation and other processes through its interaction with Tyro3, Axl, and Mer receptors, but little is known about its role in skeletal muscle. The purpose of this study was to examine the effects of Gas6‐Axl double knockout (dKO) in skeletal muscle recovery after injury. Muscle injury was produced by injecting BaCl2 into tibialis anterior (TA) muscles of wild type (WT) and dKO mice. Muscle tissue was harvested 3, 7, and 14 days after injury. Gas6 and Axl expression over the injury timecourse was characterized by western blotting and ELISA. Muscle damage and cross‐sectional fiber areas were assessed on hematoxylin and eosin (H&E) stained sections. Muscle satellite cell content and proliferation were assessed by Pax7/Ki‐67 staining. We found that both Gas6 and Axl protein expression increased substantially after injury in WT muscles, peaked at 3 days post injury, then declined toward basal levels thereafter. TA mass was lower for dKO muscles 14 days after injury, but fiber CSA was elevated in dKO muscles, suggesting a deficit in fiber number leading to compensatory hypertrophy of existing fibers. Pax7‐positive satellite cell number was unaffected by genotype in uninjured control muscles, but was ~3X higher in WT vs. dKO muscles at 3 days post injury, suggesting that the rate of satellite cell proliferation was suppressed or delayed by dKO. These results indicate that Gas6‐Axl signaling plays a role in regulating skeletal muscle regeneration after injury. Impaired Gas6 function may be linked to excess hypertrophy and loss of muscle mass after injury.

Support or Funding Information

This work was supported by a grant from the Flight Attendant’s Medical Research Institute (FAMRI) and a BYU Graduate Research Fellowship.