bluuebunny/crossref_metadata_2025 huggingface dataset:
What Is the Mechanism Underlying Pressure‐induced Chronotropy in Collecting Lymphatic Vessels?
Authors: Davis Michael J. , Li Min , Jaggar Jonathan H. , Zawieja Scott C.
Published: April 2020

The rhythmic contractions of lymphatic smooth muscle (LSM) cells enable lymph propulsion against adverse pressure gradients. These contractions are initiated by LSM action potentials (APs), generated by an ionic pacemaker, whose rate is sensitive to the transmural pressure difference. We recently showed that SM‐specific deletion of the Ca2+‐activated Cl− channel, anoctamin1 (Ano1), reduced the basal AP firing rate of mouse inguinal‐axillary lymphatic (IALs) vessels to ~25% of normal and abrogated pressure‐induced chronotopy. However, IALs are efferent vessels that show only a modest (2‐fold) increase in contraction frequency over the presumed physiological pressure range 0.5 – 8 cmH2O. In contrast, popliteal afferent lymphatics (PLs) exhibit an 8‐fold increase in chronotropy from 0.5 – 5 cmH2O and SM‐specific deletion of Ano1 attenuates but does not abolish their pressure‐induced chronotropy. Here we asked: 1) what other ionic mechanisms participate in pressure‐induced chronotropy of PLs? 2) what accounts for the residual, pressure‐independent pacemaking in IALs and PLs after Ano1 deletion?

To address these questions we measured the frequency‐pressure relationship (F‐P) of cannulated, pressurized PLs from control mice and mice deficient in key mechanosensitive and second‐messenger‐gated ion channels implicated in arterial pressure sensing. SM‐specific deletion of the mechanosensitive cation channel Piezo1 had no significant effect on F‐P. Global deletion of the osmosensitive cation channel TRPV4, or the DAG‐gated cation channels TRPC3 or TRPC6, or both, had no significant effect on F‐P. TRPM4−/− mice were not available, but the selective TRPM4 inhibitors, CBA and NBA, had minimal effects on F‐P. SM‐specific deletion of the cation channel TRPP1 (Pkd2) had no significant effect on F‐P. Deletion of T‐type Ca2+ channels Cav3.1 and/or Cav3.2 (previously implicated in lymphatic pacemaking), had no significant effect on F‐P. However, SM‐specific deletion of IP3R1, the only IP3R isoform expressed in mouse LSM, produced a phenotype similar to Ano1 deletion: a reduction in basal frequency and blunting of the F‐P relationship, suggesting a pressure‐induced IP3 generation downstream from mechanosensitive G‐protein(s). We then tested the prominent arterial SM mechanosensitive GPCR, AT1R. The inverse agonist losartan had no effect on F‐P nor did global knock out of AT1bR, the only isoform expressed in mouse LSM. In conclusion, our results point to a GPCR mechanosensing mechanism, independent of AT1R, that generates IP3 in a pressure‐sensitive manner to activate Ano1 and regulate the cycling rate of the ionic pacemaker in lymphatic smooth muscle.

Support or Funding Information

NIH‐HL‐122578
Breathing and Respiratory Muscle Function in Senescent Wild‐type and Ts65Dn Male Mice
Authors: Sheekh-Nuur Muno M. , Rodd Maeghan C. , Receno Candace N. , Burns David P. , DeRuisseau Keith C. , Cunningham Caitlin M. , DeRuisseau Lara R.
Published: April 2020

Down Syndrome (DS) results from trisomy 21 and is the most common cause of mental retardation in the US. People with DS display reduced muscle tone and cardiorespiratory complications that may contribute to breathing disturbances. Ts65Dn (Ts) mice, a model of DS, exhibit increased apnea frequency at 3 months of age (7±9 vs. 36±28; wild‐type (WT) vs. Ts) that persists at 6 (19± 7 vs. 77±58) and 12 months (5±6 vs. 64±77; p<0.001 for age, strain, and age by strain). People with DS have increased life expectancy and it is unknown how breathing changes across the lifespan in this population. We tested the hypothesis that ~20‐month‐old Ts mice would have altered ventilation compared to age‐matched WT mice. To test this hypothesis, unrestrained barometric plethysmography was used to quantify breathing frequency (breaths/min), tidal volume (VT; mL), VT/inspiratory time (VT/Ti; mL/sec), minute ventilation (VE; mL/min) and ventilatory equivalent for CO2 (VE/VCO2) in 18 to 22‐month‐old male WT (n=12) and Ts (n=13) mice. Data Sciences International Ponemah software was used to analyze flow tracings during exposure to air (20.93% O2, balanced N2). Data are expressed as mean±S.D., WT vs. Ts. A main effect of strain was uncovered using MANOVA (p=0.033). Breathing frequency (171±33 vs. 136±41 breaths/min; p=0.025) and VE (74.6±25.7 vs. 50.3±15.5 mL/min; p=0.010) approached the a priori cutoff (Bonferroni correction, p<0.01). VT (0.439±0.112 vs. 0.377±0.075 mL/breath; p=0.123) and the ratio of VE/VCO2 (45.5 ±22.0 vs. 42.4±19.8; p=0.715) were not different. VT/Ti (3.65±1.03 vs. 2.49±0.759 mL/sec; p=0.004) was reduced in Ts vs. WT, which suggests Ts mice have a reduced ventilatory drive. Since people with DS demonstrate evidence of muscle weakness, we examined diaphragm (DIA) and sternohyoid (SH; representative pharyngeal dilator) muscle function in WT (DIA n=10, SH n=9) and Ts (DIA n=7, SH n=7) mice. Twitch (2.2±1.1 vs 2.3±1.0 N/cm2; p=0.835) and tetanic (11.2±7.3 vs. 11.6±4.8 N/cm2; p=0.900) force, and the force frequency‐relationship (p=.766) for SH muscle were equivalent between groups. Similarly, no differences were observed for DIA twitch (6.0±2.7 vs. 5.2±2.1 N/cm2; p=0.438) and tetanic (20.7±6.0 vs. 19.9±2.8 N/cm2; p=0.758) force, or the force‐frequency relationship (p=0.339). Diaphragm function was preserved in aged Ts mice which is in line with similar VT between groups. These data reveal the increased propensity for apnea in Ts mice is independent of upper airway muscle weakness and may alternatively be of neural origin. Current studies are examining motor control of the upper airway in Ts mice.

Support or Funding Information

This work was funded by 1R15HD076379 (LRD), 3R15HD076379 (LRD). Additional support was granted by the Le Moyne College McDevitt Center Undergraduate Research Fellowship in Natural Science (MS‐N, MR), and the Le Moyne College Biological Sciences Department.
CHARACTERIZING THE LEPTIN‐INDUCED ACTIVATION OF PRE‐SYMPATHETIC CARDIOVASCULAR AND METABOLIC‐RELATED HYPOTHALAMIC NEURONS IN MICE
Authors: Hillard Kynlee Jo , Lindsey Stacia Jayce , Fraley Stephanie Kate , Peterson Jonathan Micheal , Zahner Matthew Ralph
Published: April 2020

Obesity has risen 75% in the United States since 1980 and an estimated 80 million American adults are considered obese. Obesity activates the sympathetic nervous system and is associated with neurogenic hypertension. Leptin is an obesity‐related neuropeptide released from fat cells which reduces appetite and increases metabolism. Leptin activates metabolic and cardiovascular responsive pre‐sympathetic neurons within the hypothalamus. Although leptin increases metabolism and curbs appetite, it is also reported to increase blood pressure and heart rate. Considering that the common goal of obesity treatments is to diminish the cardiovascular‐related co‐morbidities increases in blood pressure and heart rate are unacceptable side effects for any potential treatment. Thus, a better understanding of the role hypothalamic sites involved in obesity‐related hypertension is necessary for the development of successful treatments. In this study, we tested the hypothesis that leptin activates pre‐sympathetic hypothalamic neurons that control either metabolic (raphe pallidus‐projecting) or cardiovascular activity (RVLM, rostroventrolateral medulla‐projecting). First, to identify leptin receptor expressing neurons we created a line of transgenic reporter mice using the cre‐lox recombination system to express tdTomato under the control of the leptin (ObRb) receptor gene. Then, to determine if exogenous leptin treatment activates pre‐sympathetic metabolic or cardiovascular‐related hypothalamic neurons we performed neuroanatomical tracer studies in these mice. Metabolically‐related neurons were identified by microinjection (50 nl) of green FluoSpheres™ (505/515) into the raphe nucleus and cardiovascular‐related neurons were identified by microinjection (50 nl) of magenta FluoSpheres™ (660/680) into the rostroventrolateral medulla (RVLM). We then treated these mice with an acute dose of leptin (25 μg/mouse IP) and then performed fluorescence immunohistochemical labelling to identify leptin‐induced neuronal activation of cFos, a marker of neuronal activation. Using this approach we identified both a population of RVLM‐projecting and a population of raphe‐projecting leptin expressing neurons. These data provide additional information to suggest that the leptin‐induced increases in cardiovascular and metabolic activity is mediated in part by activation of pre‐sympathetic hypothalamic neurons.

Support or Funding Information

ETSU RDC 19‐030 (Zahner MR) NHLBI R15 HL145645‐01 (Zahner MR)
Nuclear Phosphoinositide 3‐kinase delta (PIK3CD) Increases with Conditional Deletion of Hepatocellular Integrin Linked Kinase
Authors: Martucci Nicole Jenna , Cotner Mary-Clare , Bhushan Bharat , Mars Wendy M. , Michalopoulos George K.
Published: April 2020

Introduction

The extracellular matrix (ECM) is important for survival, differentiation, and normal functioning of cells within the liver; integrins are key signaling receptors in this process. Previous data from our lab has shown that with hepatocyte specific knockout of integrin linked kinase (ILK KO), there is hepatocyte proliferation, increased matrix deposition, unorganized biliary cell/ductal proliferation, and possible transdifferentiation of hepatocytes to cholangiocytes; this led us to investigate the signaling pathways downstream of hILK KO that might be responsible for the observed phenotype, and we uncovered a potential central role for Phosphoinositide 3‐kinase (PI3K) delta (PIK3CD), a variant of PI3K that is considered to be leukocyte specific.

Methods

Mice with germ‐line loss of hepatocellular ILK were generated by crossing double floxed ILK mice (ILKfl/fl) with mice expressing cre recombinase under an afp enhancer‐albumin promoter (Cre+/−) to obtain ILKfl/fl:Cre+/− (ILK KO) and ILKfl/fl:Cre−/− (WT). Data array analyses were performed on livers from 7‐ and 14‐week old chronic ILK KO and WT mice. RNAs of interest were identified and then further examined by western blot analyses, immunohistochemical staining, and induction of acute loss of ILK using AAV8 cre in ILKfl/fl mice.

Results

Examination of the 7‐week RNA array analysis suggested a central role for PIK3CD in the observed hILK phenotype. Unexpectedly, using immunohistochemistry we detected prominent staining of PIK3CD protein in ILK KO hepatocytes. The presence of PIK3CD was verified in cultures of wild type hepatocytes where inhibition of PIK3CD resulted in a decrease in both pAKT (a downstream target) and Cyclin D1. Western blot analyses of whole livers also uncovered a size variant that is only observed in isolated nuclei. Additionally, at 7 days after the acute removal of ILK, we can observe staining for the cholangiocyte specific marker, CK19, in hepatocytes as well as strong PIK3CD staining in regions with spontaneous duct formation.

Conclusion

Our RNA and protein data from ILK KO mice have 1) revealed PIK3CD, a leukocyte specific protein, to be hepatocellular 2) shown that a size variant of PIK3CD exists in the nuclei of hepatocytes and 3) uncovered a potential relationship between PIK3CD in hepatocytes and their transdifferentiation to biliary cells. Currently, we are using an in vitro model of trans‐differentiation in an attempt to further explore these preliminary findings.

Support or Funding Information

CATER NIH T32 EB001026.
Targeting estrogen non‐genomic signaling in a three‐dimensional (3D) in vitro model of inflammatory breast cancer
Authors: Negrón Dalissa , Peterson Esther
Published: April 2020

Inflammatory Breast Cancer (IBC) is the most aggressive form of breast cancer with a low overall 5‐year survival rate of ~30–60%. To this date the molecular signature associated with IBC is not completely characterized, which lead to a lack of effective targeted therapeutics, especially for those patients that account for the 20–40% of triple‐negative (TNBC) IBC cases. Several studies established that molecular signature of IBC shows important differences from the other breast cancer subtypes, such as overexpression of ErbB receptors, RhoC GTPase and Cox‐2, E‐cadherin aberrant expression, and loss of WISP3 among others. However, these molecular alterations do not explain completely the aggressive and rapid IBC phenotype. Recently, several studies have shown that estrogen can exert non‐genomic effects in triple‐negative IBC and other TNBCs, mediated by the expression of alternate estrogen receptors, including ERα36 and GPR30. Estrogen non‐genomic signaling refers to the rapid action of these alternate estrogen receptors that upon estrogen binding activates protein‐kinase cascades in the cytoplasm. We hypothesize that estrogen can elicit a specific non‐genomic signaling cascade that promotes the acquisition of aggressive oncogenic phenotypes in IBC and that targeting this pathway can be an effective anticancer therapy for IBC. To test this hypothesis, we are going to use pharmacological inhibition of ERα36 and GPR30 using antagonists against these receptors alone or in combination with EGFR inhibition. In parallel, we are going to use siRNA to down‐regulate the expression of these receptors. The first approach is to determine if inhibiting estrogen nongenomic signaling by targeting the alternate receptors affect cell proliferation in a 3D colony formation assay using Matrigel. Preliminary observations showed that cells treated with E2 develop larger colonies (increase proliferation) when compared to vehicle control. Inhibiting GPR30 alone have a slight reduction in cell proliferation compared to E2, but when combined with an EGFR inhibitor (Gefitinib) showed a synergistic effect by decreasing proliferation further that by Gefitinib alone. Ongoing experiments include the effects of Icaritin (modulator of ERα36) and the siRNA experiments to test the effects in cell proliferation, migration and invasion. This study will provide the foundation for further characterization of the role estrogen non‐genomic signaling in the progression of IBC and to test its potential as a therapeutic target.

Support or Funding Information

Support by RISE Program Grant number: 5R25GM061151‐18
How to be a Giant: Hypermetric Scaling of the Leg in Cockroaches and Scarab Beetles Suggests Oxygen Transport to the Legs Limits Maximal Insect Size
Authors: Harrison Jon F. , Wagner Julian M. , Aivazian Vahan , Duell Meghan E. , Klok C. Jaco , Weed Michael , Munoz Elyse , Fezzaa Kamel , Socha John Jacob , VandenBrooks John M.
Published: April 2020

Understanding the causes and consequences of the evolution of large or small body size remains one of the important challenges of evolutionary biology. Insects have an unusual respiratory system, transporting oxygen in the gas phase via air‐filled tracheae. The possession of this unique system has been hypothesized to limit insect size, but how the morphology of the tracheal system changes as insects vary in size is still poorly known. Based on comparisons with vertebrates, we expect either geometric isometry in scaling (as found in lungs) or hypometric scaling (as found in vertebrate capillaries), with the latter matching the hypometric scaling of metabolic rate found for most animals. We tested these expectations by measuring the 3D morphology of tracheal systems of ten species each of cockroaches and scarab beetles. In both groups, body mass varied by more than an order of magnitude across species, which included some of the largest extant species of insect. Cockroaches were imaged at Argonne National Laboratory using synchrotron x‐ray microcomputed tomography (SR‐μCT), and scarab beetles were imaged at Virginia Tech using tabletop μCT. We measured the fraction of body volume occupied by the tracheal system for the major body compartments. In general, the tracheal system scaled isometrically in the head, thorax, and abdomen. However, within the legs, tracheal volume scaled strongly hypermetrically, resulting from an increase in number (in roaches) and size (in scarabs) of tracheae and/or air sacs. These data strongly suggest that evolution of larger species of insects requires increased relative investment in the tracheal system of the legs to overcome the challenges of long‐distance transport to muscles and nerves located in these long, blind‐ended structures, limiting the maximal size of insects.

Support or Funding Information

Supported by NSF IOS 1122157 and IOS 1558052.
Inhibition of Sodium Glucose Cotransporter‐2 Improves Cardiac Efficiency During Regional Myocardial Ischemia Independent of Sodium/Hydrogen Exchanger‐1
Authors: Baker Hana , Tune Johnathan , Mather Kieren , Blaettner Bianca , Clark Hannah , Kowala Mark , Fliegel Larry , Goodwill Adam
Published: April 2020

Recent clinical outcome studies demonstrate that inhibitors of the type 2 sodium glucose cotransporters (SGLT2i) significantly reduce major adverse cardiovascular events and heart failure outcomes in type 2 diabetes mellitus. The mechanisms underlying these clinical benefits remain to be elucidated. We tested the hypothesis that SGLT2i improves cardiac function and efficiency during acute, regional ischemia/reperfusion injury via inhibition of cardiac sodium/hydrogen exchanger‐1 (NHE‐1). Lean swine (~50 kg) were anesthetized, a thoracotomy was performed, and perivascular flow transducers were placed around the left anterior descending and circumflex coronary arteries. A pressure‐volume catheter was then inserted into the left ventricle. Swine received a 15–30 min (0.05–0.1 mL/min) infusion of vehicle (DMSO), the SGLT2i canagliflozin (30 μM), or the NHE‐1 inhibitor (NHE‐1i) cariporide (1 μM) immediately prior to a 60 min occlusion of the left circumflex artery followed by a 2‐hour reperfusion period. Mean arterial pressure decreased from ~91 ± 3 mmHg at baseline to ~62 ± 3 mmHg following 60 min of myocardial ischemia in control, canagliflozin and cariporide treated swine. Heart rate averaged ~78 beats/min at baseline and throughout the ischemic period, however, SGLT2i significantly increased heart rate during reperfusion compared to control and NHE‐1i treated swine. SGLT2i was associated with a significant increase in left ventricular end diastolic volume (P = 0.02) that corresponded with a significant increase in stroke volume (P = 0.03) and cardiac efficiency (P < 0.001) relative to control animals. End diastolic volume, stroke volume, and efficiency were not significantly affected by ischemia/reperfusion injury in either the control or NHE‐1i treated animals. Additional fluorometric assays of antiport‐deficient Chinese hamster ovary (AP‐1) cells transfected with wild‐type NHE‐1 showed dose‐dependent inhibition of NHE‐1 activity by cariporide (IC50 = 0.11 mM), while canagliflozin had minimal effect on overall activity. This investigation demonstrates that SGLT2i improves cardiac function and efficiency during acute, regional ischemia in healthy swine. However, these data argue against an important role of NHE‐1 inhibition in mediating the beneficial cardiac effects of SGLT2i.

Support or Funding Information

Eli Lilly and Company
Cyclophilin D Sequesters PPARα in Mitochondria to Impair Fatty Acid Oxidation in Cisplatin‐induced Acute Kidney Injury
Authors: Padanilam Babu J. , Noh Mi Ra , Ha LiGyeom , Kim Jinu , Jang Hee-Seong
Published: April 2020

Objective

Kidney is a highly metabolic organ and ATP depletion is critical to acute kidney injury (AKI). Mitochondrial fatty acid oxidation (FAO) is the preferred energy source of the kidney and is inhibited during AKI. A pivotal role for the mitochondrial matrix protein cyclophilin D (CypD) in regulating overall energy metabolism is being unraveled. We hypothesize that mitochondrial interaction between CypD and PPARα in renal proximal tubule (PT) where is highly susceptible to acute stimuli modulates FAO in cisplatin‐induced AKI (cisplatin AKI).

Methods

Using genetic and pharmacological intervention and protein‐protein interaction studies, we investigated whether PT‐CypD modulates FAO in cisplatin AKI through mitochondrial CypD‐PPARα binding and its sequestration.

Results

Cisplatin injury resulted in histological and functional damage in the kidney, along with downregulated mitochondrial FAO, increased intrarenal lipid accumulation and declined ATP level. However, PT‐specific deletion of CypD protected the malevolent effects. Immunoprecipitation and BioID methods demonstrated mitochondrial translocation of PPARα and its binding to CypD and sequestration. This led to inhibition of nuclear translocation of PPARα and reduced transcription of FAO genes during cisplatin AKI. Genetic or pharmacological inhibition of CypD suppressed mitochondrial CypD‐PPARα binding in cisplatin AKI, preventing the impairment of FAO and intracellular lipid accumulation.

Conclusions

These results uncover a novel mechanism by which mitochondrial interaction between CypD and PPARα impairs FAO in cisplatin AKI. Targeting their interaction may be a potential therapeutic strategy to prevent energy depletion and cell death in AKI.

Support or Funding Information

This work was supported by NIH grants DK‐116987, DK‐120533 and American Heart Association (AHA) Grant in Aid 15GRNT25080031 (BJP), AHA postdoctoral fellowship Grant 15POST25130003 (HSJ), and grants (NRF‐2016R1C1B2012080 and NRF‐2019R1F1A1041410) from the National Research Foundation of Korea (JK).
Probing the unusual functional interaction between mGluR1 and mGluR5.
Authors: Kammermeier Paul J. , McCullock Tyler W.
Published: April 2020

Metabotropic glutamate receptors (mGluRs) are class C G protein coupled receptors with widespread expression in the central nervous system. Unlike many GPCRs, mGluRs exist and function as obligate, disulfide linked dimers. While all mGluRs can form homodimers, and many can heterodimerize, we have recently published a paper demonstrating that while mGluR1 and 5 may reside in close proximity to each other, and can interact functionally, this interaction does not appear to be due to canonical heterodimerization. Pharmacological evidence supporting this conclusion is largely pharmacological. To ask whether the observed interaction was due to a physical interaction between receptors rather than integration of signaling effects, mutations in each receptor were introduced that uncouple the receptors from G protein signaling, presumably without affecting ligand binding or associated conformational changes. We found that mGluR1 co‐expressed with a double point mutant of mGluR5, Y64A F768D (which right‐shifted the glutamate response and uncouple from G proteins, respectively), the glutamate response of mGluR1 was right shifted. When mGluR1Y74A, a mutant with a 300 fold right‐shifted response, was expressed with mGluR5 F768D, the response was left shifted. Expression of the signaling dead mGluR5 F768D with mGluR1 also altered the pharmacological sensitivity profile, causing mGluR1 to lose sensitivity to 3‐MATIDA and gain sensitivity to MPEP. Together, these findings support the hypothesis that allosteric interactions between the receptors are responsible for the observed functional interactions.

Support or Funding Information

Supported by an institutional Drug Targets and Mechanisms pilot award.
Does Myocardial Steatosis Cause Left Ventricular Dysfunction?
Authors: Trojacek Darian , Oneglia Andrew , Chamseddine Houda , Jaffery Manall , Zaha Vlad , Szczepaniak Lidia , Cipher Daisha , Nelson Michael D.
Published: April 2020

Altered substrate metabolism is thought to contribute to dysfunction of obese and diabetic hearts; however, the exact mechanism leading to dysfunction remains incompletely understood. One increasingly popular theory involves lipid overstorage (termed steatosis) and lipotoxic injury to cardiomyocytes; however, these data have been derived almost entirely from pre‐clinical rodent models, with translational human research being far less developed; and often confounded by competing cardiovascular risk factors. Accordingly, we sought to study the acute influence of cardiac steatosis on cardiac function in otherwise healthy participants, using a unique 48‐hour fasting model. 7 young healthy men (24 ± 4 years)— free of cardiovascular, metabolic or neurological disease— volunteered for this study. Myocardial triglyceride content (mTG) was measured using 1H magnetic resonance spectroscopy, and left ventricular function was measured using magnetic resonance imaging. Fasting was performed under the direct supervision of research nurses in the Clinical Research Unit (CRU) at the University of Texas Southwestern Medical Center. As expected, fasting for 48 hours caused mTG to increase in all participants (pre: 0.30 ± 0.14 % vs. post: 1.31 ± 0.93 %, p = 0.040). Despite multiple efforts to maintain cardiac preload during the fasting intervention, left ventricular end‐diastolic volume was significantly reduced (pre: 172.5 ± 22.7 mL vs. post: 155.7 ± 27.9 mL, p = 0.032) following the 48‐hour fast. Despite this marked reduction in cardiac preload, left ventricular peak strain and diastolic strain rate were preserved, while circumferential systolic strain rate (pre: −99 ± 11 %/s vs. post: −114 ± 5 %/s, p = 0.003), peak left ventricular twisting rate (pre: 47.8 ± 10.7 °/s vs. 71.0 ± 12.3 °/s, p = 0.012), and peak torsion (pre: 2.73 ± 0.58 °/cm vs. post: 3.36 ± 0.43 °/cm, p = 0.002) were all significantly elevated following the 48‐hour fast. We interpret these data to reflect heightened cardiac contractility, secondary to fasting induced activation of the sympathetic nervous system. Future studies are needed to test this hypothesis and control for this potential confounding variable, which may be otherwise masking steatosis induced cardiac dysfunction.
Sex differences in MAP kinase activation in the periaqueductal gray after morphine treatment
Authors: McCarty Ashley , Ram Akila , McDermott Max , Bobeck Erin N.
Published: April 2020

Morphine is a potent opioid analgesic, but its long term use can lead to negative side effects, including tolerance, which is a decrease in the effectiveness of the opioid. An area of active interest is looking into the molecular effects of chronic morphine treatment in the Periaqueductal gray (PAG), a brain region that controls descending pain modulation. One such molecular target within the PAG is extracellular‐signal regulated kinase 1/2 (ERK). Previous studies have shown that pharmacological inhibition of ERK enhanced morphine tolerance, indicating that ERK activity is associated with better responsiveness to morphine. The PAG is known to contain a heterogenous population of neurons including GABA and glutamate subtypes. However, which neurons ERK is activated in within the PAG following morphine tolerance is unknown. Further, there are known differences in PAG activity between male and female mice. These sex‐differences have not been well studied after morphine tolerance using acute pain tests. The purpose of this research is to investigate differences in ERK activation following morphine tolerance in male and female mice. We treated wild‐type male and female mice with morphine (10 mg/kg, i.p.) or saline for 5 days to induce morphine tolerance, following which both behavior and protein immunofluorescence were assessed. We observe sex‐specific differences in ERK activation levels and morphine antinociceptive tolerance in mice. We also assessed co‐localization of ERK with GABA and glutamate neurons after morphine tolerance. The study will help us understand the cell‐type specificity of kinase activation following morphine tolerance. Further this will give us more information about the nature of neurons that are contributing to sex‐differences in opioid functions within the PAG.
Role of Creatine Supplementation in Alleviating Hepatotoxicity Caused by Doxorubicin
Authors: Aljobaily Nouf A. , Viereckl Michael , Aljobaily Hend A. , Albertson Jammie A. , Han Yuyan , Hydock David
Published: April 2020

Background and aim

Doxorubicin is a chemotherapeutic agent that is widely used for several malignancies. It is known to have severe side effects such as hepatotoxicity, cardiotoxicity and increase in nitric oxide concentrations in skeletal muscles. On the other hand, creatine monohydrate is a supplementation used as a therapeutic agent and plays a significant role as regenerative agent. So far, there is not enough information about the effect of doxorubicin on the overall liver functionality and how creatine monohydrate helps in easing the hepatotoxicity; therefore, our aim is to investigate the role of creatine monohydrate supplementation in alleviating the hepatotoxicity caused by doxorubicin.

Methods

Sprague‐Dawley rats were fed rodent chow, 2% creatine, 4% followed by 2% creatine supplementation for our weeks; and 15 mg/kg doxorubicin was given once the day before they were sacrificed. Peripheral blood and liver were harvested and snap frozen. Serum chemistry was obtained to evaluate the liver function by looking at the levels of Lipemia, T‐Bilirubin, AST, ALP and ALT. Liver damage was evaluated by using Hematoxylin and Eosin staining; whereas liver fibrosis was evaluated by Sirius Red staining. RT‐PCR was performed to assess the gene expression of fibrotic genes, proliferative genes, oxidative stress and apoptosis‐related genes at the transcription level, and western blotting was performed to measure the gene expression at the protein level.

Results

There was no significant difference found in the serum chemistry in the treatment groups when compared to the control group. However, the histology showed an increase in inflammation and fibrosis in the doxorubicin group when compared to the control, where there was a decrease observed in the group treated with 4% creatine followed by 2% creatine. A significant increase in the gene expression of Bax, CK‐19 and Col1‐a1 has been observed in the 2% creatine + doxorubicin. Further, there was a decrease in the protein expression of CASP3 in the 2% creatine + doxorubicin and 4%/2% creatine + doxorubicin when compared to the doxorubicin group.

Conclusion

Based on the collected data and observed behavior of groups, we conclude that the gradual dose of creatine monohydrate supplementation does alleviate the hepatotoxicity caused by doxorubicin.

Support or Funding Information

Graduate Student Association at the University of Northern Colorado
The Effects of Incremental Maxillomandibular Advancement Surgery on Airway Morphology
Authors: Wolak Liliana , Shimizu Michael , Tassi Ali , Galil Kahdry , Beveridge Tyler S. , Wilson Timothy D.
Published: April 2020

Obstructive sleep apnea (OSA) is a growing epidemic, affecting up to 7% of the population. Characterized by noctural airway obstruction, OSA is associated with long‐term health effects. To treat this, maxillomandibular advancement surgery (MMA) may be used to expand the dimensions of the pharynx by advancing the maxilla and mandible. Despite positive outcomes, the underlying MMA mechanisms are poorly understood, nor are the ideal advancement parameters defined. The objective of the current study is to investigate the relationship between MMA advancement and resulting airway changes. MMA surgery was performed on five cadaveric specimens. The mandible and maxilla were incrementally advanced using KLS Martin distraction devices. Beginning at baseline, specimens underwent a CT scan at each subsequent 2mm advancement to 14mm (Siemens Medtronic O‐Arm Imaging System; 0.415×0.415×0.833mm,120 kVp, 32 mA, 240 mAs). Segmentation and analysis of changes in airway dimensions were performed in AMIRA (5.3.3, Mercury Computer Systems/3D Viz Group). Linear dimensional changes were measured using a digital ruler at pre‐defined anatomical borders. Superior and inferior boundaries of the airway were defined as the level of the hard palate and base of epiglottis respectively. Length was measured between these boundaries. Anterior/posterior (AP) limits were delineated by a plane perpendicular to the Frankfort horizontal at the base of the tongue and lateral (LAT) dimensions were determined by averaging widths at 25, 50, 75% of AP distance in the same plane. Airway volume was calculated at each 2mm advancement from baseline to 14mm. Normalized results, presented as percent change from baseline, indicate that at 14mm overall mean volumetric increase was 176.5±11.7% mean overall AP increase of 89.5±7.7%, and mean LAT increase was 105.4±8.0%. Maximal incremental changes for volume and AP occurred at 4mm advancement (34.2±23.7% and 21±17%, respectively). In contrast, maximal incremental change for LAT occurred at 10mm (26.5±29.8%). Simultaneously, total length decreased on average, 12.2±0.9% with the maximal decrease at 2mm (‐3.2±1.7%). Incremental morphological changes in the human airway during MMA indicates overall volumetric increase at 12.6% per mm advancement with maximal volumetric enhancement rates happening early in the surgery (17.1%/mm at 4mm). The mechanism of this change is bimodal with maximal rate of AP expansion occurring early (10.5%/mm at 4mm) while later maximal LAT expansion (13.3%/mm at 10mm). Ongoing analysis examines pharyngeal shape change and airflow resistance to better understand functional consequences of MMA.
Acute Pressor Responses in Mice Exposed to Early Life Stress
Authors: McPherson Kasi C. , Becker Bryan K. , Rhoads Megan K. , Jin Chunhua , Pollock David M. , Pollock Jennifer S.
Published: April 2020

Exposure to adverse childhood experiences, also known as early life stress, is an independent risk factor for the development of cardiovascular disease in adulthood. Previous studies have indicated ELS in rodents may induce impaired blood pressure regulation in response to various pro‐hypertensive stimuli. In the current study, we used maternal separation and early weaning (MSEW), a mouse model of early life stress, to determine the effects of acute behavioral stress on blood pressure regulation (n = 9/group). We utilized exposure to cage switch stress (CSS), a model of acute behavioral stress that subjects mice to a cage previously inhabited by other male mice. MSEW involves maternal separation 4h/day from postnatal days (PD) 2–5 and 8h/day from PD 6–16 then weaned at PD 17. Normally reared litters weaned at PD 21 were used as controls (CON). In normally reared CON and MSEW C57/BL6 male mice, telemetry transmitters were surgically placed with catheters in the carotid artery at 9 weeks of age. After recovery from surgery for 7–10 days, baseline measurements were recorded for 72 hours. Continuous 24 hour systolic and diastolic blood pressures (SBP and DBP) were similar between CON and MSEW mice (SBP: 125 ± 1 vs 121 ± 5 mmHg, p = 0.64; DBP: 89 ± 1 vs 91 ± 1 mmHg, p = 0.24). Additionally, exposure to MSEW did not significantly affect baseline day and night heart rate. We then subjected to CSS for 4 hours. In response to CSS, the peak change in SBP was significantly greater in MSEW mice compared to CON (39 ± 2 vs 27 ± 4 mmHg, respectively, p = 0.02). However, the total pressor response determined by area under the curve for SBP and DBP during the 30 minutes following onset of CSS and 30 minutes following the end of the 4 hour CSS exposure were similar between MSEW and CON mice. Heart rate significantly increased in both MSEW and CON mice compared to baseline (MSEW: 777 ± 10 vs 562 ± 20 bpm, p < 0.0001; CON: 776 ± 4 vs 575 ± 25 bpm, p < 0.0001) and thus the peak change in heart rate was not significantly different between groups (215 ± 18 vs 201 ± 23 bpm, p = 0.64). However, area under the curve analysis indicated MSEW exhibited a blunted heart rate recovery 30 minutes following the end of the 4 hours of exposure to CSS compared to CON (17998 ± 159 vs 18751 ± 287, respectively, p = 0.05). Given the changes in SBP and heart rate following CSS, these data indicate that MSEW mice have an exaggerated pressor response during exposure to acute stress. In conclusion, ELS may predispose exaggerated cardiovascular responses to stress later in life.

Support or Funding Information

Funded by NIH grants HL136267 and HL69999 to D.M.P. and J.S.P. and AHA grant 17SFRN33670697 to J.S.P.
Differential expression of renal urate transporters in male and female mice
Authors: Halperin Kuhns Victoria L. , Hoque Kazi Mirajul , Woodward Owen M.
Published: April 2020

Elevated urate (UA) levels in the serum (hyperuricemia, HUA) can contribute to the development of many diseases, including kidney stones, chronic kidney disease, cardiovascular disease, metabolic syndrome, and gout, the most prevalent form of inflammatory arthritis. Men are 5‐fold more likely to have HUA than women, however, this risk increases in women 4‐fold after menopause. This suggests premenopausal women are protected against developing HUA, but the mechanisms have yet to be elucidated. These differences are echoed in our hyperuricemia mice models. Previously, we found insertion of the orthologous human pathological mutation Q141K (Q140K in mice) in the UA transporter ABCG2 causes significant hyperuricemia in the male mice but not in the female mice. Paradoxically, we also observed female mice have lower fractional UA excretion than males, suggesting complex differences in the renal handling between males and females. We hypothesized that these differences were likely due to either UA transporter expression or regulation. To better understand the gene expression architecture of UA transporters in the mouse kidney, we compared gene expression in male and female wild type (WT) and Q140K ABCG2 mice. RNA‐Seq was performed followed by DESeq2 analysis to determine differentially expressed genes between males and females of each genotype. Targets of interest included the 12 transporter and 3 transcription factor (TF) genes that associate with serum UA levels in humans in recent genome wide association studies. We found small (0.49 – 2.1‐fold) but statistically significant changes in all the UA associated transporter genes, except ABCG2and SLC2A9. Specifically, female mice had lower transcript expression of UA transporters SLC22A12, SLC22A6, SLC17A1, and ABCA1. These genes have variants that associated with lower UA levels in human populations, consistent with the female mice potentially more efficiently excreting surplus UA. Females also had significantly lower levels of the 3 key urate associated transcription factors HNF1A, HNF4A, and HNF4G, providing a potential mechanism of differentiation. Interestingly, the Q140K mice DESeq2 analysis reveals 486 alterations in male Q140K kidney gene expression as compared to WT, including 5 potentially compensatory SLC transporters. None of these changes were observed in female Q140K mice, indicating that changes are the result of the elevation in SUA and not due to mutant ABCG2 protein. These findings strongly suggest female sex may confer protection from developing HUA and associated conditions. Further understanding this mechanism should lead to improved understanding of UA homeostasis and new insights into HUA treatment.

Support or Funding Information

NIDDK R01DK114091
Structure Activity Relationship Studies of Psychostimulant Synthetic Cathinones Reveal Nanomolar Antagonist Potency of α‐Pyrrolidinohexiophenone (α‐PHP) at Human Muscarinic M2Receptors
Authors: Chen Yiming , Canal Clinton E.
Published: April 2020

Synthetic cathinones (SCs), psychostimulant drugs of abuse, are β‐keto analogs of amphetamine that primarily target monoamine transporters as inhibitors or substrates. Certain pyrrolidine‐containing SCs, such as α‐Pyrrolidinohexiophenone (α‐PHP) and methylenedioxypyrovalerone (MDPV), are potent monoamine transporter inhibitors that appear to produce distinct toxic, autonomic—e.g., tachycardia and arrythmia—and neuropsychiatric—e.g., delirium and cognitive impairments—adverse events compared to N‐alkylated SCs and amphetamines, which are monoamine transporter substrates. Although the apparent differences in toxicity could be caused by unique pharmacodynamic effects at monoamine transporters, we hypothesized there is another mechanism. We focused on muscarinic receptors (MRs), as numerous MR antagonists are well‐characterized deliriants that also cause tachycardia. We conducted pharmacological assays assessing human M1, M2, M3, M4, and M5R activity of 10 pyrrolidine‐containing SCs, systematically evaluating the effect of the α‐carbon chain length of methylenedioxy‐containing and unsubstituted α‐propiophenone analogues. [3H]Scopolamine competition binding assays revealed that increasing the α‐carbon chain length of unsubstituted, but not methylenedioxy derivatives led to a stepwise enhancement in affinity (Ki) at all MR subtypes, with highest affinities typically observed at M2Rs. Relative to all other SCs tested, α‐PHP had the highest affinity at M2Rs, with a calculated pKi of 6.28 (Ki= 520 nM). We next assessed the M2R functional effects of α‐PHP using M2R—cAMP and M2R—β‐arrestin recruitment assays; α‐PHP behaved as an M2R competitive antagonist in both assays. α‐PHP antagonized oxotremorine‐elicited cAMP inhibition with an apparent pKb of 7.29 (Kb= 50 nM) and antagonized oxotremorine‐elicited β‐arrestin recruitment with an apparent pKb of 6.22 (Kb= 602 nM). Toxicology studies report that serum concentrations of commonly abused pyrrolidine‐containing SCs, including α‐PHP, can range from 713 to 2,619 nM. Thus, α‐PHP’s potency at M2Rs is physiologically relevant and likely contributes to its toxicity. Given the dominant function of M2Rs to slow heart rate, α‐PHP may cause tachycardia, in part, by inhibition of cardiac M2Rs. This inhibitory effect on the parasympathetic nervous system, in combination with sympathomimetic effects caused by monoamine transporter inhibition could explain the serious adverse cardiovascular events associated with certain pyrrolidine‐containing SCs.

Support or Funding Information

Funding: NIDA R21DA040907
Z‐line‐like Structures Are Distinct from α‐actinin‐rich Dense Bodies in Nematode Obliquely Striated Muscle
Authors: Ono Shoichiro , Nickoloff Emily , Ono Kanako
Published: April 2020

Obliquely striated muscles are present widely in soft‐bodied invertebrates and differ from cross‐striated muscles in that thin and thick filaments are staggered and obliquely aligned. Distinct Z‐lines or Z‐discs are absent from obliquely striated muscles. In general, thin filaments in obliquely striated muscles are considered to be anchored at the dense bodies, although there is no direct evidence of a linkage between the ends of thin filaments and dense bodies. In this study, we used body wall muscle of the nematode Caenorhabditis elegans as a representative obliquely striated muscle and found that the barbed ends of sarcomeric actin filaments are not localized to the dense bodies, but instead aligned to linear Z‐line‐like structures. In C. elegans, α‐actinin (ATN‐1) is one of the major components of the dense bodies. However, sarcomeric actin, as probed by anti‐actin antibody, phalloidin, or GFP‐actin, was only marginally co‐localized with ATN‐1. To specifically localize the barbed ends of sarcomeric actin filaments, we expressed GFP‐tagged CAP‐1, an α subunit of heterodimeric actin capping protein. GFP‐CAP‐1 was localized in a striated pattern filling gaps between dense bodies but was not co‐localized with ATN‐1. These “Z‐line‐like” structures of GFP‐CAP‐1 assembled normally in atn‐1‐knockout worms, indicating that ATN‐1 is not required for linear alignment of the actin barbed ends. In atn‐1‐knockout worms, major cytoplasmic portions of the dense bodies are absent, and only moderate disorganization of sarcomeric actin filaments are observed (Mulder et al., 2010). However, RNA interference of cap‐1 caused larval lethality and strong disorganization of sarcomeric actin filaments in embryonic body wall muscle cells. These results indicate that the barbed ends of sarcomeric actin filaments are aligned to Z‐line‐like structures, which assemble independently of dense bodies, and that capping protein plays an essential role in early sarcomeric assembly.

Support or Funding Information

Supported by NIH 5R01AR048615‐18.
The Cryptococcus neoformans capsule polysaccharide glucuronoxylomannan stimulates endothelial barrier permeability and the activation of Src and RhoA
Authors: Khan Mahrukh , Buttan Sagar , Masri Abigail , Martinez Luis , Dores Michael Robert
Published: April 2020

The human pathogenic yeast Cryptococcus neoformans is capable of crossing the blood brain barrier (BBB), causing over 220,000 cases of fungal meningitis annually. The BBB is comprised of specialized endothelial cells that control the flow of nutrients, signaling molecules and pharmacological agents into the brain. We have identified a novel mechanism for C. neoformans endothelial barrier disruption through the shedding of its capsule polysaccharide glucuronoxylomannan (GXM). GXM stimulates the breakdown of adherens junctions between endothelial cells and stimulates actin stress‐fiber formation, however the molecular mechanisms that control these cellular responses are not known. GXM is proposed to activate toll like receptors expressed on the surface of endothelial cells. Here we demonstrate that exposure of human brain endothelial cells (HBECs) to GXM induces the activation and internalization of TLR‐2 and TLR‐4. In addition, GXM stimulation leads to the activation of Src kinase, a signaling factor that is known to stimulate VE‐cadherin internalization and adherens junction disruption. We show that treatment of HBECs with GXM activates RhoA, a signaling effector that regulates actin stress fiber formation. Our current research seeks to identify new components of these signaling pathways and to determine whether inhibition of these signaling pathways can prevent GXM‐mediated endothelial barrier permeability.

Support or Funding Information

NIH NIAID: 1R01AI145559‐01A1
West‐Nile virus replicon particle entry requires the C‐terminal half of the carbohydrate‐recognition domain of the Dendritic cell‐specific ICAM‐3 grabbing non‐integrin related protein
Authors: Ogno Domenic G. , Menting Cody , Martinez Osvaldo
Published: April 2020

West‐Nile virus (WNV) is an arbovirus usually transmitted to humans via a mosquito vector. Infections commonly result in febrile symptoms while rare severe neuroinvasive cases may result in encephalitis or meningitis. Studies have shown that WNV infection efficiency is enhanced by expression of DC‐SIGNR on target cells, which normally do not express DC‐SIGNR. To investigate WNV tropism, we established 293T kidney epithelial cell lines that stably express vector, DC‐SIGNR and mutants of DC‐SIGNR that lack the entire carbohydrate‐recognition domain (CRD) or lack the C‐terminal half of the CRD. We demonstrate successful surface expression of DC‐SIGNR and its mutants from stably‐transfected 293T cells, but not vector‐transfected 293T cells. Further, we show that monoclonal antibody 120604 which binds specifically to the DC‐SIGNR CRD binds to DC‐SIGNR expressing 293T cells, but not to vector nor any of the DC‐SIGNR mutants expressing cells. Virus replicon particles (VRPs), replication‐incompetent viral particles containing necessary structural proteins for infection and a viral plasmid including a GFP reporter are used to safely and conveniently study viral entry. Entry assays using WNV (NY99) VRPs as well as a variant of WNV (NY99) which contains the beta‐lactamase enzyme show significant entry into DC‐SIGNR expressing cell lines, but not in controls that do not express DC‐SIGNR. Additionally, we show that WNV VRPs do not enter DC‐SIGNR expressing cells that lack the CRD or the C‐terminal half of the CRD suggesting that the C‐terminal half of the CRD is required for successful entry of WNV via DC‐SIGNR. In the future, WNV particle entry into DC‐SIGNR mutation (150(STOP), S139A, S108A) expressing cells will be tested.

Support or Funding Information

Winona State University student research and travel grants, Winona State University Foundation Special Projects