bluuebunny/crossref_metadata_2025 huggingface dataset:
Investigation of type 1 angiotensin II receptor activation induced gene expression changes in rat vascular smooth muscle cells
Authors: Hunyady Laszlo , Balla András , Szalai Laura , Gém Janka , Porkoláb Edit , Kovács Kinga , Szakadáti Gyöngyi
Published: April 2020

Angiotensin II (AngII) mainly acts through type 1 angiotensin II receptor (AT1R) to promote a broad variety of biological effects. The vascular smooth muscle cells are one of the main target of AngII and its stimulation activates numerous signaling pathways which could result in gene expression changes in these cells.

We carried out Affymetrix GeneChip experiments to analyze the effects of AngII stimulation on gene expression. More than 200 genes were upregulated in response to AngII stimulation of primary rat vascular smooth muscle cells in our experimental setup. We have selected several genes (DUSP5, DUSP6, DUSP10, Lmcd1, HbEGF, and endothelin) to further investigate the kinetics of the gene‐expression changes, and the possible signal transduction processes which lead to the altered expression changes after AngII stimulation. The results of the quantitative PCR measurements confirmed the AngII‐induced upregulation of the investigated genes. The maximal induction of the most genes appeared two hours after the AngII stimulation. Our data show that the upregulated genes can be stimulated through several, possibly parallel signal transduction pathways. We also examined the role of epidermal growth factor receptor (EGFR) transactivation in the upregulation of the investigated genes. According to our data the EGF receptor transactivation is not critical in response to AngII stimulation since silencing of EGF receptor did not significantly alter the AngII induced gene expression changes. Our pharmacological approach revealed that dasatinib, which is a potent inhibitor of ABL and Src family soluble tyrosine kinases, can significantly inhibit several gene expression changes. Interestingly, when we applied ABL kinase inhibitors, which have less potency toward Src tyrosine kinases such as imatinib or nilotinib did not evoke similar effects as dasatinib.

Our data can help to understand the details of AngII‐induced long term effects and the pathophysiology of AT1R, moreover it can help to develop potential interventions in symptoms when this receptor over functions such as vascular remodeling or atherosclerosis.

Support or Funding Information

This work was supported by the National Research, Development and Innovation Fund (NKFI K116954 and NVKP_16‐1‐2016‐0039).
Red Blood Cell‐resolved 3D Computational Modeling of Blood Flow in Physiologically Realistic Microvascular Networks
Authors: Bagchi Prosenjit , Ebrahimi Saman
Published: April 2020

Microvascular networks represent geometrically complex architecture due to constantly bifurcating and merging vessels. Moreover, blood vessels are not necessarily straight, and often highly winding. Mathematical models of blood flow in such networks have treated these vessels as 1D straight segments neglecting the geometric complexity and details. These models also do not consider the deformation and flow of individual red blood cell, and instead use empirical relations to prescribe blood viscosity and red blood cell distribution at a vascular bifurcation. As such, these models cannot predict hemodynamic variations over short time and length scales. To overcome this limitation, we have recently developed a high‐fidelity 3D, first‐principle based, fluid‐‐structure interaction model of blood flow in physiologically realistic microvascular networks. The model retains the exact geometric details of the vasculature, e.g., 3D nature of the blood vessels and bifurcations, and vessel curvature. Deformation and flow of each individual red blood cell as they flow through the vessels are exactly modeled. The model is based on the minimal assumptions: it does not assume any empirical viscosity law or bifurcation law; rather, these laws are predicted by the model. The model provides microscale details of hemodynamic quantities that are not readily available in experiments, and cannot be predicted by the traditional 1D network models, such as wall shear stress gradient and time‐dependent red blood cell partitioning at bifurcations.

Of particular interest is the role of red blood cell deformability on hematocrit distribution in the networks. Cell deformability is known to reduce in many diseases, such as sickle cell disease, malaria and diabetes. By using in vivo images of retinal microvasculature, we have performed simulation of network blood flow under varying cell deformability in such networks. The model shows that altered cell deformability significantly changes the cell distribution in the network, and subsequently the flow resistance in vessels. While some vessels show a significant increase in hematocrit, some show a significant decrease. Such a redistribution of hematocrit for diseased red blood cells may have a significant impact on tissue oxygen delivery. The simulation results are used to mechanistically explain the observed redistribution of hematocrit and vascular resistance.

Support or Funding Information

Funded by NSF
Use of Mass Spectrometry‐Based Proteomics to Investigate Protein Regulation Associated with COPD‐Related Muscle Dysfunction
Authors: Schiltz Sarah B. , Balnis Joseph , Jaitovich Ariel , Vincent Catherine E.
Published: April 2020

Chronic Obstructive Pulmonary Disease (COPD) is an umbrella term used to encompass several progressive lung diseases. A common comorbidity of COPD is skeletal muscle dysfunction with muscle mass loss, a condition shown to decrease quality of life and increase mortality rates independently of the level of pulmonary disease. Interestingly, rehabilitation exercises for the recovery of skeletal muscle integrity has been associated with improved disease outcomes. There are two mouse models that have been developed to mimic the COPD phenotype and demonstrate skeletal muscle wasting that recapitulates the disease seen in humans. A comprehensive mass spectrometry‐based proteomics study in one of these models (a mouse conditionally over‐expressing interleukin‐13, IL13TG) yielded a set of potential protein biomarkers for the degree of muscle dysfunction in skeletal muscle, including haptoglobin, hemopexin, and ceruloplasmin. Our present study will use mass spectrometry‐based proteomics to interrogate protein regulation in the EDL muscle of the other mouse model (a mouse that has lung‐specific expression of human matrix metalloproteinase‐1 (MMP‐1), an interstitial collagenase). We hypothesize that we will find a set of regulated proteins in common between the two studies, providing a set of conclusive biomarkers for COPD‐related skeletal muscle dysfunction that are independent of genetic modification. If our hypothesis is confirmed, we will then have a list of potential targets that can help facilitate early detection and/or potential interventions for COPD in humans to achieve improved patient outcomes.

Support or Funding Information

Part of the results reported herein have been funded Hartwick College and by NHLBI of the National Institutes of Health under the award number K01‐HL130704 (AJ), and by the Collins Family Foundation Endowment (AJ); NIH/NHLBI 5R01HL049426 (HAS); NIH/NHLBI PO1 HL114501(JAE); R01 HL115813 (CGL); NIH/NIGMS grant 1R01GM124133 (APA)
Melatonin plus Folic Acid Treatment Ameliorates Reserpine‐Induced Fibromyalgia: Evaluation of Pain, Oxidative Stress and Inflammation
Authors: Fusco Roberta , Siracusa Rosalba , D’Amico Ramona , Peritore Alessio Filippo , Cordaro Marika , Gugliandolo Enrico , Crupi Rosalia , Impellizzeri Daniela , Cuzzocrea Salvatore , Di Paola Rosanna
Published: April 2020

Background

Fibromyalgia is a chronic condition characterized by increased sensory perception of pain, neuropathic/neurodegenerative modifications, oxidative and nitrosative stress. An appropriate therapy is hard to find, and the currently used treatments are able to target only one of these aspects.

Methods

The aim of this study was to investigate the beneficial effects of a melatonin plus folic acid administration in a rat model of reserpine‐induced fibromyalgia. Sprague–Dawley male rats were injected with 1 mg/kg of reserpine for three consecutive days and later administered with melatonin, folic acid or both for twenty‐one days.

Results

Administration of reserpine led to a significant decrease in nociceptive threshold as well as significant increase in depressive‐like symptoms. These behavioral changes were accompanied with increased oxidative and nitrosative stress. Lipid peroxidation was significantly increased, as well as nitrotyrosine and PARP expression, while superoxide dismutase, nonprotein thiols and catalase were significantly decreased. Endogenously produced oxidants species are responsible of mast cells infiltration, increased expression pro‐inflammatory mediators and microglia activation.

Conclusion

Melatonin plus acid folic administration was able to ameliorate the behavioral defects, oxidative and nitrosative stress, mast cells infiltration, inflammatory mediators overexpression and microglia activation induced by reserpine injection with more efficacy than them separate administration.
Promoter Sequence Determinants of Transcription Initiation Rates in Zymomonas mobilis
Authors: Schiffman Allison , Ghosh Indro , Vera Jessica , Landick Robert
Published: April 2020

A modification to a naturally occurring ethanologenic pathway in a gram‐negative bacterium called Zymomonas mobilis has been engineered to produce isobutanol, an advantageous biofuel. However, low isobutanol yields remain an issue. Optimizing this pathway can be aided by a selective use of strong and weak promoters, but the determinants of promoter strength in Z. mobilis are unknown. Thus, a comprehensive database of promoter strengths will enable more accurate engineering of the isobutanol pathway and other synthetic pathways in Z. mobilis. Here, two libraries based on the rpoB‐P1 promoter with modifications to the −10 and −35 elements were designed. One of the libraries contained modifications at the bases that are highly conserved and the other contained modifications at the bases that are less conserved. In total, 20,480 different promoter sequences were assembled into plasmids and transformed into Z. mobilis where they drive expression of a synthetic RNA containing a randomized 15‐base pair barcode. DNA sequencing data confirm that the libraries have assembled as expected and that ~70% of transformants into Z. mobilis are from the designed libraries. This result has established the database of promoter sequences and corresponding barcodes. The quantity of barcoded‐RNA produced will now be used as a readout for strength of the associated promoter. Promoters selected from the complete library of promoter strengths will then be used to optimize the isobutanol pathway in Z. mobilis.
Structural and Functional Characterization of Phosphoglycosyl Transferases from Human Pathogens
Authors: Dodge Gregory , O’Toole Katherine , Bernstein Hannah , Seebald Leah , Allen Karen N. , Imperiali Barbara
Published: April 2020

Bacterial virulence and viability are strongly dependent on extracellular glycoconjugates including lipopolysaccharide (LPS), capsular polysaccharide (CPS), wall teichoic acid (WTA) and glycoproteins. While these glycoconjugates are diverse in structure and function, they share a common biosynthetic logic. For example, in Gram‐negative bacteria the biosynthesis of a variety of glycoconjugates are initiated on the cytoplasmic face of the bacterial inner membrane, with a phosphoglycosyl transferase (PGT) catalyzing the first membrane‐committed step of the pathway. PGTs transfer a phosphosugar from a nucleotide‐activated donor substrate onto a polyprenol phosphate such as undecaprenol phosphate (UndP). PGTs comprise two distinct superfamilies – polytopic, with multiple membrane spanning helices, and the more recently described monotopic, which contain an unusual re‐entrant membrane intercalating helix. To date, monotopic PGTs have only been observed in bacteria, and remain underexplored antimicrobial targets. In an effort to understand NDP‐sugar specificity and inform inhibitor design, a library of monotopic PGTs from human pathogens was expressed, purified, and subjected to structural and functional characterization. Both detergent and styrene maleic acid (SMA) copolymers were assessed for solubilization towards the purification and stabilization of these challenging protein targets. These efforts have led to purified samples of diverse monotopic PGTs for activity analyses using the UMP‐Glo ™ assay and crystallization screens, which have afforded reproduceable crystals for further investigation.

Support or Funding Information

R01GM131627, F32GM134576
Gut microbiome induces leaky gut and inflammation by activating miRNAs which in turn reduces tight junction proteins
Authors: Mishra Sidharth P. , Nagpal Ravinder , Razazan Atefeh P. , Wang Bo , Yadav Hariom
Published: April 2020

In obesity and diabetes, abnormal gut microbiota and increased gut permeability (leaky gut) are common risk factors which in turn contributes in increasing chronic low grade inflammation. However, precise mechanism(s) whether and how gut microbiota contributes in induction of leaky gut remain largely unknown. Here we, demonstrated that obese/ diabetic gut microbiota not only associated with leaky gut and inflammation, but it plays a causative role to induce leaky gut and inflammation. Using fecal microbiota transplantation, intestinal organoids and human epithelial Caco2 cell systems, we demonstrated that obese microbiota dramatically reduces intestinal barriers such as tight junction proteins (TJPs), that are associated with increased leaky gut and inflammation. Intestinal epithelial TJPs are important structural proteins that holds epithelial cells tight enough to regulate non‐specific permeability of microbial/ food and other antigens from intestinal lumen, thereby, keep a check on inflammatory reactions in the mucosal immune system. We also demonstrated that gut microbiota metabolites that are enriched in obese/ diabetic gut contributes in reducing the expression of TJPs and increasing leaky gut. To decipher the mechanism, we found that obese/diabetic microbiota derived metabolites increases an array of miRNAs (including miR‐10a and miR101a‐3p) that downregulates TJPs expression, which in turn results leaky gut and chronic inflammation in diabetes and obesity. Results demonstrating that obese/diabetic microbiota and its metabolites are causative to increase leaky gut and inflammation by activating miRNAs that disrupts TJPs, are not only of great significance to understand the underlying biology of leaky gut and inflammation in obesity and diabetes, but will also pave the ways to develop novel strategies to prevent/ treat these debilitating public health diseases.

Support or Funding Information

We thank funding from the Department of Defense, National Institutes of Health and Wake Forest School of Medicine, and wonderful support from other team members.
Employing Eye Tracking Technology to Understand How Novices Learn Neuroanatomy
Authors: Nguyen Angela , Cheung Beata , Leclair Rebecca , Saini Jessica , Lyons Jim , Wainman Bruce , Heisz Jennifer , Brewer-Deluce Danielle
Published: April 2020

Introduction

Neurophobia is the fear of neuroscience experienced by students who are learning and applying content. Although the cause remains unclear, neurophobia presents a major barrier to student success in neuroanatomy. A preliminary study suggested that individuals with low working memory capacity (WMC) benefit from using high‐contrast anatomical images when learning neuroanatomy. However, the reason for the improvement is unknown.

Aim

This study uses eye tracking technology to better understand how individuals of varying WMC and expertise study neuroanatomical images. We aim to: 1) assess differences in gaze patterns between students of high and low WMC when viewing high and low contrast brain slices and 2) compare novices and experts to uncover potential competency‐related differences.

Methods

Undergraduate students with no prior anatomical education (n=120) and neuroanatomical experts were recruited. During an eye tracking session, participants were given 5 minutes to study twelve structures on digital images of 4 brain slices (either coronal or transverse slices in high or low contrast) and were then tested on their ability to identify the learned structures on similar low‐contrast images. This procedure was repeated such that all participants were exposed to a set of coronal/transverse and high/low contrast images in a randomized fashion. All participants’ eye tracking data and test accuracy were recorded. After testing, participants completed the Automated Operation Span Task (OSPAN) to quantify WMC.

Results

Preliminary results show that students with high WMC are more accurate in identifying neuroanatomical structures when compared to low WMC students [F(1, 32), p =.088]. Dwell time was a significant predicting factor for accuracy [r(32)=40, p =.02]. High WMC students fixated longer on neuroanatomical structures when compared to low WMC students [F(1, 32)=6.50, p =.02]. Data collection from expert participants is ongoing.

Discussion/Conclusion

Overall, these results offer a quantitative measure of how neuroanatomical information is viewed and learned to gain insight into neurophobia and influence future teaching practices in neuroanatomy.
Perfluorooctanoic Acid (in the Presence of Fetal Bovine Serum) Induces Proliferation in ERα Positive and ERα Negative Breast Cancer Cell Lines
Authors: Gaw Victoria , Glenn Manderrious , Cannon Jennifer
Published: April 2020

Perfluorooctanoic acid (PFOA) is a synthetic chemical belonging to a larger group of fluorotelomers. These compounds have been used in the production of both industrial and consumer products as surfactants and due to their chemical nature are environmentally persistent pollutants. Individuals can be exposed to PFOA through ingesting PFOA‐contaminated water or food. PFOA has been shown to have a half‐life of approximately 4 years in humans. While the long‐term effects of PFOA are largely unknown, there is increasing evidence suggesting it to be an endocrine disruptor. Studies have shown that PFOA binds to and activates peroxisome‐proliferator‐activated receptor α (PPARα), which can regulate the expression of other genes and receptors such as the other PPAR isoforms as well as estrogen receptor α (ERα).

Previous experiments in our lab demonstrated that PFOA treatment of MCF‐7 breast cancer cells (an ERα positive cell line) decreased expression of ERα mRNA and protein, and decreased cell viability by ~20% within 48h of treatment compared to DMSO controls. However, these cells were treated in the absence of fetal bovine serum (FBS). When we repeated these experiments without serum withdrawal, we initially noted a tendency towards increased proliferation in MCF‐7 cells treated with 50μM and 100μM PFOA at both 24h and 48h compared to control. To further examine the role of ERα in this PFOA‐induced proliferation, we carried out additional experiments in MCF‐7 cells along with experiments in another ERα‐positive cell line, T47D, as well as an ERα‐negative cell line, MDA‐MB‐231. There was a tendency for increased viability in all three cell lines when treated with PFOA, although only the MDA‐MB‐231 cells and the T47D cells showed a statistically significant (~20%) increase at 48h with 50μM PFOA and 100μM PFOA, respectively. These data suggest that the PFOA‐induced increase in cell viability in these cell lines is not dependent on ERα expression. In addition, the opposing effects of PFOA on proliferation in MCF‐7 cells in the presence and absence of FBS demonstrates the importance of accurately and completely reporting cell culture and treatment conditions.

Support or Funding Information

Augusta University Department of Biological Sciences, Augusta University Center for Undergraduate Research and Scholarship
Alterations in Structure and Neurotrophic Factors of Hippocampus and Prefrontal Cortex following Perinatal Dietary Protein Deficiency in F1 and F2‐generations of Rats
Authors: Ebuehi Osaretin Albert Taiwo , Abey Nosarieme Omoregie , Imaga Ngozi O.
Published: April 2020

Reports have shown that neurogeneration is on the increase and has been linked to transgenerational consequences. Neurotrophic factors play a key role in development, synaptogenesis, survival of neurons as well as the process of adaptation to external influences. This study provides insight into potential consequences of persistent early life dietary protein deficiency in F1 and F2 generations of Sprague Dawley rats in brain and glial cell – derived neurotrophic factors (BDNF and GDNF), at the hippocampal and prefrontal sections of the brain. Rats in four groups were fed different rations of protein diet (PD): 21% PD, 10%PD, 5%PD and control diet, from adolescence through to gestation and lactation, next generations were weaned to the maternal diet groups. Histopathology was carried out on the hippocampal and prefrontal sections (PFC), the expression levels of BDNF and GDNF were quantified using Promega Elisa kit. Photomicrograph reveals that the 21%PD and control groups had prefrontal cortex with normal cells well placed within the neuropils, the hippocampus had clear delineation of the pyramidal layer at F1 and F2. However, the protein deficient groups (5% and 10%PD) were characterized with degenerated pyramidal cells in the pyramidal layer of hippocampus and reduction in cellular density of F1‐generation PFC followed by halo‐spaced neuropils which became prominent in F2‐generation. The PFC levels of BDNF and GDNF in F1 and F2 ‐ generations were significantly reduced in the 5% protein diet group, while Hippocampal BDNF level in F2 was significantly reduced, with no statistical differences in the GDNF levels. Alterations in activation and functions of the brain may represent reorganization and structural shift in critical sections as an important signature to how the brain attempt to compensate for the nutritional deficiency.
Exploring the Role of Immunological Biomarkers in the Association Between Mental Health Status and Executive Function in Early Adulthood
Authors: Moore Jessy , Maguire Aindriu R. R. , Dempster Kylie S. , Gagnon Madison K. , Henry Megan A. , O’Leary Deborah D. , MacNeil Adam J. , Wade Terrance J.
Published: April 2020

By the time Canadians reach 40 years of age almost 50% have, or have had, a mental illness, with the majority of mental health problems first manifesting in adolescence and early adulthood. Poor mental health is the leading cause of disability in Canada and the disease burden is higher than all cancers put together, highlighting the importance of understanding this condition. People with higher levels of depressive and anxiety symptomatology, as measures of mental health status tend to have lower life satisfaction, feel less able to handle daily stresses and have a shorter life expectancy. Poor mental health is most prevalent in working‐age Canadians and is known to affect mood, thinking, and behavior. Recent evidence suggests an altered immunological state among individuals with higher depressive symptoms. It has also been found that there may be a link between an altered immune state and cognitive functioning as many cytokines play a role in communicating with the brain. The aim of the current study was to explore a potential immunological link between depressive and anxiety symptomatology with executive function in early adulthood. This study included a sample of young adults from the Niagara Longitudinal Heart Study (NLHS). Participants completed a self‐reported questionnaire that included questions about depression, anxiety, and executive function. Blood samples were collected, and serum was used to analyze levels of various immune biomarkers. Several immune markers, including interleukins and interferons were found to be associated with changes in depression and anxiety from childhood to early adulthood. These same immune biomarkers were strongly correlated with measures of executive function in early adulthood. Furthermore, participants who reported feeling depressed or anxious reported lower levels of executive function. Thus, we propose an association between mental health status, executive function, and an altered immunological state in early adulthood. This represents a potential physiological state which could help explain both poor mental health status and lower levels of executive function. These immune biomarkers may also represent a new target for research aimed at reducing the burden of mental illness and poor mental health status in early adulthood.

Support or Funding Information

The NLHS is funded by the Canadian Institutes of Health Research (CIHR #s 363774, 399332). ARRM is supported by the Ontario Graduate Scholarship (OGS) program. KSD is funded by a CIHR Doctoral Research Award – Frederick Banting and Charles Best Canada Graduate Scholarship (RFN#167014).
Effect of Range of Motion in the Various Eccentric Exercise Intensities – A Preliminary Study
Authors: Sadacharan Chakravarthy Marx
Published: April 2020

Purpose of the study

To assess the functional limitations in the activities of daily living (ADL) and the level of muscle pain in individuals after completing small range of motion eccentric exercises with and without yoga poses.

Methods and Materials

Twelve research participants (untrained active healthy men and women) aged between 18 and 30 years were recruited from the students of the University of New England and were randomly assigned into groups 1 and 2. Four muscle groups (elbow and knee flexors and extensors) were selected (one muscle group per limb) from each participant and maximal voluntary isometric contraction (MVIC) were calculated using a hand‐held dynamometer. Participants performed eccentric exercises protocol (5 × 10 repetitions) with 60° of active range of motion (AROM) using their 15% of MVIC on day 1 of first week, 25% of MVIC on day 1 of third week, and 35% of MVIC on day 1 of fifth week in the elbow and knee joints. To control the initial 60‐degrees of AROM in the elbow and knee joints, dynamic adjustable AROM controlled splints were used. Participants did not perform any exercise protocol during the weeks 2 and 4. In weeks 1, 3, and 5, after the eccentric exercise protocol (day 1), from day 2 to 6, the subjective ADL difficulty scale, visual analogue scale (VAS)) and objective assessments (pain pressure threshold (PPT), arm and thigh circumference, and AROM were carried out. Group 1 performed only exercise protocol whereas group 2 performed prior yoga poses for upper and lower limbs along with exercise protocol. Linear regression and two‐way variance of ANOVA statistical analysis were used.

Results

Group 2 had a minimal pain and ADL difficulty when compared to group 1. Group 1 that performed exercise protocol at week 5 with 35% MVIC had a mild pain (pain scale ranged 1–2 out of 10) in the elbow extensors and knee flexors and ADL difficulty (scale ranged 1–2 out of 10) in pull a heavy object, carry an object with arm above shoulder, washing opposite armpit and back, ascending stairs, bend to pick up from the floor when compared to group 2. There was no significant difference in the AROM and, arm and thigh circumference between both groups during entire exercise protocol.

Conclusion

The small range of motion eccentric exercise with low intensity and yoga poses may have great effect in reducing muscle pain. Current preliminary study can be used as an essential research for sedentary older adults with chronic conditions.

Support or Funding Information

Funded by the Biology Department, College of Arts and Sciences (CAS), University of New England, ME, USA ‐04005
Sleep homeostasis in the rat following the induction of a torpor‐like state
Authors: Hitrec Timna , Cerri Matteo , Del Vecchio Flavia , Luppi Marco , Martelli Davide , Occhinegro Alessandra , Piscitiello Emiliana , Squarcio Fabio , Tupone Domenico , Amici Roberto
Published: April 2020

Soon after the exit from a state of torpor/hibernation, a non‐REM sleep (NREMS) bout with high Delta electroencephalographic activity (EEG) power occurs, suggesting that the time passed in torpor/hibernation is a period of sleep deprivation (Deboer et al., J Comp Physiol A., 1994). The homeostatic nature of the after‐torpor Delta rebound has been challenged (Strijkstra & Daan, Am J Physiol. R, 1998) in the past. According to this alternative view, the Delta enhancement is a consequence of a non‐specific reactivation of cortical activity.

We recently found that in the rat, a non‐hibernator, the induction of a torpor‐like state, achieved by multiple injections of muscimol (GABA‐A agonist) into the rostral ventromedial medulla (RVMM), is also followed by a Delta rebound, when the animal recovers from the hypothermic state (Cerri et al., J Neurosci., 2013). The aim of the study was to understand whether the Delta rebound observed in our rats is homeostatically driven or unspecific.

We used twelve male Sprague Dawley rats (300–350 g) implanted, under general anaesthesia (Diazepam, 5mg/kg, i.m., Ketamine, 100 mg/kg, i.p.) with: i) electrodes for
EEG, and nuchal (nEMG) and diaphragm (dEMG) electromyogram recording, for Wake‐Sleep states scoring; ii) a thermistor for deep brain temperature (Tbrain) recording; iii) a wireless catheter for arterial pressure (AP) and heart rate (HR) recording; iv) a microcannula in the RVMM for drug delivery. Animals were induced into a torpor‐like state by multiple injections of muscimol (1mM, 120 nl) into the RVMM at an ambient temperature (Ta) of 15°C starting at 11 AM. At 5 PM, after 6h of torpor‐like condition, the animals were placed at Ta 28°C in order to favor the rewarming process. After the return to normothermia (8 PM) the animals were either sleep deprived by gentle handling for 6h or left undisturbed (n=6 each).

In comparison to their baseline, animals exposed to sleep deprivation showed a powerful increase in Delta power during NREMS soon after the end of the deprivation period. This increase was significantly larger in sleep deprived rats (281±13%) than in non‐deprived animals (237±19%, p<0.05).

The size of the Delta rebound in sleep deprived rats indicates that such rebound is not only the consequence of the previous sleep deprivation period, but also of the sleep debt accumulated during the torpor‐like state. Thus, t
he Delta rebound which immediately follows a torpor‐like condition is not due to a non‐specific reactivation of cortical activity, but is the expression of a sleep need.
Effects of a mitochondria targeted antioxidant on skeletal muscle mitochondrial function with cigarette smoke exposure and Chronic Obstructive Pulmonary Disease
Authors: Layec Gwenael , Thurston Taylor S. , Decker Stephen , Huecksteadt Thomas , Hoidal John R. , Sanders Karl , Kwon Oh Sung
Published: April 2020

Reactive oxygen species (ROS) are important signaling molecules in cells. However, the exaggerated free radical production accompanying mitochondrial dysfunction with Chronic Obstructive Pulmonary Disease (COPD) has been suggested to play a crucial role in the development of metabolic abnormalities in these patients. Consequently, selective inhibition of mitochondrial ROS could be a potential therapeutic strategy to restore mitochondrial function and subsequently attenuate exercise‐induced metabolic disturbance during exercise in COPD patients. The goal of this study was therefore to determine the effects of a mitochondria targeted antioxidant, MitoQ, on free radical levels, mitochondrial function, and skeletal muscle metabolism in six patients with moderate to very‐severe COPD and a smoking mouse model (C57/Bl6 exposed to cigarette smoke for 8 months) using electron paramagnetic spectroscopy (EPR), 31P/1H magnetic resonance spectroscopy (1H/31P MRS), high‐resolution respirometry in permeabilized skeletal muscle fiber. 4 weeks of MitoQ supplementation (MQ) decreased blood free radical levels measured by EPR (control, CTL: 4.3 ± 3.3 a.u.; MQ: 1.8 ± 1.7 a.u.; P<0.05). Nevertheless, the exercise‐induced changes in intracellular O2 partial pressure, high‐energy phosphate, and pH measured by 1H/31P MRS in the plantar flexor muscles of patients with COPD were not significantly altered with MitoQ. Also, the peak rate of oxidative ATP synthesis measured by 31P‐MRS was lower with MitoQ (CTL: 19 ± 6 mM.min−1; MQ: 14 ± 4 mM.min−1, P<0.05), suggesting an impaired mitochondrial function in vivo. This finding was further supported by the lower muscle respiratory capacity measured in permeabilized skeletal muscle fibers incubated with MitoQ (−43%, P<0.05) from a smoking mouse model. Specifically, complex I (−42%) and free‐fatty acid (−51%) linked respiration were significantly decreased with MitoQ (P<0.05). Together, these findings suggest that decreasing ROS levels using a mitochondria targeted antioxidant may, in fact, be detrimental to oxidative phosphorylation capacity in the skeletal muscle of COPD patients.

Support or Funding Information

This work was funded in part by grants from the Flight Attendant Medical Research Institute (FAMRI), NIH National Heart, Lung, and Blood Institute (HL125756)
Vascular Endothelial Growth Factor Production by Interleukin‐17C‐Stimulated Colorectal Cancer Cells Is Dependent on the STAT3/miR‐23a‐3p Signaling
Authors: Im Eunok , Lee Yunna , Kim Su Jin , Choo Jieun , Heo Gwangbeom , Rhee Sang Hoon
Published: April 2020

Recent studies have shown that interleukin‐17C (IL‐17C) is associated with tumor initiation and progression of colorectal cancer (CRC). However, the detailed mechanism of action remains to be elucidated. We aimed to investigate the effect of IL‐17C on angiogenesis and delineate the direct target genes of IL‐17C signaling in CRC. Aims were tested by using angiogenesis assays, murine xenograft experiments, immunohistochemical staining, gene silencing, quantitative real time PCR, ELISA, and western blot analysis. IL‐17C induced angiogenesis of intestinal endothelial cells, subsequently enhancing cell invasion and migration of DLD‐1 cells. IL‐17C‐conditioned DLD‐1 cells enhanced angiogenesis by secreting vascular endothelial growth factor (VEGF). Moreover, IL‐17C significantly increased xenograft tumor growth via VEGF production, but treatment with the VEGF receptor 2 inhibitor Ki8751 ameliorated its tumor promoting activity. IL‐17C also increased levels of PECAM and VE‐cadherin in tumor tissues, which was suppressed by Ki8751. Furthermore, IL‐17C increased miR‐23a‐3p expression via STAT3 phosphorylation. Accordingly, miR‐23a‐3p suppressed semaphorin 6D (SEMA6D) expression, enabling VEGF induction. Therefore, our data showed that IL‐17C promotes tumorigenesis and angiogenesis through VEGF production via the STAT3/miR‐23a‐3p/SEMA6D axis, suggesting its potential as a novel target for anti‐CRC therapy.

Support or Funding Information

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2019R1A2C1010536 to E.I.).
Molecular determinants of the mechanosensitivity of G protein‐coupled receptors
Authors: Mederos y Schnitzler Michael , Storch Ursula , Erdogmus Serap , Danner Laura , Becker Jasmin , Winter Michaela , Ziegler Nicole , Wirth Angela , Offermanns Stefan , Hoffmann Carsten , Gudermann Thomas
Published: April 2020

G‐protein coupled receptors (GPCRs) are broadly tuned cellular sensors for chemical stimuli, and also serve as mechanosensors e.g. in vascular regulation and cardiac hypertrophy. The molecular mechanism underlying mechanically induced GPCR activation is still only partially understood. Here we show that mechanosensitive histamine H1 receptors (H1Rs) are endothelial sensors of fluid shear stress and contribute to flow‐induced vasodilation. At the molecular level, H1Rs undergo stimulus‐specific patterns of conformational changes supporting the notion that mechanical forces and agonists induce distinct active receptor conformations. GPCRs devoid of C‐terminal helix 8 (H8) lack mechanosensitivity. Conversely, transfer of H8 to non‐responsive GPCRs renders them sensitive to mechanical cues. Disrupting H8 structural integrity by amino acid exchanges impairs mechanosensitivity. Thus, H8 is the essential structural motif endowing GPCRs with mechanosensitivity. These findings provide a mechanistic basis for a better understanding of the roles of mechanosensitive GPCRs in health and disease.
The existence of a Cryptic Allosteric Site on Plasmodium falciparum Malate Dehydrogenase
Authors: Botros Natalie , Bell Ellis , Bell Jessica
Published: April 2020

Approximately one million people die from malaria each year, and thus this disease is a prevalent and pressing matter. Plasmodium falciparum, the protozoan parasite responsible for malaria has been subject to intensive study with the goal of developing specific drugs for malaria treatment. Plasmodium falciparum has a distinctive Malate Dehydrogenase (MDH), being tetrameric compared to the canonical dimeric forms of MDH, yet shares the same catalytic and substrate binding site motives as human mitochondrial and cytosolic forms. To investigate the possible existence of a cryptic allosteric site in the P falciparum enzyme, we have used sequence and structural bioinformatics and identified several short sequences,(G175‐L176, Q211‐M216) each containing highly conserved (in Plasmodium and related species) amino acids that are different in the Plasmodium falciparum enzyme than the human mitochondrial or cytosolic forms. To investigate the possibility that these regions form a cryptic allosteric site in Plasmodium falciparum we expressed, purified, using NiNTA Affinity chromatography, and characterized all three forms (Plasmodium falciparum, human cytosolic and human mitochondrial malate dehydrogenases). In terms of kinetic parameters the Plasmodium falciparum enzyme resembled the mammalian mitochondrial isoform, exhibiting similar substrate inhibition with oxaloacetate and weaker affinity for NADH than the mammalian cytosolic isoform. To determine the potential that the Plasmodium falciparum enzyme had a cryptic allosteric site we designed two mutations, one in each of the two significant sequence differences between the human isoforms and the Plasmodium falciparum enzyme (.D176N and R214E ). Each mutation was constructed using Quikchange mutagenesis, transformed into XL Gold cells and expressed. The purified mutant proteins were characterized using enzyme kinetics and size exclusion chromatography and their overall secondary structures compared to the wildtype enzyme using circular dichroism spectroscopy. Stability was compared using CD thermal melts at 222nm and a Fluorescence based Thermal Shift assay. While both mutations showed altered specific activities compared to wildtype, the D176N mutant showed a 90% lower activity than wildtype Plasmodium falciparum malate dehydrogenase. Both mutations significantly lowered affinity for the cofactor NADH, >20fold decrease in affinity. Taken together these results suggest that Plasmodium falciparum malate dehydrogenase may have a highly conserved cryptic allosteric site distinct from any such site in the mammalian isoforms. This Cryptic Allosteric Site could be exploited to develop inhibitors of Plasmodium falciparum malate dehydrogenase that would not impact the human isoforms.

Support or Funding Information

This work was supported by NSF Grants 1726932 and 0448905.
Flow regulation of WNK1 localization in the cortical collecting duct (CCD)
Authors: Carrisoza-Gaytan Rolando , Subramanya Arohan R. , Ray Evan C. , Kleyman Thomas R. , Satlin Lisa M.
Published: April 2020

A high K diet (HKD) x 10 d increases (i) luminal flow rate in the distal nephron and (ii) expression of apical immunodetectable L‐WNK1 in CCD intercalated cells (IC), which we propose enhances apical BK channel activity measured as flow‐induced K+ secretion (FIKS) (Webb et al, 2015). We previously demonstrated that fluid shear stress (FSS) x 30 min induces expression of ERK and p‐38, both BK channel modulators, in a CCD principal cell (PC) model (Carrisoza‐Gaytan et al, 2014). The objective of this study was to test the hypothesis that an increase in tubular fluid flow rate rapidly induces apical localization of L‐WNK1 in the CCD.

CCDs isolated from NZW rabbits fed a HKD x 10 d were microperfused at slow (n=4) or fast (n=4) luminal flow rates x 1 hr, fixed on the rig, immunoperfused with antibodies (Abs) directed against L‐WNK1 (+ A488‐fluorescent 2o Abs) and rhodamine‐conjugated peanut lectin (PNA) or Dolichus biflorus lectin (DBA), which bind to apical surfaces of IC and PC, respectively, and examined by confocal microscopy. MDCK cells were subject to no (static), low or high FSS x 1 hr (n=2 each condition) and fixed for immunodetection of L‐WNK1 and BKα in situ or harvested for semi quantitative immunoblotting of isolated plasma membranes.

The expression of IC apical L‐WNK1 relative to that in the total cell was 40.0±1.1 % greater in CCDs perfused at fast flow compared to those perfused at slow flow rates (p<0.001). In plasma membrane preparations of MDCK cells subject to low and high FSS, L‐WNK1 abundance was 6.0±0.8 and 11.9±2.5 fold greater, respectively, to that measured in the absence of flow. In static MDCK cells, BKα did not colocalize with L‐WNK1 at the apical membrane; however, the two proteins colocalized in cells subject to low or high FSS.

In conclusion, (i) apical expression of L‐WNK1 in the microperfused CCD IC is rapidly stimulated by increases in luminal flow rate and (ii) FSS favors apical colocalization of L‐WNK1 with BKα, responses that may facilitate BK channel‐mediated FIKS in the CCD.

Support or Funding Information

NIH NIDDK: R01 DK038470‐27A1, P30 DK079307, R01 DK119252, R01 DK098145; NIH NHLBI R01 HL147818.
Dimerization of the Transmembrane Domain of Mucin 16
Authors: Murphy Robert James , Butch Nicole , Li Edwin
Published: April 2020

Mucin 16 (MUC16) is a transmembrane protein produced by epithelial cells, and its main role is to contribute to the formation of a protective mucous barrier to inhibit debris, pathogens and viruses from entering the cells. However, when overexpressed, MUC16 can lead to cancers of the breast and ovary. Recent studies have shown that in cancer cells, MUC16 is trafficked to the nucleus, but little is known regarding the mechanism of this trafficking. One possible explanation may involve dimerization of MUC16, as shown in the case of mucin 1 (MUC1). Overexpression of the transmembrane MUC1 is also associated with various forms of cancer. Recent studies have shown that MUC1 is trafficked to the nucleus, where it may act as a transcription factor regulating the expression of genes associated with cell growth. The nuclear trafficking of MUC1 is dependent on the formation of MUC1 dimers, formed through inter‐disulfide bonds by two cysteine residues near the end of the transmembrane domain (TMD) of MUC1.

The TMD of MUC16 (FWAVILIGLAGLLGVIT
CLI
C) has two cysteine residues that reside in the end of the TMD, which led us to the hypothesis that MUC16 may also form cysteine‐mediated dimers. The ToxR assay, a technique to measure transmembrane domain dimerization, was used to investigate the roles of the cysteine residues in MUC16 TMD dimerization. Our preliminary results show that the cysteine residues in the TMD play a role in dimerization, but not as significant as in the case of MUC1 TMD. These results suggest that noncovalent interactions may be important for MUC16 TMD dimerization. Understanding the formation of MUC16 dimers, similar to MUC1, is important because dimerization may serve as a precursor to the nuclear trafficking of MUC16.
Regulation of Junctophilin‐2 Expression by Triiodothyronine in Adult Rat Cardiomyocytes
Authors: Gilani Nimra , Wang Kaihao , Muncan Adam , Peter Jerrin , Gerdes A. Martin , Stout Randy , Ojamaa Kaie Martin
Published: April 2020

Introduction

Cardiac junctophilin‐2 (Jph2) plays a central role in myocardial contractility by anchoring transverse tubules (TT) to the junctional sarcoplasmic reticulum (jSR), essential for excitation‐contraction (EC) coupling and calcium signaling. Decreases in the expression levels of Jph2 have been observed in a variety of pathological conditions including hypertrophic cardiomyopathy, arrhythmias, and progression of disease in failing hearts. Studies have shown that T3 regulates SR Ca2+ ATPase (SERCa2), phospholamban (PLN), L‐type Ca2+ channels and ryanodine receptors (RyR2), that are responsible for regulating intracellular Ca2+ and maintaining normal systolic and diastolic function. We previously reported that T3 improves cardiac contractile function after induction of myocardial infarction (MI) by maintaining TT network integrity and by regulating expression of myofibrillar and Ca2+ handling proteins. We hypothesize that T3 regulates Jph2 expression and its localization within the jSR‐TT complex to maintain functional TT structures and thus normal Ca2+ transients and contractility.

Methods

Thyroid deficiency was induced in female Sprague Dawley rats (~220g) by ingestion of 0.025% PTU (propylthiouracil) in drinking water. Control (EU) rats had access to untreated water. After 8 weeks, a subgroup of PTU treated rats received T3 (10ug/kg/d) in PTU‐containing water for an additional 2 weeks. RNA was extracted from left ventricular (LV) tissue to measure Jph2, SERCa2, PLN, LTCC and RyR2 mRNA by RT‐qPCR. RNAseq analysis interrogated 15,000 genes. LV myocytes were isolated by collagenase digestion and cultured with and without T3 (10−7 M). Cells were harvested at 24, 48, and 72 hrs for RT‐qPCR analysis of Jph2, SERCa2, and Bridging Integrator‐1 (Bin1). Isolated myocytes from all study groups were immunolabeled with RyR2, Cav1.2 and Jph2 for nanoimaging using STORM.

Results

RT‐PCR analysis showed significant upregulation of Jph2 expression compared to thyroid deficiency (1.404±0.73 vs 0.322±0.17 fold change). RNAseq revealed 567 differentially expressed genes with 377 genes up‐regulated and 190 down‐regulated in T3‐treated hearts compared to TH deficient hearts. Results showed up‐regulation of Jph2 (p<0.001) and SERCa2 by >1.5‐fold (p<0.00001). Cultured EU and PTU cardiomyocytes similarly showed significantly (p<0.05) increased Jph2 expression in T3 treatment vs non‐treated cells at 48 and 72 hr time points. STORM imaging showed increased clustering of LTCC (Cav1.2) within the RyR/Cav1.2 clusters of TT‐jSR complexes in the T3‐treated vs TH deficient cardiomyocytes (0.53±0.13 vs 0.36±0.03).

Conclusions

T3 regulates expression of Jph2 which in turn is a critical regulator of the jSR‐TT organization in cardiomyocytes allowing for optimal calcium transients and contractile function.