bluuebunny/crossref_metadata_2025_split huggingface dataset:
Physical Inactivity‐induced Endothelial Dysfunction : The Role of Toll‐like Receptor 4
Authors: Guerrero Maria , Sun Chang , Kwon Oh Sung 
Published: April 2020

BackgroundA hallmark characteristic of physical inactivity is not only the rapid deterioration of cardiovascular function and whole body insulin sensitivity, but also the accumulation of free fatty acids (FFAs) which contribute to vascular dysfunction, specifically endothelial cell dysfunction as a consequence of a mismatch between the synthesis and degradation of nitric oxide (NO). Although a mechanism that may contribute to physical inactivity‐induced endothelial dysfunction is unclear, the accumulation of the sphingolipid ceramide within endothelial cells may activate Toll‐like receptor (TLR) 4, which finally leads to vascular dysfunction. Therefore, this study sought to investigate the role of TLR4 signaling in physical‐inactivity (hindlimb unloading) induced endothelial dysfunction.MethodsAdult female wild type (WT) mice were separated in three groups ; control (CON) (n=8), hindlimb unloading for 14 days (HU) (n=8), and hindlimb unloading with toll‐like receptor 4 inhibitor, TAK‐242 (4mg/kg) (HUT) (n=8). Following 14 days, using an in vitro preparation, endothelium‐dependent and ‐independent vasodilation were assessed in arteries in response to three stimuli 1) flow‐induced shear stress, 2) acetylcholine (ACh), and 3) sodium nitroprusside (SNP). Additionally, L‐NG‐monomethyl arginine citrate (L‐NMMA) was utilized to identify the impact of blocking NO synthase on these responses. Endothelial NOS (eNOS), TLR4, NFkB protein expression were analyzed via Western blot analysis.ResultsAfter 14 days of physical inactivity, HU group showed that progressive physical inactivy‐induced reductions in endothelial‐dependent vasodilatory function were present in response to the intraluminal flow (CON: 81 ± 3; HU: 40 ± 2%, P < 0.05) and ACh (CON: 93 ± 4, HU: 44 ± 3 %, P < 0.05). However, TLR4 inhibition using TAK‐242, significantly restored endothelial function following physical inactivity; intraluminal flow (HUT: 73 ± 2%, P < 0.05) and ACh (HU: 85 ± 3 %, P < 0.05). All groups had no significant effect on endothelium‐independent vasodilation (SNP). P‐eNOS/eNOS protein expression was significantly decreased with physical inactivity but TAK‐242 prevented HU induced eNOS reduction. In addition, HU significantly activated an inflammation marker, NFkB protein expression, but TAK‐242 prevented physical inactivity‐induced NFkB protein expression.ConclusionsOverall, these findings suggest that physical inactivity induces endothelial dysfunction and endothelial TLR4 signaling may play a critical role in physical inactivity‐associated endothelial dysfunction. Therefore, TLR4 signaling could be a novel therapeutic target to restore endothelial function and NO bioavailability.
Comparing Lipid and Detergent Environments as Models for Studying Renal Transmembrane Proteins
Authors: Pavkov Isabela , Strickland Kerry , Cottrill Kirsten , Imhoff Barry R. , McCarty Nael A. 
Published: April 2020

BackgroundMutations in transmembrane proteins cause kidney diseases such as Dent’s disease, Bartter syndrome, and polycystic kidney disease.
1 Understanding the structure and function of renal membrane proteins reveals how they contribute to kidney function as well as kidney disease. Recent studies have shown the importance of generating a native lipid environment when studying membrane proteins.
2 However, membrane proteins are often studied in the presence of detergent, which may modify their natural structure and lead to decreased function. Nanodiscs, a novel lipid bilayer system, provide a more native environment for protein reconstitution.MethodsIn this study we used two proteins found in the kidney, the Na+/K+‐ATPase pump and CFTR ion channel, to determine whether nanodiscs or detergent micelles serve as the better model for studying renal transmembrane proteins. To do this, we expressed and purified our own Membrane Scaffold Protein (MSP), a base protein for nanodiscs, in Escherichia coli. We generated empty nanodiscs with three different lipid types: 1,2‐dimyristoyl‐sn‐glycero‐3‐phosphocholine (DMPC), 1‐palmitoyl‐2‐oleoyl‐glycero‐3‐phosphocholine (POPC), and 1‐Palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphoethanolamine (POPE).ResultsWe have and are continuing to reconstitute CFTR and Na+/K+‐ATPase into nanodiscs with different lipid compositions. We evaluate membrane protein stability using ATPase activity in detergent and different lipid nanodisc environments. Preliminary data show improved ATPase activity for CFTR in nanodiscs versus detergent. Higher ATPase activity of proteins reconstituted in nanodiscs would indicate that detergent structurally modifies these proteins and consequently decreases their function. Nanodiscs provide a more stable environment in which to study these and other proteins found in the kidneys, allowing for more accurate assessment of structure and function.
Role of miR‐130b/301b cluster in macrophage polarization and obesity
Authors: Jiang Shaoning , McBride Aubrey , Luo Wenyi , Chernausek Steven 
Published: April 2020

Macrophage activation status, such as M1 classic and M2 alternative activation, plays an important role in the development of obesity and other metabolic diseases. The molecular mechanisms regulating macrophage M1/M2 polarization are not fully understood. MicroRNAs are regulatory RNAs that suppress the translation and stability of the mRNA targets. In the present studies, we show that compared to wild type mice, knockout mice of miR‐130b/301b cluster exhibited decreased adiposity, increased whole body energy expenditure, and increased oxygen consumption in response to high fat diet. miR‐130b/301b knockout attenuated the LPS‐induced pro‐inflammatory activation of bone marrow macrophages, while promoted IL‐4 induced alternative macrophage activation. In addition, miR‐130b/301b knockout mice had increased AMP‐activated protein kinase (AMPK) activation in white adipose tissue, which can explain the increased energy expenditure. Together, the present study identified miR‐130b/301b cluster as a novel regulator of macrophage polarization and energy metabolism, which contributes to the development of obesity.Support or Funding InformationThe study is supported by OCAST (PI: Shaoning Jiang) and OK‐INBRE (RPI: Shaoning Jiang) grants.
Proteomic and Functional Characterization of STARD9
Authors: Clutario Kevin 
Published: April 2020

The centrosome is a non‐membrane bound structural organelle that is crucial in spindle assembly, as the centrosomes act as the microtuble organizing centers at the spindle poles during mitosis. Deviation from normal spindle assembly can lead to activation of the spindle assembly checkpoint (SAC). Some of the most successful antimitotic drugs activate the SAC in order to arrest cancer cells in mitosis to induce subsequent apoptosis. Recently, kinesins and kinases implicated in spindle assembly have gained attention as possible therapeutic targets, as their functions rely on their ability to hydrolyze ATP. Our lab’s previous proteomic and genetic studies identified steroidogenic acute regulatory protein‐related lipid transfer domain containing 9 (STARD9) as a kinesin involved in spindle assembly. Its depletion invokes multipolar spindles in dividing cancer cells and most surprisingly, STARD9‐depleted cancer cells display increased Taxol sensitivity. STARD9 plays a functional role in the stabilization of pericentriolar material (PCM) during centriole maturation, but its mechanism of action and its regulation are not yet fully understood. To elucidate these mechanisms we have used proteomic and chemical‐genetic approaches to study both STARD9’s major domains and endogenous STARD9 protein. These approaches have identified putative interacting proteins that may play regulatory roles in PCM stabilization. We are currently focused on determining the importance of these STARD9 interactions for PCM stabilization. Our work looks to further the knowledge of STARD9’s function within centrosome assembly and more broadly cancer cell division.Support or Funding InformationUSHHS Ruth L. Kirschstein Institutional National Research Service Award #T32 CA009056
Molecular pathological mechanism of blue ray induces murine photoreceptor cell 661W apoptosis
Authors: Chang Wei-Min , Chen Chun-Yen , Li Li-Jie , Chan Ming-Hsien , Hsiao Michael 
Published: April 2020

Modern life is full of artificial light in day and night. Smart phones, laptops, televisions, and high definition monitors provide unnatural human light source into human eye and cause ocular defects in modern human being. Understanding the molecular mechanism of the interplay between artificial light and ocular cells may help researcher society understanding the methods that preventing artificial light‐induced visual impairment. Here, we reported a short‐term exposure at short wavelength light source from blue and green light‐emitting diode (LED) could cause the murine photoreceptor cells 661W cells death, but not in red LED. However, the molecular mechanism remaining unclear. Using the molecular probe of caspase 3 reporter, we found the blue and green LED caused caspase 3 activation even as short as 10 minutes exposure and subsequently cell death at 16 hours later. We further confirmed 661W cells apoptosis by cleavage form caspase 3 and PARP formation. Moreover, we found the decreasing of mitochondria membrane potential and increasing of cellular relative‐oxygen species in 661W cells both indicate early cell apoptosis under short‐term exposure. Up to our knowledge, there was no transcriptome analysis which revealed immediately early gene expression in light‐induced cell death. Thus, we analyzed the 661W transcriptome at 2 hours after 10 minutes’ short wavelength light exposure and validated the candidate genes expression by RT‐Q‐PCR. Using the ingenuity pathway analysis, we found valuable up‐stream regulators, pathways, and potential mechanisms in short wavelength induced photoreceptor cell death and may provide novel therapy niches. In conclusion, our preliminary results prove that short wavelength light exposure can cause the photoreceptor cells death and the potential molecular clues. Further pathohistological analysis of key protein expression in the animal models may help us to prove the mechanism inside the eye damage from short wavelength light in future.Support or Funding InformationThis study was support by Shu Zen College of Medicine and Management with grant number (SZL10800012) and Academia Sinica
FoxO1: a potential target of cinnamon extract on the PI3K cascade
Authors: Borgman Rachael , Pearce Kathryn , Aulthouse Amy , Stockert Amy 
Published: April 2020

Cinnamon has long been used medicinally for digestive discomforts. Literature highlights anti‐diabetic properties of cinnamon in cell culture and human clinical studies, suggesting a potential role for type 2 diabetes therapy. Despite these studies and patients’ use of cinnamon, the therapeutic mechanism is still poorly understood. Transcription factor PPAR‐gamma has been identified as one potential target of the cinnamon extract components given the effects of cinnamon on morphological changes in the 3T3‐L1 cells during treatment. We confirmed the presence of PPAR‐gamma in 3T3‐L1 cells using immunohistochemistry providing evidence for the spontaneous differentiation of cells when grown in three‐dimensional culture. Furthermore, we have characterized morphological changes in cinnamon‐treated cells while grown in monolayer and sought to determine what expression changes occur under these growth conditions. Due to the changes in expression that we observed, we identified FoxO1 as a potential target of the cinnamon extract. FoxO1 is a metabolic regulator that has been implemented in lipid localization, aging, and cell death. In addition to PPAR‐gamma, expression and localization of GLUT4 transporters is up‐regulated with cinnamon treatment in both muscle and adipose. Interestingly, FoxO1 is involved with both PPAR‐gamma and GLUT4 expression and activities, making it a reasonable target of the extract. Together, these data suggest that cinnamon not only affects multiple targets, but is also likely involved at several stages in the PI3K cascade. In addition to examining the expression levels of FoxO1 in treated and untreated cells, we sought to explore the localization of the FoxO1 transcription factor as it is proposed to shuttle between the nucleus and cytoplasm depending on conditions. We have initiated immunocytochemistry studies to determine the relative localization of the transcription factor when treated with cinnamon extract as compared to no treatment in order to correlate the location to the expression data.Support or Funding InformationOhio Northern University Raabe College of Pharmacy Bower, Bennett and Bennett Research Award
Analysis of Antidepressant Agonist Activity at LPA1, LPA2 and LPA3 lysophosphatidic acid receptors
Authors: Olianas Maria C. , Dedoni Simona , Marras Luisa , Ingianni Angela , Onali Pierluigi 
Published: April 2020

Besides inhibiting monoamine reuptake, antidepressants have been shown to affect a number of neurotransmitter receptor systems, at which they most often act as antagonists. We have previously shown that in neuronal and non‐neuronal cells different antidepressants, predominantly belonging to the tricyclic and tetracyclic classes, activate the LPA1 lysophosphatidic acid (LPA) receptor to induce proliferative and prosurvival signaling. In the present study we further characterize this unique agonist activity of antidepressants by examining their ability to activate two additional members of the LPA receptor family, LPA2 and LPA3, which display high sequence similarity to LPA1. The human LPA receptors were stably transfected in HEK293 cells (HEK293‐LPA1–3 cells), and their activation was monitored by measuring the phosphorylation/activation of ERK1/2 by Western blot. Exposure to LPA caused a concentration‐dependent increase of phospho‐ERK1/2 levels with EC50 values of 145, 210 and 45 nM in HEK293‐LPA1, ‐LPA2 and ‐LPA3 cells, respectively. As previously observed in other cell types endogenously expressing LPA receptors, amitriptyline, imipramine, clomipramine, desipramine, mianserin and mirtazapine effectively increased ERK1/2 phosphorylation in HEK293‐LPA1 cells. In HEK293 cells stably transfected with a LPA1 mutant (Q3.29A), which was unresponsive to LPA, the stimulatory effects of amitriptyline, clomipramine and mianserin were greatly suppressed, indicating the dependence on receptor structural integrity. Tricyclic antidepressants failed to stimulate ERK1/2 phosphorylation in HEK293‐LPA2 cells, whereas the tetracyclic compounds mianserin and mirtazapine displayed agonist activity, although with relative efficacies lower than those showed in HEK293‐LPA1 cells. Conversely, both tricyclic and tetracyclic antidepressants induced ERK1/2 activation in HEK293‐LPA3 cells. These data demonstrate that some classes of antidepressants can exert agonist activity at more than one LPA receptor, thus extending their spectrum of pharmacological activity. In particular, the present finding that antidepressants can activate LPA3, which is expressed in human hippocampus, amygdala, frontal cortex and in different peripheral tissues, suggests that this receptor can be a potential target for the therapeutic and adverse effects of these drugs.Support or Funding InformationUniversity of Cagliari FIR 2019
Understanding the Cell‐Cycle Dependence of Mus81‐Mms4 Recruitment and its Reliance on Slx4
Authors: Siavoshi Mehrnaz Ashley , Le Lindsey , Buo Sorinna , Fischhaber Paula 
Published: April 2020

DNA damage repair pathways are a key element in maintaining genomic stability and preventing cancer and other diseases, particularly given the prevalence of DNA damaging agents. One form of DNA damage is double‐strand breaks (DSBs), the repair of which utilizes numerous repair proteins regardless of which subpathway is implemented. One such repair protein in S. cerevisiae is Mus81, which forms a complex with Mms4, that functions as a structure‐specific endonuclease that cleaves flapped DNA structures present in intertwined chromosomes that have undergone DSB repair in late S and G2 phases.Here, we seek to understand the cell cycle dependence of Mus81‐ recruitment to DSBs and determine if this recruitment is Slx4‐dependent. This exploration requires complete cell cycle synchronization with □‐factor followed by release back into cell cycle following □‐factor removal. As they progress through the cell cycle, cultures are monitored by fluorescence microscopy and flow cytometry to properly correlate fluorescence data with discrete stages of the cell cycle. The yeast strain under study contains a Mus81 protein tagged with yEmRFP which enables its tracking during the fluorescence microscopy portion of the experiment. Preliminary microscopy experiments on a Mus81‐yEmRFP labeled strain have demonstrated that Mus81 is present in low abundance, making it difficult to track the RFP signal throughout the cell cycle. This creates the need for increased expression of Mus81 if it is to be investigated via fluorescence microscopy. Therefore, a plasmid has been constructed that incorporates an ADH1 promoter in front of the Mus81‐yEmRFP gene to enable overexpression. The use of this modified strain will allow for experimental correlation of Mus81‐Mms4 activity with a specific stage of the cell cycle.Support or Funding InformationNational Institutes of Health (NIH) (Grant SC1GM127204)NIH BUILD Program (NIH Grant 5TL4GM118977)
Teaching venous return without throwing students a curve
Authors: Jawad Rasha , McCabe Richard D. 
Published: April 2020

Here we develop a comprehensive presentation of venous return.Guyton’s venous return curve describes venous return in terms of right atrial pressure, mean circulatory filling pressure, and sympathetic activity.
1 This work demonstrated the key role of venous return in cardiac function, and it has been part of undergraduate medical education since that time. However, experience shows that venous return curves are difficult to present or understand, and have limited direct clinical application.
2,
3Venous return remains of central importance to physiology and the pathophysiology underlying many clinical disorders.
4,
5 We propose a simple, intuitive model to explain venous return that can be progressively presented to explain both venous filling and circulatory mobilization of blood. The latter concept encompasses venoconstriction as well as other mobilizing factors.The presentation centers around venous return for students familiar with the role of central venous pressure (CVP) in cardiac preload. The model is presented in the following progression:

Start with a simple circuit consisting of the heart, arteries, a set of capillaries, and veins at stop‐flow condition with a specified filling pressure.
Illustrate the development of normal CVP and mean arterial pressure (MAP), starting from stop‐flow to normal cardiac output.
Introduce positive or negative external filling sources that control filling pressure (i.e. gastrointestinal [GI], urinary, respiratory and integumentary systems).
Add transcapillary forces as the factors controlling internal filling, via shifting volume between interstitial fluid (ISF) and plasma.
Model venules ‐ added in parallel in the diagram ‐ as mobilizers of blood under the control of sympathetic nerves.
Add other mobilizing factors (i.e. GI/splenic blood, muscular pumping, muscular filling) as additional parallel elements under appropriate controls.The model is qualitative, while the venous return curve is quantitative. However, we anticipate it being more easily understood. It is also more comprehensive and more easily applied to clinical medicine. Additional regulatory and pathophysiological factors are easily incorporated into this model. We plan to compare students’ understanding of venous return following presentation of this model versus their understanding following presentation of Guyton’s venous return curve.
Novel Binge Drinking Model Causes Perturbations in Muscle Metabolism with Refeeding
Authors: Steiner Jennifer , Tice Abigail , Laudato Joseph , Johnson Bonde 
Published: April 2020

IntroductionIt is well known that acute alcohol intoxication impairs metabolic signaling within skeletal muscle including suppression of protein synthesis which is at least partially due to inhibition of mTOR activity as assessed by phosphorylation of its downstream substrates p70 ribosomal protein S6 Kinase 1 (S6K1), ribosomal protein S6 (rpS6) and eukaryotic initiation factor 4E (4E‐BP1). The current mouse model used to mimic binge drinking employs either oral gavage or intraperitoneal (IP) injection, both of which are stressful to the animal and in the case of the injection, lack physiological relevance as it bathes all organs in a high dose of alcohol while bypassing normal intestinal metabolism. Therefore, we wanted to determine if an acute ‘dose’ of alcohol incorporated into the food would be sufficient to mimic the binge drinking effects previously reported.MethodsC57BL6 male (n=9) and female mice (n=10) were randomized to receive either control liquid diet food (# 710027, DYETS, Bethlehem, PA), or a calorie (kcal) matched liquid diet containing 32% kcal from alcohol (# 710260, DYETS, Bethlehem, PA). Following a 1 week acclimation period to the control liquid diet, mice were fasted for 16 hours during their light cycle and then, at the onset of the dark cycle, were provided with liquid diet (Con or EtOH) for 30 minutes. The gastrocnemius (GAS) and blood were collected 1.5 hour after the beginning of the refeeding period. Western blots were performed on GAS for protein expression. T‐tests (2 tailed, unpaired) for males (Con vs. EtOH), and females (Con vs. EtOH), or between sexes of the same dietary treatment group, were used for analysis (p<0.05).ResultsDespite the high concentration of alcohol in the liquid diet and its novel introduction to the EtOH group, male mice consumed the same volume of food regardless of treatment group. Control females though, consumed significantly more food than EtOH females (4.5 mL vs. 3.6 mL). Alcohol intake per kg body weight was higher in females than males; however, total alcohol intake (g), and blood alcohol levels (213 vs. 187 mg/dL), were not different between males and females. Alcohol intake decreased AKT S473 phosphorylation in GAS, along with phosphorylation of mTOR S2448 in males and females. Downstream substrates of mTOR were also suppressed by alcohol, including phosphorylation of S6K1 T389 and rpS6 S240/244.ConclusionDespite lower blood alcohol levels (compared to injection), alcohol reversed or prevented the increase in mTOR signaling typically induced by an anabolic stimulus (i.e. nutrient intake). Further, novel findings included the consumption of a high percentage of alcohol in liquid diet form without any prior exposure to a lower dose or acclimation period to alcohol. Future work will continue to characterize this model and lead to optimization of models of alcohol intoxication that best mimic human practice.