bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effect of concentration and staining duration of iodine contrast applied to study the masticatory muscle of Wistar rats in micro‐CT images
Authors: Piau Fabio Giardiello , Haddad Juliana , Rossi Ana Cláudia , Prado Felippe Bevilacqua , Freire Alexandre Rodrigues 
Published: April 2020

The challenge to study the anatomy of the muscular system is to replace destructive methods such as macroscopic dissection with non‐destructive methods involving magnetic resonance imaging and computed microtomography (micro‐CT). The aim of this study was to evaluate the effect of concentration and staining duration of iodine contrast applied to study the masticatory muscle of Wistar rats in micro‐CT images. Fifteen male Wistar rats (Rattus norvegicus albinus), 2 months old (200–250g) from CEMIB‐UNICAMP were used. After euthanasia, the animals were randomly assigned to the experiments in 3 groups of 5 rats each, with the iodine contrast solutions of 3%, 5% and 10%. Each group was subdivided into 3 subgroups according to the staining duration: 7 days, 15 days and 30 days. The pieces were scanned on a 1174 Skyscan microtomograph (Bruker, Belgium) with a voltage of 50 Kv and amperage of 800 μA at the Piracicaba Dental School‐UNICAMP. The scanning of the pieces was done with a pixel dimension equal to 31 μm. After scanning, the images were exported to the software NRecon Reconstruction (Bruker, Belgium) in which they were reconstructed three‐dimensionally in axial sections presenting the x‐ray attenuation coefficients with values relative to the muscle structure reinforced by contrast. The reconstructed images were exported to the software Materialise MIMICS v18 (Materialise, Belgium) to evaluate the anatomical characteristics as well as the density of masticatory muscles. The muscles observed were: Temporal, Masseter (superficial part), Masseter (deep part), External pterygoid, Internal pterygoid and Zigomaticomandibular. These parameters were evaluated in each piece to compare the effects of different contrast concentration and staining duration. The 5% concentration showed better characterization of muscle groups, regardless of the period. At 5%, the 30‐day period showed better definition of muscle fibers. The 3% concentration presented only muscle mass and its contours, but there was no definition in the image that could differentiate each muscle. In the group with a concentration of 10%, contrast enhancement was observed only on the muscular surfaces. Thus, the solution concentration was the most effective parameter to stain the muscle fibers. The preparation and application protocol of iodine‐based contrast solution was most effective at 5% concentration with 30 days action.Support or Funding InformationNational Council for Scientific and Technological Development (PIBIC/CNPq).
Lymphocyte Specific Protein‐1 suppresses TCPOBOP induced hepatocellular proliferation
Authors: Koral Kelly , Bhushan Bharat , Stoops John , Orr Anne , Mars Wendy , Michalopoulos George 
Published: April 2020

Lymphocyte specific protein‐1 (LSP‐1), an f‐actin binding protein expressed in hematopoietic cells as well as hepatocytes, is the gene with the most copy number variations detected in human HCC. Our previous work has demonstrated that LSP‐1 suppresses hepatocellular proliferation following partial hepatectomy as well as in primary hepatocyte cultures. LSP‐1 acts as a scaffold for the Ras/MEK/ERK MAP kinase pathway and along with kinase suppressor of Ras (KSR) inhibits ERK phosphorylation and thus its activation preventing downstream signaling to induce cellular proliferation and migration. To determine if LSP‐1 functions to attenuate hepatocellular growth in models which induce proliferation without loss of tissue or liver injury, we aimed to explore if LSP‐1 can modulate TCPOBOP induced hepatocellular proliferation and hepatomegaly. TCPOBOP (1,4‐Bis [2‐(3,5‐Dichloropyridyloxy)] benzene), a constitutive androstane receptor (CAR) agonist, has been shown to induce hepatocellular hyperplasia through a CAR dependent mechanism. Since LSP‐1 has been shown to suppress hepatocyte proliferation during liver regeneration following partial hepatectomy and in culture, we hypothesized that LSP‐1 will attenuate TCPOBOP driven hepatocellular proliferation. In this study, we administered TCPOBOP to both a global LSP1 knockout mouse model as well as a hepatocyte specific LSP‐1 transgenic (TG) mouse model. Livers were harvested at various time point after TCPOBOP gavage and proliferation was measured using Ki67 and PCNA immunohistochemistry as well as liver to body weight ratios. Increased LSP‐1 expression in the transgenic livers caused decreased Ki67 staining on day 2 after TCPOBOP administration as well as no measurable change in liver to body weight ratios as compared to baseline. However, by day 5, the TG liver to body weight ratios increased above those of the WT but the percentage of Ki67 positive hepatocytes was decreased in the TG as compared to the wild type controls. Loss of LSP‐1 expression led to a statistically significant difference in the liver to body weight ratios between the KO and WT mice on day 2 after TCPOBOP administration as well as increased hepatocellular proliferation on both days 2 and 5 after gavage in the KO livers. CAR target genes and downstream signaling pathways that are modulated by LSP‐1 expression will be analyzed.ConclusionLSP‐1 attenuates TCPOBOP induced hepatocellular proliferation and loss of expression leads to increased hyperplasia supporting the role of LSP‐1 as a growth suppressor.Support or Funding InformationSupported by the Department of Defense grant CA160119, the University of Pittsburgh Menten Endowment, and the Cleveland Foundation Morningstar Fund.
Investigation of the Signaling Mechanisms of the Mu‐Delta Opioid Receptor Heterodimer
Authors: Sieffert Madison Monique , Keresztes Attila , Barker Natalie , Langlais Paul , Streicher John 
Published: April 2020

It has been hypothesized that the mu‐ and delta‐opioid receptors (MOR and DOR) can form a heterodimer (MDOR) and that the resulting heterodimer has a distinct pharmacology. Limited evidence from the literature suggests that MDOR‐selective antagonists could be a novel therapy to enhance opioid efficacy while decreasing side effects. However, due to the lack of selective tools, it is very difficult to study this complex system. We thus recently developed D24M, a novel, first‐in‐class selective MDOR antagonist. When D24M was combined with oxymorphone in vivo, we found potentiation of antinociception and reduction of withdrawal symptoms, validating the hypothesis of the MDOR as an anti‐opioid negative feedback loop and that MDOR antagonists could be novel therapeutics. These striking results prompted us to investigate the underlying signaling mechanisms of the MDOR. First, we treated male and female CD‐1 mice with oxymorphone and/or D24M, analyzed the brainstem using quantitative phospho‐proteomic analysis. Out of the thousands of hits, we found dozens of potential targets that became phosphorylated or dephosphorylated upon exposure to both oxymorphone and D24M. Based on this phospho‐proteomic database, we selected the signaling kinases CaMKII and Src to further investigate in vivo. We ran Western blot and tail‐flick anti‐nociception experiments in male and female CD‐1 mice in the presence or absence of specific kinase inhibitors (KN93 and Src I1) combined with oxymorphone and/or D24M. We found that both Src and CaMKII inhibitors blocked the enhanced anti‐nociception caused by D24M, while not impacting baseline opioid response. These results suggest that Src and CaMKII are both repressed by the MDOR to repress opioid anti‐nociception, which is reversed by D24M treatment. However, Western blot analysis did not show phosphorylation differences in the canonical activation loop sites of either kinase, suggesting that alternate phosphorylation sites identified in our proteomic analysis may be responsible for the signaling effects of these kinases downstream of the MDOR. Our results have thus identified 2 key nodes in the MDOR signal transduction cascade by which the MDOR blocks opioid anti‐nociception, and begun to explore their molecular signaling mechanisms. Future work will expand on the signaling cascades of the MDOR, as well as further establish MDOR antagonists as novel opioid therapeutics.Support or Funding InformationThese studies were funded by NIH R21DA044509 and UG3DA047717 to JMS. The authors have no relevant conflicts of interest to declare.
Epigenetically Regulated DNA Rereplication as a Precursor to Inherited Gene Amplification
Authors: Black Joshua 
Published: April 2020

Genome‐wide analysis of copy number variation (CNV) in cancers has identified chromosomal regions with higher frequencies of gain or amplification. However, we do not understand the origins of many of these CNVs or whether it is possible to prevent their development. We previously demonstrated that epigenetic enzymes can regulate CNVs during S phase by inducing Transient Site‐specific Gains (TSSGs) through DNA rereplication. Regions undergoing TSSG are frequently amplified in tumors suggesting that TSSG events could precede stable inherited amplifications. These previously undetected and unanticipated transient CNVs alter how we think about tumor heterogeneity and the development of inherited CNV.To understand the origins of environmentally induced gene amplifications, we used the classical gene amplification model of the metallothionein (MT) genes. The MT locu sis amplified in cells selected by exposure to copper, zinc or cadmium. Increased expression and amplification of the MT locus correlates with poor overall survival in breast cancer. However, the mechanisms that lead to MT amplification are poorly understood.Here we describe derivation of cadmium resistant breast cancer cells that exhibit progressive amplification of the MT locus. Surprisingly, early focal, transient amplification of the MT locus (MT TSSG) precedes larger inherited gene amplifications. Using novel sequencing technology for detecting DNA rereplication, we demonstrate that the focal transient MT locus amplifications correlate with DNA rereplication. Importantly, regions of DNA rereplication resemble MT amplifications observed in breast cancer patients.We demonstrate that TSSG of the MT locus is epigenetically regulated and long‐term inhibition of epigenetic silencing pathways allows transition from focal MT TSSG to inherited gene amplifications in the absence of selective pressure from cadmium. Furthermore, we demonstrate that transition from focal transient amplifications to stable inherited MT amplifications requires the homologous recombination pathway. Our results describe a new paradigm for the origin of targeted gene amplifications where epigenetically regulated DNA rereplication precedes stable inherited gene amplification.
Intermittent access to cocaine self‐administration impairs performance in a visual attention task in rats: effects of psychostimulants.
Authors: Martin Thomas J. , Kim Susy A. , Eure Mary M. , Blough Bruce E. , Nader Michael A. 
Published: April 2020

Cocaine abuse leads to a number of deficits in cognition, including attention. Several rodent models for attention have been developed, including the 5 choice serial reaction time task. We recently developed a variant of this task in rats (5CTV) that systematically adjusts task difficulty within sessions to match subject performance. Task difficulty is altered by adjusting the visual cue duration dynamically such that the cue duration decreases in trials following correct responses and increases in trials following incorrect responses or omissions. Animals were trained in this task and then permitted to self‐administer infusions of 1.5 mg/kg of cocaine (COCSA) under different access conditions. 5CTV sessions were conducted each weekday 2 hr prior to COCSA sessions. COCSA for 6 hr/d, but not 3 hr/d, produced a progressive impairment in 5CTV performance over 3 weeks, which returned to normal over 2 weeks of forced abstinence. Access to COCSA for 6 hr/d every other day (3 d/wk) however produced a sustained increase in MCD in two‐thirds of the subjects, while one‐third were resistant to this effect. Administration of cocaine, L‐amphetamine, or phenmetrazine prior to 5CTV sessions failed to improve performance in disrupted animals, while caffeine pretreatment resulted in significant improvement. COCSA produces significant disruption in the 5CTV visual attention task in rats, and adenosine antagonists but not catecholaminergic psychostimulants may improve visual attention with ongoing periodic cocaine use.Support or Funding InformationSupported by NIDA P50‐DA006634
Beneficial Effects of Mild Acidosis and Mild Alkalosis Follow the Mirror‐J‐Nonlinearity Curve
Authors: Vičić Ivan , Kovač Zdenko 
Published: April 2020

BackgroundPhysiological agents are strictly regulated within the reference range. Any deviation from that range is considered to be a disruption of homeostasis and a potentially harmful process. However, it has been reported that finite alterations of several physiological agents may behave according to a nonlinearity described as the mirror‐J‐shaped curve. Namely, small deviations from the reference range produce a positive (beneficial) effect, whereas larger deviations of the same physiological agents produce a negative (harmful) effect. Certain clinical trials and basic research data support such concept. These hormetic effects have been described for the physiological agents oscillations of glucose, oxygen, calcium, calories, osmolality, etc. It thus appeared as a more general physiological phenomenon.HypothesisMild acidosis and mild alkalosis may produce a beneficial physiological effect.MethodsResearch of published data that may support the concept of beneficial effects of mild acidosis and mild alkalosis.ResultsMild acidosis has beneficial effects in various experimental and clinical settings, whereas stronger acidosis is regularly toxic. On a cellular level mild acidosis has been proven to prolong human neutrophil lifespan by induction of phosphorylation of Akt, ERK, and p38MAPK, and therefore stabilizing mitochondrial transmembrane potential, delaying mitochondrial permeability transition and apoptosis. The same mechanism was found in ischemia/reperfusion injury models in which induced acidosis during reperfusion attenuated mitochondria‐dependent apoptosis and reduced reperfusion injury. The positive effect of a mild acidosis was also found in models of ventilator‐induced lung injury where permissive hypercapnia exerted positive effects. Mild alkalosis may be physiologically beneficial. The positive effects of mild alkalosis are mostly found in human exercise studies. It has been shown there to potentiate lactate efflux and by doing so improving cellular metabolism. Extracellular alkalosis during exercise contributed to the preservation of intracellular‐to‐extracellular K+ gradient and therefore the stability of the muscle membrane, and overall performance. Mild metabolic alkalosis was also found to increase muscle glycolytic flux and H+ efflux and increased total work done along with decreased exercise‐induced muscle fatigue in humans.ConclusionMild acidosis and alkalosis follow the principle of the mirror‐J‐physiology. This interpretation of counterintuitive studies results could be a step forward in our understanding of integral homeostasis and body adaptation. Expectedly, moderate and strong acidoses and alkaloses have been found harmful/toxic.
Effects of Potassium Supplementation and the Accompanying Anion on Cardiovascular Parameters
Authors: Little Robert , Poulsen Søren B. , Murali Sathish K. , Matchkov Vladimir , Fenton Robert A. 
Published: April 2020

In general, a high NaCl intake is associated with increased blood pressure (BP), whereas dietary potassium intake has a negative correlation with BP, cardiovascular (CV) outcomes and reduces the ‘salt sensitivity’ of BP. High K+ intake reduces activity of the sodium‐chloride cotransporter NCC, promoting natiuresis, which is a proposed major mechanism for mediating the effects of K+ on BP. However, the role of the accompanying anion during K+ supplementation is a matter of debate, especially considering that low plasma chloride levels are associated with better CV outcomes. This study sought to establish an independent role of the anion during chronic potassium supplementation on cardiovascular parameters. Male mice implanted with telemetric BP monitors were initially normalized to a control diet of NaCl (0.3% Na+) and KCl (1.05% K+), and subsequently maintained on this control diet or a high NaCl (1.57% Na+) diet for 7 weeks. Subsequently, mice were stratified to receive either a high KCl (5.25% K+) or a high K‐citrate (KCit, 5.25% K+) diet during control or high NaCl intake for a subsequent 3 weeks. Finally, a subset of mice were switched to a zero KCl diet with either control or high NaCl level. Throughout the experiment BP, heart rate, activity, urinary ion excretion and plasma Na+/K+/Cllevels were monitored.Fractional excretion (FE) of K+ was significantly increased in both high K+ diets after 3 weeks and was independent of NaCl load. KCl and KCit feeding significantly increased FE of Na+ similarly but did not augment an already increased FE Na+ in mice fed high NaCl. High NaCl intake alone had no significant effect on BP, and chronic high K+ diets did not significantly reduce BP. However, overall plasma K+ levels inversely correlated with BP. Interestingly, plasma Cl− levels also inversely correlated with BP. KCl fed mice exhibited lower heart rate relative to KCit fed animals. Regression analysis points to KCit fed mice having lower BP, of about 5–10mmHg, if at the same heart rate as KCl fed animals. In contrast, 2 weeks of low K+ diet increased BP, more notably in high NaCl fed animals (BP increase of 7mmHg). NaCl‐KCit fed mice exhibited a significant 14% reduction in heart weight tibia length ratio (HW:TL) compared to NaCl‐control fed animals. No significant reduction in HW:TL was evident from NaCl‐KCl fed mice.In summary, in the timeframe of this study, potassium supplementation had limited effects on BP in normotensive mice during normal or high NaCl intake despite increasing the FE Na+. However, chronic KCit feeding may have some benefits to cardiovascular health above those promoted by KCl. The molecular mechanisms underpinning these effects are under investigation.Support or Funding InformationThis work was funded by grants from Novo Nordisk Foundation, Danish Independent Research Council and the Leducq Foundation.
Effects of Anti‐Cancer Therapy on Human Microvascular Function ‐ a Longitudinal Study
Authors: Norwood Toro Laura E. , Hader Shelby N. , Didier Daniela , McIntosh Jennifer J. , Freed Julie K. , Kong Amanda , Beyer Andreas M. 
Published: April 2020

RationalCardiovascular toxicities that result from anti‐cancer therapy (CT) significantly contribute to the morbidity and mortality of breast cancer survivors. While the negative impact of CTs such as trastuzumab or doxorubicin on cardiomyocytes is well‐established, significantly less is known about how the circulatory system is affected. Impaired microvascular (MV) function as measured by coronary flow reserve is a recognized predictor of future adverse cardiovascular events. Our goal is to understand how CT damages the microcirculation and how that damage contributes to acute and long‐term cardiovascular toxicities.MethodsTo study the direct effect of CT on the MV function, we are conducting a longitudinal study of neoadjuvant breast cancer patients. Patient adipose samples are collected before, during, and after CT treatment. Endothelial‐dependent dilation to flow (FMD) and acetylcholine (Ach) and smooth muscle‐dependent dilation to papaverine were evaluated in freshly isolated microvessels via video microscopy. NO and mitochondrial ROS were evaluated via florescence microscopy. Data are presented as a percentage of maximum diameter and are analyzed with ANOVA RM with statistical significance set at *p<0.05; data are expressed as mean ± standard error. Additionally, angiogenic potential was evaluated using a matrigel plug assay. Data are presented as a percentage of wells with capillary sprouts. Cardiac function was monitored via standard clinical echocardiogram.ResultsVessels from breast cancer patients showed normal FMD and Ach responses and a normal increase in NO production to flow stimulus before CT; however, CT resulted in impairment of FMD (pre‐CT 80.9±4.2%; during‐CT 21.8%±3.1%*; post‐CT 30.3±4.5%*) and dilation to Ach (pre‐CT 81.3±9.1%; during‐CT 23.1%±1.8%*; post‐CT 38.6±6.5%*). Smooth muscle‐dependent dilation (to papaverine) was not affected by CT treatments. Angiogenic potential was impaired in adipose samples collected during CT (6.5%±6.5%) compared to pre‐CT (47.3%±10.4%). Surprisingly, despite significantly decreased endothelial function, angiogenic potential completely recovered one month after final CT treatment (59.8%±16.4%). No clinical signs of reduced cardiac function were observed in any of our study participants.ConclusionOur study shows for the first time a longitudinal evaluation of human microvascular function in response to anti‐cancer therapy. We observed significant reductions in MV function during treatment with CT in the absence of a clinically relevant reduction in cardiac function. Our findings suggest that CT‐induced MV dysfunction precedes and may contribute to future adverse CV events.Support or Funding InformationThis work was supported by NIH R01 HL133029, We Care Foundation Grant; MCWCardiovascular Center Pre‐PPG grant, Advancing a Healthier Wisconsin – Redox Biology Grant.
Aldosterone mediates a mineralocorticoid receptor‐mediated increase in prostate cancer cell migration
Authors: Feldman Ross , Ding Qingming , Chorazyczewski Jozef , Gros Robert 
Published: April 2020

Aldosterone is a well‐established regulator of renal and cardiovascular functions. However, its role in regulation of cancer growth and spread has only begun to be appreciated. Studies from our laboratory have demonstrated that aldosterone stimulates renal cancer cell migration in vitro and metastatic spread in vivo (Feldman et. al. FASEB J. 2016 30(6)2086–96). However, the generalizability of these findings to other cancers is unknown. Therefore, we assessed the effect of aldosterone on prostate cancer cell migration and the receptor mechanism involved utilizing three widely‐used prostate cancer cell lines, viz. TRAMP C1, LNCaP and DU 145 cells.RT‐PCR analysis detected robust expression of the mineralocorticoid receptor (MR) in all cell lines. GPER expression was detectable in TRAMP C1 and LNCaP cells but not in DU145 cells. In LNCaP and TRAMPC1 cells, aldosterone mediated a concentration‐dependent increase in migration to a maximum of 141±7% of control at 1000 nM (n=5) in LNCaP cells and 125±3 % at 10 nM (n=6) in TRAMP C1 cells, similar to the maximal extent of stimulation by testosterone [146±9% (n=9) in LNCaP cells and 136±9% (n=3) in TRAMP C1 cells]. The effects of aldosterone were inhibited by the MR antagonist eplerenone, but NOT by the GPER antagonist G15. In contrast, the GPER agonist, G1, INHIBITED migration in both cell lines‐effects that were blocked by G15. In DU 145, cells neither testosterone nor aldosterone stimulated migration.In TRAMP C1 cells, infection with an adeno shMR vector blocked aldosterone‐mediated stimulation of proliferation/migration without effects on G1‐mediated inhibition, whereas infection with an adeno shGPER vector selectively blocked G1‐mediated inhibition of proliferation and migration, without effects on aldosterone’s actions.These findings suggest that in TRAMP C1 and LNCaP cells aldosterone stimulates migration via an MR‐dependent pathway. In DU145 cells, resistance to the effect of aldosterone paralleled resistance to the effect of testosterone. These data support the hypothesis that inhibiting aldosterone secretion/effects may be a novel adjunctive therapeutic approach‐‐at least in some forms of prostate cancer.Support or Funding InformationHeart and Stroke Foundation of Canadaand Molson Foundation