bluuebunny/crossref_metadata_2025 huggingface dataset:
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.

Conclusion

LSP‐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 Information

Supported 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 Information

These studies were funded by NIH R21DA044509 and UG3DA047717 to JMS. The authors have no relevant conflicts of interest to declare.
Modulating Motor Activity and White Matter Tracts Through Deep Brain Stimulation Surgery
Authors: Hirt Lisa , Ojemann Steven G. , Thompson John A.
Published: April 2020

Essential tremor (ET) is the world’s most common movement disorder and is more prevalent than Parkinson’s Disease and Epilepsy. Even though there is a high proportion of people with ET, recent evidence suggests that refined diagnostic criteria are needed to better classify or treat ET. Deep Brain Stimulation surgery (DBS) is a highly efficacious therapy for several movement disorders, including ET, that modulates pathologic brain activity in deep brain structures. Although DBS has been used to manage motor symptoms in ET, much remains unknown about how DBS activity in ET target affects down and upstream targets. The FDA approved DBS target for ET within a motor relay nucleus called the ventral intermediate nucleus of the thalamus (VIM). Within VIM, DBS stimulation parameters induce unique volume of tissue activation (VTA) within each patient. By quantifying VTA, clinicians can estimate the percent of VIM that is activated which could elucidate how the activity is spreading to other brain areas. The aim of the current study is twofold, to explore the localization of the VTA (1) within the area of VIM, and (2) use diffusion tensor imaging (DTI) tractography to trace white matter tracts transversing through the VTA to examine what cortical areas are being subsequently stimulated. Preliminary results (N = 11) from IRB approval number 16‐1060, were analyzed using MATLAB and LeadDBS. Calculating voxel data from co‐registered preoperative MRI and postoperative CT data indicates that the percent of VIM activated from VTA is on average 50.33%. DTI data has been collected and is actively being analyzed in MATLAB. This study will overlay brain maps formatted in MNI space to patient DTI data to map cortical areas activated by VTA. Future studies will examine how different electrode types affect and modify VTA, white matter tracts, and patient outcomes.
Creating Computed Tomography Angiography Stereoscopic Models to Enhance the Learning of the Hepatobiliary Region
Authors: Garza Andrew W. , Yang Gongchao , Meyer Edgar R. , Cui Dongmei
Published: April 2020

Current anatomical education continues to increase the use of three‐dimensional (3D) models rendered from radiographic imaging to assist student’s anatomical spatial awareness. Such orientation is valuable and clinically essential, especially when such knowledge is used in conjunction with diagnostic imaging modalities that can further guide treatment plans. Therefore, in programs that rely on cadaveric dissection, it is crucial to efficiently orient novice students to utilize imaging modalities early in their educational careers so that they can recognize any pathological or anomalous structures that other educational 3‐D resources may have limitations in displaying. Some 3‐D rendered models obtained from computed tomography angiography (CTA) have already been clinically derived to facilitate in orienting students to the intracranial neurovascular anatomy. However, there are a limited amount of CTA derived 3‐D models to aid in the study of other anatomical regions, in particular, the hepatobiliary region of the abdomen. In an effort to enhance the continued education of graduate and medical professional students, we have developed a 3D stereoscopic model rendered from CTA‐derived data that are consistent with the pancreatic hepatobiliary region with accompanying vasculature and associated gastrointestinal (GI) tract. Although 3‐D stereoscopic models usually use voxel‐based CTA data, our model uses semi‐auto combined rendering with complementary volume and surface rendering methods. Our ultimate goal is to utilize this virtual model presentation to help students clearly identify anatomical structures, both on cadaveric specimens and through imaging modalities. Future studies will consist of comparing students who are undertaking a gross anatomy course to students who are enrolled in the course but who also view the virtual 3D hepatobiliary model presentation to further support their anatomical education.
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 Information

Supported 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

Background

Physiological 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.

Hypothesis

Mild acidosis and mild alkalosis may produce a beneficial physiological effect.

Methods

Research of published data that may support the concept of beneficial effects of mild acidosis and mild alkalosis.

Results

Mild 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.

Conclusion

Mild 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 Information

This 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

Rational

Cardiovascular 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.

Methods

To 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.

Results

Vessels 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.

Conclusion

Our 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 Information

This 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 Information

Heart and Stroke Foundation of Canadaand Molson Foundation
Reduction of Leishmania activity by N‐acetyl ‐L‐cysteine: in vivo study
Authors: Gugliandolo Enrico , Crupi Rosalia , Siracusa Rosalba , Impellizzeri Daniela , Cordaro Marika , Di Paola Rosanna , Britti Domenico , Cuzzocrea Salvatore
Published: April 2020

Leishmaniasis is a emergent disease characterized by different clinical manifestations in both humans and dogs. The clinical manifestation of this disease comprises a widespread range of expressions, such as skin ulcers at the set of the infection or dissemination in visceral organs followed by leucopenia, anemia, fever and weakness. Predominant clinical features of cutaneous leishmaniasis are ulcerative painless skin lesions. Several data reported that pain is associated with human and dog leishmaniasis, out with areas of painless ulcerative lesions per se. Actually, current medications used for leishmaniasis management are characterized by several side effects and, in addition, some cases of the disease are refractory to the treatment. On this background it is mandatory the identification of new and safe candidates for designing less toxic and low‐cost remedies. Therefore, the search for new leishmanicidal compounds is indispensable. In the present paper we investigated how orally N‐acetyl ‐L‐cysteine (NAC) supplementation affected parameters of inflammation and pain in subcutaneous Leishmania (L). amazonensis infected BALB/c mice, to better understand the contribution of this treatment in the pathogenesis and progress of leishmaniasis. The findings of our study provided the scientific data demonstrating that L. amazonensis infection induces inflammation and pain in BALB/c mice that are reversed by administration of NAC.
Increased Brown Adipose Tissue Mediates Healthful Aging as Reflected by Enhanced Exercise Performance and Protection Against Glucose Intolerance
Authors: Oydanich Marko , Babici Denis , Zhang Jie , Vatner Dorothy E. , Vatner Stephen F.
Published: April 2020

Increased brown adipose tissue results in improved metabolism and protection against cold exposure in humans. The goal of this investigation was to determine if increased brown adipose tissue can improve exercise performance and protect against glucose intolerance. This was accomplished by surgical removal of interscapular brown adipose tissue from c57 wild type mice and transplanting it to other c57 wild type mice. Following transplantation it was necessary to determine how long it would take for any changes to develop. 3 days following transplantation both recipients and donors were exercise tested using a treadmill. No differences were observed between recipients and donors in terms of both running distance (372 ± 14m vs. 377 ± 12m) and work to exhaustion (17.7 ± 1.0 J vs. 16.5 ± 1.4 J). However, at 8 weeks following brown adipose tissue transplantation, the recipient mice demonstrated enhanced exercise performance, p<0.05, in terms of running distance (507 ± 16m) and work to exhaustion (24.3 ± 1.4J), whereas exercise capacity did not change in brown adipose tissue donors. Indirect calorimetry was measured during exercise, with recipients exhibiting improved metabolism, p<0.05, resulting in 7% higher levels of peak oxygen consumption (VO2), 15% higher levels of peak carbon dioxide production (VCO2), and 9% higher levels of peak energy expenditure (EE). The improved exercise metabolism observed in the c57 recipients was accompanied by improved glucose tolerance. Following 8 weeks after brown adipose tissue transplantation, c57 recipients exhibited improved glucose tolerance as demonstrated by glucose tolerance test. Glucose (1g/kg), was administered IP and measurements of blood glucose were made at 30 minute intervals until blood glucose returned to baseline. Glucose tolerance was measured as the area under the curve, which was lower, p<0.05, in brown adipose tissue recipients (7515 ± 676 A.U.) as compared to c57 donors (11051 ± 1035 A.U.). Therefore, increased brown adipose tissue induces healthful aging, both in terms of enhanced exercise performance and improved metabolism resulting in protection against diabetes.
Frequent non‐nutritive sweetener intake alters the expression of endocannabinoid receptors in liver and pancreas in mice
Authors: Mercado-Beltrán Alejandra , Contreras Irazú , Estrada José Antonio
Published: April 2020

The endocannabinoid system plays a major role in the regulation of systemic energy balance, including the modulation of hepatic and pancreatic homeostasis, which is relevant for energy metabolism. Nutritional alterations such as consumption of hypercaloric diets associated with development of obesity promote alterations in hepatic and pancreatic functions and also affect endocannabinoid‐dependent signaling. Non‐nutritive sweeteners have been extensively used to decrease calorie intake from diet and inhibit development of chronic non‐transmissible diseases like obesity and type II diabetes; nonetheless, there is accumulating evidence that these compounds may have additional metabolic effects of clinical significance. The objective of this study is to determine alterations in the expression of CB1 and CB2 endocannabinoid receptors in liver and pancreas of adult mice supplemented with the commercially available non‐nutritive sweeteners sucralose and steviol glycosides, daily, for 6 weeks. After supplementation, hepatic and pancreatic expression of CB1 and CB2 receptors is evaluated by western blot and immunofluorescence. Preliminary results show increased expression of CB1 receptors in male animals supplemented with sucralose, whereas a similar effect is observed in female mice supplemented with sucrose, compared to non‐supplemented controls. In contrast, CB2 expression is increased in male animals supplemented with steviol glycosides and no significant changes have been observed for CB2 expression in females. So far, our results suggest that frequent consumption of non‐nutritive sweeteners alter the expression of endocannabinoid receptors in liver and pancreas in vivo, which may affect the activity of metabolic routes involved in the regulation of systemic energy balance.
Elucidating the biosynthetic pathways of human milk oligosaccharides through a systems biology approach
Authors: Lewis Nathan E. , Richelle Anne E. , Kellman Benjamin E.
Published: April 2020

Human Milk Oligosaccharides (HMOs) are abundant and functionally important components of milk, with major impacts on the health and development of the breastfed infant. Despite their discovery more than half a century ago, their biosynthesis in the human mammary gland remains incompletely characterized. To address this we have developed a framework for predicting glycosyltransferases responsible for synthesizing HMOs and the structures of poorly characterized HMOs. This approach builds nearly 49 million possible pathway maps that could synthesize the 16 most abundant HMOs. From this, we identified the most likely pathways, enzymes, and HMO structures when analyzing the pathway maps in the context of transcriptomic data and HMO measurements from two cohorts of women. We subsequently validated the predicted enzymes through assays. Through this we were able to unravel the molecular basis for HMO biosynthesis including enzymes responsible, and therefore this knowledge can now be used to guide new strategies for HMO synthesis for academic and nutritional use.
A gardian of gut epithelial barrier from inflammation: the elastase inhibitor ELAFIN
Authors: Deraison Celine , Jaeger Marie Charlotte , Guiraud Laura , Rolland Claire , Bellot Audrey , Rolland Corinne , Sebbag Mireille , Bermudez Luis , Langella Philippe , Vergnolle Nathalie
Published: April 2020

In the gastrointestinal tract (GIT), proteolytic activity is tightly regulated to prevent inappropriate and potentially harmful proteolysis. In IBD this regulation is broken and our recent work has demonstrated that elastolytic activity is significantly increased in colonic epithelium from IBD patients. We have shown that the expression of the only elastase released by epithelial cells, ELA2 is up‐regulated; and the selective elastase inhibitor ELAFIN is severely down regulated in colonic epithelium from IBD patients. We showed that oral treatments with ELAFIN‐expressing L. Lactis bacteria restored proteolytic balance in the inflamed gut and was protective against acute and chronic colitis. ELAFIN is a low‐molecular weight molecule (10‐kDa), which specifically inhibits elastases. At N‐terminal terminus, a cationic helix showed anti‐microbial activities and could interact with phospholipids. Moreover ELAFIN is also able to inhibit NFkB and AP‐1 activation but this domain is not localized on protein. Therefore, several mechanisms of action are thus possible to explain ELAFIN’s protective property.

Objectives

If ELAFIN‐recombinant L. lactis has to be considered as a possible treatment for IBD in human, there is an absolute need to define the mechanisms by which ELAFIN delivery protects against intestinal inflammation.

Methods

We generated and expressed in LAB ELAFIN’s mutants allowing investigation the biological properties of ELAFIN individually. These different forms of ELAFIN‐recombinant LAB were co‐culture with thapsigargin (10mg/ml) and ELAFIN (107 CFU/well). After 6H, cells were additionally exposed to gentamicin (100 mg/ml) to limit bacterial development. The barrier function was measured using FITC‐dextran passage through the epithelial layer. The elastolytic activity and CXCL8 release were quantified from the culture media. Expression of inflammatory mediators was quantified by qPCR.

Results

Addition of thapsigargin for 24H on Caco2 cells induced ER‐stress and disruption of elastolytic balance. ELAFIN expression was decreased with up‐regulation of ELA2, leading to increase of elastolytic activity in medium, disclosed in IBD patients. Concomitantly, barrier function was reduced, and expression of CXCL8, CXCL10 and hBD2 was increased. Co‐culture with ELAFIN expressing‐LAB normalized all these parameters confirming anti‐inflammatory properties of ELAFIN. Neutralization of a‐helix (mutant K44D/K50G) did not modify ELAFIN’s properties. However, disruption of protease inhibitory function (mutant A62G/M63G) led to unability of ELAFIN mutant to modify permeability and inflammatory phenotype induced by exposure of intestinal epithelial cells to thapsigagin.

Conclusion

This suggests that anti‐inflammatory activity of ELAFIN is based on the control of elastase activity released by epithelial cells.
Identification of Key Factors in Cardiomyocyte Development by Single‐Cell Transcriptome Analysis
Authors: Zhu Jun , Wu Dingfeng , Gao Wenxing , Chen Wanning , Fu Qianzheng , Jiao Na , Zhu Lixin , Zhu Ruixin
Published: April 2020

Background

A promising intervention for heart diseases is the application of cardiomyocytes differentiated from pluripotent stem cells. However, the driving factors of the differentiation process still remain unclear. This study aims to map the transcriptional landscapes of directional differentiation of pluripotent stem cells at multiple stages, using single‐cell sequencing data, in order to identify the key drivers of the differentiation process.

Method

Single‐cell sequencing data from pluripotent stem cell‐derived cardiomyocytes at age day 0, day 2, day 5, day 15 and day 30 were included and normalized. UMAP algorithm was then used to reduce the dimensionality of the transcriptome profiles, followed by community detection to group the cells and cell type identification using marker genes. Graph‐autocorrelation analysis was implemented to identify differentially expressed genes among cell groups with Gene Ontology term enrichment. Next, we calculated the pseudotimes of each cardiomyocyte to construct the pseudotime trajectories. Finally, knowledge based dynamic changes in gene expression were analyzed to identify the key factors driving the cardiomyocyte development.

Result

We identified 9 different cell types in the process of cardiomyocyte development, including cardiomyocytes, induced pluripotent stem cells, cardiogenic mesoderm cells, cardiovascular progenitor cells, embryonic stem cells, endoderm cells, epithelial progenitor cells, cardiomyocyte progenitor cells and multipotent progenitor cells. Identification of cardiomyocyte was confirmed with specific gene expression profiles such as significant enrichment in the biological processes of cell adhesion and supramolecular fiber organization. The expression of NKX2‐5, which encodes a homeobox‐containing transcription factor, was found to be at a lower level in the early stages and continually increased as cardiomyocyte develop, suggesting that NKX2‐5 was one of the driving factors of cardiac development, acting through promoting myocardial maturation and development.

Conclusion

Single cell sequencing data revealed the dynamic changes of gene expression during cardiomyocyte development differentiation. NKX2‐5 was identified as a key factor in cardiac development. Our finding contributes to the understanding of the mechanism behind the cardiomyocyte development, and may serve as the basis for stem cell based therapies.

Support or Funding Information

This work was supported by National Natural Science Foundation of China 81774152 (to RZ) and 81770571 (to LZ).
Secreted insect transferrin‐1 with strong and reversible iron‐binding has potentially tissue specific roles in immunity and iron transport
Authors: Weber Jacob J. , Park Yoonseong J. , Gorman Maureen
Published: April 2020

Iron is biologically essential and potentially toxic. To maintain healthy physiological systems, iron must be properly sequestered, transported and stored in animals. The transferrin family of proteins plays a major role in maintaining this homeostasis in vertebrate animals. However, little is understood about iron homeostasis and transferrin’s role in invertebrates. In mammals there are two well understood secreted iron‐binding transferrins: (1) serum transferrin (STF) binds two ferric‐iron ions in the blood and transports them into cells by receptor mediate endocytosis; (2) lactoferrin (LTF) functions as an immune protein by sequestering two ferric‐iron ions in secreted fluids to inhibit bacterial growth. Insect Transferrin‐1 (Tsf1) is found in insect hemolymph and secreted fluids and is homologous to STF and LTF; however, its function in the iron transport and immunity of invertebrates is not clear. As a first step towards elucidating its function, I have purified recombinant Drosophila melanogaster Tsf1 (DmTsf1) and hemolymph derived Manduca sexta Tsf1 (MsTsf1). Comparing the spectroscopic and kinetic data of DmTsf1 and MsTsf1 to STF and LTF shows that these Tsf1s coordinate iron differently and have a relatively lower but still potent iron binding affinity at pH 7.4 (DmTsf1 log K′= 17, MsTsf1 log K′= 17 and STF log K′ = 20). The Tsf1s release iron over the pH range of approximately 6 to 4, which is nearly identical to STF and indicates the possibility of iron release in the endosome. Current work is aimed toward determining how iron from Tsf1 is transported into cells. One hypothesis is that Tsf1 is endocytosed and iron is released in the endosome—similar to the iron transport mechanism of STF in mammals. I tested this hypothesis using immunohistochemistry and confocal imaging of Drosophila tissues. Immunostaining shows that DmTsf1 colocalizes with an early endosome marker (Rab5) in the developing oocyte, supporting my hypothesis of iron delivery. DmTsf1 was also found in the tracheal tissue, but instead of clearly colocalizing with Rab5, it appears that Tsf1 is secreted into the tracheal lumen and not taken back up. Overall, these results indicate that Tsf1s may have tissue specific roles: iron delivery to the oocyte and iron sequestration in the tracheal lumen. Future work will be completed to test if Tsf1 is endocytosed in other tissues and if it plays an important role in supplying iron to these tissues.

Support or Funding Information

NSF: 1656388
Gemcitabine Exposure Induces Epigenomic Remodeling in Pancreatic Cancer Cells during Resistance Development
Authors: Urrutia Guillermo , Dusetti Nelson , Iovanna Juan , Urrutia Raul , Lomberk Gwen
Published: April 2020

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer‐related deaths in the United States with a devastating overall five‐year survival rate of less than five percent. Chemotherapy is a mainstay for PDAC treatment, with Gemcitabine (Gem) as a part of a first‐line regimen. Although Gem has shown clinical benefit, it possesses limited control over cancer progression, due to the major obstacle of chemoresistance. Consequently, there is an urgent need to further improve our understanding of molecular mechanisms underlying resistance development to identify innovative therapeutic targets. Our laboratory is focused on the use of epigenomic drugs as novel therapeutic approaches. Our experimental design involves developing chemoresistance in patient derived PDAC cells to identify epigenomic alterations and gene expression changes that can participate in the initiation and establishment of resistance to chemotherapeutic agents. Identifying such modifications can provide valid druggable targets to enhance the effect or reverse resistance to anti‐cancer drugs. In the present study, we induced resistance to Gem on a naïve patient‐derived PDAC cell line and evaluated epigenomic landscape remodeling through changes of histone post‐translational modifications. In addition, we characterized phenotypic changes in response to Gem exposure. Using the Incucyte® imaging system, we determined that exposure to increasing concentrations of Gem induced resistance on naïve patient‐derived PDAC cells. Gem resistant cells showed a reduced basal proliferation rate and apoptotic response compared to parental cells, as measured by activation of caspases 3 and 7. Additionally, cells developed morphological changes towards an epithelial state, coincident with the increase of Keratin 17/19 and E‐Cadherin. Moreover, treated cells demonstrated reduced sensitivity to the well‐known DNA damaging agents cisplatin and 5‐Fluorouracil. Evaluation of histone post‐translational modifications on these cells revealed reduction in the global amount of repressive marks (H3K9Me1, 2 and 3; H3K27Me2 and 3), as well as an increase of the activating histone mark H3K27Ac, associated with super enhancers. Furthermore, immunofluorescence‐based microscopy showed re‐distribution of the H3K9Me1 and H3K27Me3 repressive marks from the nuclear periphery to the whole nucleus, suggesting epigenomic remodeling of pancreatic cancer cells in response to the development of Gem resistance. In summary, these data demonstrate that Gem treatment induces chromatin remodeling and that targeting key epigenomic pathways may hold promise for novel strategies toward re‐sensitizing or preventing resistance of pancreatic cancer to chemotherapeutic agents.
Distinct projections from the prelimbic cortex modulate active avoidance
Authors: Valentín-Valentín Viviana P. , Diehl María M. , Iravedra-García Jorge M. , Morán-Sierra Jonathan , Rojas-Bowe Gabriel , Gonzalez-Díaz Fabiola N. , Quirk Gregory J.
Published: April 2020

Avoiding threatening stimuli is a survival mechanism that can become maladaptive when avoidance interferes with other goal‐directed behaviors. For example, when an individual is seeking food, a threatening stimulus may be encountered, leading to an avoidance response, which comes at a cost of obtaining food. To model this behavior, we used an active avoidance task in which food‐restricted rats learn to step onto a platform to avoid a tone‐signaled shock, at the cost of obtaining a sucrose pellet. Activity in prelimbic cortex (PL), basolateral amygdala (BLA), and ventral striatum (VS) are necessary for the expression of platform‐mediated active avoidance (PMA) (Bravo‐Rivera, et. al., 2014), suggesting that these structures form a neural circuit to regulate PMA. We recently reported that inhibitory tone responses in PL signal the avoidability of shock, and opposing these responses using 4Hz photoactivation impaired avoidance (Diehl, et. al., 2018). Here, we want to know if PL inhibition signals downstream structures that are necessary for avoidance (i.e. VS and BLA), using an optogenetic approach. Photoactivating PL‐VS projections using Channelrhodopsin (ChR2) impaired avoidance (eYFP 88% (n=12) vs. ChR2 22% (n=8), p<0.0001), whereas photosilencing PL‐VS projections using Archaerhodopsin (ArchT) had no effect, suggesting that inhibition of this pathway promotes avoidance. Additionally, photoactivating PL‐BLA projections accelerated avoidance (eYFP 75% (n=9) vs. ChR2 88% (n=8), p=0.017), and photosilencing PL‐BLA projections impaired avoidance (eYFP 77% (n=9) vs. ArchT 40% (n=7), p=0.026), suggesting that excitation of this pathway promotes avoidance. Overall, these findings suggest that distinct PL projections have opposing roles in controlling avoidance by driving or suppressing avoidance.

Support or Funding Information

This work was supported by NIH grants R37‐MH058883 and P50‐MH086400 (Conte Center) to GJQ; F32‐MH105185 to MMD; 5T34‐GM00782140 to GRB, 5R25‐GM097635 to JIG and VVV; 5R25‐GM061151 to GRB, FDG and JMS.