bluuebunny/crossref_metadata_2025 huggingface dataset:
Locally formed Peroxynitrite inhibits endothelial TRPV4 channels and elevates blood pressure in obesity
Authors: Ottolini Matteo , Hong Kwangseok , Cope Eric Lee , Daneva Zdravka , Johnstone Scott R. , Isakson Brant , Sonkusare Swapnil K.
Published: April 2020

Impaired endothelium‐dependent vasodilation is a hallmark finding of obesity‐induced hypertension. Recognition that Ca2+ signaling in endothelial cells promotes vasodilation has led to the hypothesis that endothelial Ca2+ signaling is compromised during obesity, but the underlying abnormality is unknown. Here, we show that Ca2+ influx through TRPV4 channels at myoendothelial projections (MEPs) to smooth muscle cells lowers resting blood pressure in non‐obese mice, a response that is diminished in obese mice. Counter‐intuitively, release of nitric oxide (NO) from endothelial cells attenuates TRPV4 channel activity and TRPV4‐mediated vasodilation in the obese animals. Increased activities of iNOS and NOX1 correlated with elevated levels of NO and superoxide radicals at the MEPs in obese mice, ultimately reacting to form peroxynitrite. Subsequent findings revealed that peroxynitrite causes cysteine oxidation of the regulatory protein AKAP150 to inhibit TRPV4 channel activity at the MEPs. TRPV4 channel activity and vasodilation are also impaired in the arteries from obese patients, and are restored by strategies that lower peroxynitrite levels. These data suggest that defective TRPV4 channel–mediated vasodilation may contribute to obesity‐induced hypertension, and imply that inhibiting peroxynitrite may represent a strategy to normalize endothelial TRPV4 channel activity, vasodilation and blood pressure during obesity.

Support or Funding Information

HL121484‐01, HL138496
Preliminary Characterization of the 125I‐Angiotensin 1‐7 Binding Site in Rodent Liver
Authors: Stoyell-Conti Filipe Fernandes , Chabbra Alesa , Speth Robert C.
Published: April 2020

Angiotensin 1–7 (Ang 1‐7) is a product of the renin‐angiotensin system that acts centrally and peripherally to reduce blood pressure and improve baroreflex sensitivity. For more than a decade Mas has been viewed as the receptor for Ang 1‐7, however, Ang 1‐7 binding has not been pharmacologically characterized in tissue membrane preparations. In this study, we show that 125I‐Ang 1‐7 binds to a site for which Ang 1‐7 has low affinity. To assess the possibility that the low affinity of Ang 1‐7 to compete for 125/127I‐Ang 1‐7 binding was due to its susceptibility to metabolism by peptidases in the tissue membrane homogenate, the integrity of Ang 1‐7 as well as 127I‐Ang 1‐7 (iodoAng 1‐7) was evaluated by HPLC analysis. Both Ang 1‐7 and iodoAng 1‐7 were substantially metabolized in liver membrane homogenates at room temperature (RT). However, when incubated on ice (~0 C), 125I‐Ang 1‐7 binds with moderately higher affinity (~3 nM versus 10–20 nM at RT) to a binding site in rat liver membranes that is displaceable by iodoAng 1‐7 at nanomolar concentrations, but Ang 1‐7 still shows a low affinity to compete (1.4μM for Ang 1‐7and 69.4nM for iodoAng 1‐7). At RT 125I‐Ang 1‐7 binding is partially displaceable by metalloprotease (MMP) and A Disintegrin And Metalloprotease (ADAM) inhibitors, e.g., marimastat, batimastat, and GM‐6001 at nanomolar IC50 values, similar to their IC50 values for MMP activity, suggesting that 125I‐Ang 1‐7 binds to MMPs and/or ADAMs as well as other tissue elements at RT. However, when incubated at ~0 C, the MMP and ADAM inhibitors had micromolar IC50 values, suggesting that 125I‐Ang 1‐7 binding to these peptidases is temperature dependent. Our results continue to suggest that the addition of an iodine molecule to the tyrosine in position 4 of Ang 1‐7 drastically changes the characteristics of this peptide making it unsuitable for characterization of Ang 1‐7 receptors. Continuing studies are underway to identify the peptidase(s) or other binding moieties to which 125I‐Ang 1‐7 binds to determine their potential therapeutic importance as druggable targets.
Enalapril‐associated combined training is most effective in reducing SBP in spontaneously hypertensive rats
Authors: de Araujo Amanda Aparecida , Shecaira Tânia Plens , Paixão Camila , da Silva Dias Danielle , Irigoyen Maria Cláudia , De Angelis Kátia
Published: April 2020

High blood pressure is the leading risk factor to development of cardiovascular disease worldwide. Diuretic and angiotensin‐converting enzyme blockers treatment is widely used for its efficiency in reducing blood pressure (BP). Combined physical training (aerobic + resistance) is recommended for hypertensive patients, however, it is not yet clear whether the combination of antihypertensive treatment with this training modality results in additional benefits. Therefore, the aim of this study was to compare the effects of combined training with different drug treatments on systolic BP (SBP) of spontaneously hypertensive rats. Twenty‐eight SHR rats were divided into four groups (n=7/group): HSM (hydrochlorothiazide sedentary males), HTM (hydrochlorothiazide trained males), ESM (enalapril sedentary males) and ETM (enalapril trained males). Drugs were administered in drinking water at the following doses: enalapril 10 mg/kg/day and hydrochlorothiazide 30 mg/kg/day. All groups underwent exercise testing before and after 2 months. The trained groups performed combined training 3 times a week, being aerobic and resistance at same session, moderate intensity. The SBP was analyzed by caudal plethysmography before starting the drugs and 24h after the last training session. As a result, only HTM group increased the body weight (HTM vs. HSM, p=0.001). Both trained groups increased maximal aerobic exercise test (p<0.0001) and resistance exercise test (HTM vs. HSM, p=0.02; ETM vs. ESM, p<0,0001) versus the group control. We observed that there was no difference in the initial SBP between the groups (HSM 176±4.3, HTM 175±3, ESM 173±1.4 and ETM 183±3 mmHg) and there was a reduction in the HTM (161±1.3 mmHg), ESM (159 ± 4.4 mmHg) and ETM (147 ± 3.3 mmHg) groups. In addition, the trained group associated with enalapril treatment further reduced SBP when compared to the hydrochlorothiazide‐trained group (ETM vs. HTM, p=0.02). Therefore, the data support that the combination of drug treatment with enalapril and combined physical training in the same training session is more effective in reducing blood pressure than the combination with hydrochlorothiazide. It is noteworthy that the training proposed in this study was performed three times a week, getting closer to human reality and being enough for additional benefits in the control of blood pressure of spontaneously hypertensive rats.

Support or Funding Information

CAPES
GPER Contributes to the Development of Pulmonary Hypertension in Female Rats
Authors: Ahmadian Rosstin , Sheak Joshua R. , Weise-Cross Laura , Jernigan Nikki L. , Arterburn Jeffrey B. , Prossnitz Eric R. , Snow Jessica B. , Resta Thomas C.
Published: April 2020

Studies from our group and others demonstrate a protective role of estrogen in attenuating the progression of chronic hypoxia (CH)‐induced pulmonary hypertension. Physiological responses to estrogen are mediated by the nuclear estrogen receptors a and b, as well as by the membrane‐bound G protein‐coupled estrogen receptor (GPER). However, the role of GPER in the development of pulmonary hypertension resulting from CH is unknown. We hypothesized that GPER acts to limit the severity of CH‐induced pulmonary hypertension.

To test this hypothesis, we evaluated indices of pulmonary hypertension in control and CH (4 wk, PB = 0.5 atm) female rats administered the GPER antagonist G36 via osmotic pumps (1 mg/kg/day, s.c.) during the entire 4 wk exposure period (n=7–8/group). Right ventricular systolic pressure (RVSP) was measured using a fluid‐filled catheter advanced through the right jugular vein into the right ventricle (RV) during isoflurane anesthesia. Additionally, RV hypertrophy was determined from Fulton’s index [RV/(LV+S)], and the polycythemic response to CH was assessed by measuring hematocrit.

CH increased RVSP, Fulton’s Index, and hematocrit in vehicle‐treated rats (P<0.0001), indicative of pulmonary hypertension and associated polycythemia. Unexpectedly, RVSP was lower in G36‐treated CH rats compared to control animals (P<0.05), with a downward trending Fulton’s Index (not significant). However, hematocrit was unaltered by GPER inhibition. We conclude that GPER mediates a novel effect to facilitate the development of pulmonary hypertension resulting from CH exposure.

Support or Funding Information

NIH Grants R01 HL132883, T32 HL007736, and P20 GM103451. AHA Grant 18AIREA33960020
Mathematical model of oncolytic virotherapy with Myxoma virus infection in cancer cells
Authors: Ortegon Sofia , Franco Lina S. , McFadden Grant , Cordovez Juan
Published: April 2020

Oncolytic virotherapy is an innovative alternative to more conventional cancer therapies that is based on the ability of oncolytic viruses to specifically target and kill tumor cells. This therapy can activate the innate and adaptive immune response of the body achieving the destruction of cancer cells by the immune system. Myxoma virus is a poxvirus that infects in European rabbits, can also replicate in a variety of human cancer cells lines, which makes it an ideal candidate for oncolytic virotherapy. However, the dynamic mechanisms between the virus and the cancer cells are not completely understood. Mathematical models are useful tools to provide relevant insights about the dynamics that govern the interplay between normal and cancer cells and their viral parasites. In this study murine melanoma cancer cells were infected with a Myxoma virus expressing GFP (vMyx GFP) at low multiplicity of infection and pictures were taken in a fluorescence microscope every 30 minutes to determine infected cells. To understand the process of infection, namely the susceptible to infected conversion rates and the spatial effects of cell culture, we developed a mathematical model parameterized with the experiments described above that spatially explicitly simulates the process of infection. The model is an stochastic spatially explicit SI formulation that considers cancer cells and viral particles. With the model we were able to quantify and better understand this complex network of interactions. We were able to estimate important parameters concerning infection, proliferation and spatial dependency. We hope that the model will contribute to accelerate the development of a functional and effective oncolytic virotherapy with Myxoma virus.

Support or Funding Information

Office of the vice‐presidency of research at los Andes University
Role of Mitochondrial Ca2+‐Activated Cl− Channel anoctamin‐1 and mitochondrial fusion protein OPA1 in pulmonary arterial hypertension
Authors: Cypress Michael W. , Vang Alexander , Fernandez-Nicolas Ana , Mancini Thomas , Jhun Bong Sook , Clements Richard T. , Choudhary Gaurav , O-Uchi Jin
Published: April 2020

Activation of a Ca2+‐activated chloride channel, anoctamin‐1 (Ano1), is associated with enhanced cell proliferation in multiple cell types, such as endothelial cells (ECs). Recent proteomics studies indicate that Ano1 may interact with mitochondrial proteins in addition to plasma membrane (PM)‐localized proteins. However, the expression levels of Ano1 and its functions in other cellular compartments including mitochondria remain unclear. We previously showed that Ano1 is overexpressed in pulmonary artery ECs isolated from patients with idiopathic pulmonary arterial hypertension (PAH) and that Ano1 activation promotes apoptosis of pulmonary artery ECs from patients with idiopathic PAH, likely via depolarization of mitochondrial membrane potential and increased mitochondrial reactive oxygen species (ROS) generation. Therefore, we hypothesized that Ano1 exists not only at PM, but also in the mitochondria, which may contribute to a hyperproliferative phenotype of ECs under PAH. Here, we determined the mitochondrial expression of Ano1 and further investigated its role in the mitochondria. Subcellular localization of Ano1 was determined by immunostaining of Ano1 in Rat lung microvascular ECs (RLMVECs) and by live cell imaging of HEK293T cells expressing GFP‐tagged Ano1. The mitochondrial and PM localization of Ano1 was also confirmed by biochemical assays using protein fractionation from RLMVECs. Moreover, co‐immunoprecipitation assay showed that Ano1 interacts with a mitochondrial fusion protein OPA1 which is the critical regulator of mitochondrial bioenergetics and cristae formation and possesses a trans‐membrane domain at the inner mitochondrial membrane (IMM). These results indicate that 1) Ano1 expresses at the IMM in addition to the PM and 2) Ano1 and OPA1 interactions may be partly involved in the molecular mechanism underlying the hyperproliferation and apoptosis resistance of pulmonary artery ECs during PAH
TMEM184A: Evidence for a Potential Link Between HSPGs and Mechanotransduction
Authors: Talotta-Altenburg Leanna M. , Ciabattoni Grace O. , Laessig Minuette A. , Lowe-Krentz Linda J.
Published: April 2020

Heparin is a known anticoagulant that, with cellular uptake, contributes to a variety of physiological conditions, including experimentally observed decreases in vascular cell proliferation and inflammation. The fact that heparin is endocytosed and decreases cellular responses rather than blocking them entirely suggested a receptor mediated model, which has been previously identified in our laboratory as TMEM184A. Our data suggest that TMEM184A associates with vascular endothelial‐cadherin (VE‐Cadherin) and heparan sulfate proteoglycans (HSPGS), modulating mechanotransduction events in response to heparin treatment and fluid shear stress (FSS), which replicates the stress induced by blood flow under physiological conditions. TMEM184A seems to play a role in trafficking HSPGs such as syndecan‐1 (SDC1) and glypican‐1 (GPC1), both of which have been characterized as mechanosensing proteoglycans. In recent studies it has been demonstrated that VE‐Cadherin may interact with HS chains. Data in the literature have shown that when HS chains are degraded by Heparinase III treatment, the strain induced decrease in VE‐Cadherin is abolished, indicating that the HSPGS are crucial in sensing the strain. Given these findings, as well as our preliminary evidence that TMEM184A associates with HSPGs, we hypothesize that TMEM184A acts as an interface in the mediation of mechanosignaling events on the extracellular surface of adherens junctions. Co‐immunoprecipitation pull down of TMEM184A followed by subsequent western blotting and immunostaining showed both VE‐Cadherin and TMEM184A in the bound supernatant, indicating that the two may colocalize at endothelial adherens junctions and associate in a mechanosensing complex. Using a GFP‐TMEM184A plasmid construct to overexpress TMEM184A in bovine aortic endothelial cells (BAOECS), our research team observed increased surface levels of VE‐Cadherin in adherens junctions under static flow conditions. These overexpressing cells were subjected to FSS, which increases TMEM184A associations with HSPGs. Our team observed an additive increase in VE‐Cadherin in adherens junctions when compared to overexpression alone in static conditions. An additional experiment done to elucidate the role of endogenous heparin (HSPGs) revealed that BAOECs washed with a concentrated salt buffer to remove HPSG‐protein interactions demonstrated decreased colocalization of VE‐Cadherin, TMEM184A, and various vesicle associated membrane proteins. The addition of heparin rescued these colocalization events, implicating HSPGs in this complex. Our ongoing work aims to elucidate how associations of TMEM184A with VE‐cadherin and HSPGs facilitate activation of mechanically induced signals and mediate the trafficking of adherens junctions’ surface proteins.

Support or Funding Information

HL54269 to LJLK
Investigating the Role of Introns in Transcription‐Associated Mutagenesis in Budding Yeast
Authors: Lansangan Cedric Kier , Kim Jane
Published: April 2020

A key feature required for cell survival is genomic stability, which is the ability of a cell to prevent mutations in its genome and repair them if they do occur. Genomic mutations can be generated in several ways, including transcription of a gene via transcription‐associated mutagenesis (TAM). Introns are known to be able to enhance transcription in vivo, but the influence of introns on TAM is not well‐understood. Here, our work investigates whether introns modulate the rate of TAM in a URA3 reporter gene using budding yeast as a model organism. Our preliminary results suggest that the presence of introns in the natively‐intronless URA3 gene is associated with elevated mutation rates of that gene. Interestingly, there appears to be an intron‐length‐dependent effect on this trend (i.e. the yeast strain with a long intron in the URA3 reporter demonstrated elevated URA3 gene mutation rates compared to the other two strains with either a short intron or no intron). Our findings suggest that the length and presence of introns in a gene are associated with increased mutagenesis of that gene. Furthermore, the long‐intron‐containing strains in our study grew slower in the absence of uracil than the short‐intron strains and no‐intron controls, possibly indicating an effect of intron length on splicing of the URA3 primary transcript. We are currently investigating if elevated transcription rate of a URA3 gene containing an intron causes an increase in TAM rate. In addition, URA3 reporter gene expression levels are being quantitated in each strain. We expect that URA3 mRNA transcript levels will be elevated in the intron‐containing URA3 yeast strains relative to those of the no‐intron controls. In future work for this study, we will determine the effect of the presence of an intron located near the promoter of an induced URA3 gene on the rate of TAM. Our future results will contribute to better understanding the impact of introns on the mutagenesis of highly‐expressed genes, which may play a role in mechanisms underlying mutagenesis in the human genome.

Support or Funding Information

Dr. Jane Kim’s research is supported by an NIH SCORE Grant (SC3GM127198).
Characterization of Extracellular Vesicle LINE‐1 mRNA, Protein, and Reverse Transcriptase Activity Profiles
Authors: Bowers Emma , Cavalcante Alexandre , McKay Brian , Ramos Kenneth
Published: April 2020

Lung cancer has the highest mortality rate among all cancers, making early detection of this malignancy imperative for survival. Ongoing studies in our laboratory have focused on developing Long Interspersed Nuclear Element‐1 (LINE‐1) in circulating lung extracellular vesicles (EVs) as a novel mechanism‐based ‘liquid biopsy’ for lung cancer. LINE‐1 is a mammalian retroelement that can ‘copy and paste’ itself and other DNAs into different loci via a reverse transcriptase‐mediated mechanism. While LINE‐1 is epigenetically silenced in most healthy cells, its activity can be initiated by harmful environmental exposures and is strongly associated with cancer risk and mortality. Evidence is presented here that LINE‐1 mRNA and protein are present in EVs isolated from H460 lung cancer cells and human plasma and are segregated into distinct EV populations. Because reverse transcription (RT) of LINE‐1 requires formation of a ribonucleoprotein complex containing LINE‐1 mRNA and proteins, we hypothesize that this segregation has been engineered to control RT. In support of this hypothesis, we found that RT activity is only present when these two EV populations are lysed and combined. We then isolated EVs from ostensibly healthy plasma donors and found substantial inter‐individual variability with respect to LINE‐1 protein, mRNA, and RT activity. The variability between individuals and the ease of measurement from clinical samples further supports the use of these LINE‐1 metrics as health‐related biomarkers. After characterizing EV LINE‐1, we sought to determine whether EV LINE‐1 levels could serve as a proxy of cellular LINE‐1 content, which is critical to our overall goal of using isolated lung EVs as a liquid biopsy of the lung epithelium. Using a panel of six different lung epithelial cell lines, we compared baseline LINE‐1 expression between cells and EVs. We also treated lung epithelial cells with different concentrations of benzo(a)pyrene (BaP), a cigarette smoke carcinogen known to induce LINE‐1, and compared LINE‐1 levels in cells and EVs. Thus far we discovered that, in both baseline and toxicant‐induced scenarios, EV LINE‐1 mRNA levels mirror cellular levels. Together, these data demonstrate that EV LINE‐1 content and RT activity can be quantified in cultured cells and clinical samples and supports the use of EV LINE‐1 cargo as a biomarker of lung epithelial cell status.

Support or Funding Information

This work was funded in part by grants from the University of Arizona Health Sciences, the Southwest Environmental Health Sciences Center (P30 ES006694), the National Cancer Institute Cancer Center Support Grant (P30CA023074), the UAZ Bio5 Fellowship and GURI Texas.
A Flexible and Reproducible Framework for Modelling Renal Tubules
Authors: Nickerson David Phillip , Sarwar Dewan , Noroozbabaee Leyla , Kalbasi Reza , Atalag Koray , Hunter Peter
Published: April 2020

We have developed a new framework for defining renal tubule models utilising a repository of existing models from the literature. The CellML format (
https://cellml.org) is an XML format for encoding mathematical models ‐ typically used for lumped parameter, non‐spatial models of physiological systems. We have previously demonstrated how models of epithelial transport processes can be discovered in public model repositories such as the Physiome Model Repository (
https://models.physiomeproject.org) and reused in building novel models at the epithelial cellular spatial scale.

In this work we present a novel Python‐based framework in which we extend our previous work to now look at solute transport and flow in generic tubule segments. The Python code captures the standard equations for flow and solute transport and we link to CellML descriptions of transport mechanisms to define the transport of solutes between the lumen, intracellular space, and interstitial fluid.

By linking to models of, for example, epithelial transport proteins encoded in the CellML format we are able to configure the generic tubule segment to represent known physiology of the various nephron segments in the kidney. As the number and variety of suitable models in the model repository grows, we will be able to “automatically” discover and utilize them in this framework.

In future work, we will are attempting to utilize clinical data captured in semantic knowledge frameworks, such as openEHR, to guide the discovery and parameterization of the epithelial cellular transport processes based on clinical or experimental observations.

Support or Funding Information

We gratefully acknowledge funding from the Aotearoa Foundation, Medical Technologies Centre of Research Excellence, and the Auckland Bioengineering Institute.
Regional Effects of Monoamine Transporter Blockers on Extracellular Monoamine Levels
Authors: Harris Sharonda , Green Sara M. , Garber Lexi , Urs Nikhil M.
Published: April 2020

Monoamine transporter inhibitors are used in the treatment of a number of psychiatric disorders, including major depression and ADHD, and have high abuse potential. These drugs increase extracellular levels of monoamines by blocking the reuptake of monoamines through their respective transporters. Although selective norepinephrine (NE), dopamine (DA) and serotonin (5‐HT) transporter inhibitors increase NE, DA and 5‐HT, respectively, through blockade of their cognate transporter, there is substantial evidence showing that these selective inhibitors can also block the reuptake of monoamines from non‐cognate transporters. For example, studies have shown that norepinephrine transporter (NET) inhibitors can increase extracellular NE and DA levels in the PFC and hippocampus. Studies have also shown increased extracellular NE, DA and 5‐HT levels in the PFC by administration of a selective serotonin reuptake inhibitor (SSRI). Moreover, studies from dopamine transporter (DAT) and serotonin transporter (SERT) KO mice have also shown that selective NET and SERT blockers can increase extracellular DA levels in both the PFC and striatum. Although these non‐selective effects have been reported, there has not been a systematic investigation of brain‐region specific effects of selective monoamine transporter inhibitors on extracellular monoamine levels. We therefore sought to determine the effects of selective inhibitors of monoamine transporters on extracellular monoamine levels in the prefrontal cortex (PFC), nucleus accumbens (NAcc) and dorsal striatum (dStr). We used an ex vivo neurotransmitter release assay to determine the dose‐dependent effects of a NET inhibitor, desipramine, a DAT inhibitor, GRB 12909 and a SERT inhibitor, fluoxetine, on extracellular NE, DA and 5‐HT from PFC, NAcc, and dStr slices of wild‐type (WT) mice. We also examined the dose‐dependent effects of amphetamine on extracellular monoamine levels in the brain regions of interest of WT mice. The findings from WT mice were subsequently compared to those obtained from DAT and NET knockout mice. The results show that selective blockade of NET, DAT and SERT increase extracellular levels of NE, DA, and 5‐HT respectively, as well as alter the extracellular levels of non‐selective monoamines in a brain‐region and genotype specific manner. These findings will guide future studies exploring how region‐specific changes in monoamine levels affect the activity of neuronal circuits, physiology and behavior.

Support or Funding Information

BBRF/NARSAD Young Investigator award to Nikhil Urs
Connexin/gap junctions and cytoskeletons synergistically control lens fiber cell morphogenesis and metabolism
Authors: Gong Xiaohua , Li Rachel , Xia Chun-hong
Published: April 2020

We have investigated the underlying mechanisms for how a loss of the Gja3 gene (encoding Cx46) leads to variable nuclear cataracts between the C57BL/6J (B6), 129SvJae (129) and B6/129 strain backgrounds, and determined how the genetic variances of periaxin (encoding a cytoskeletal scaffold protein) and CP49 (encoding an intermediate filament protein) modulate the cataract formation. We hypothesize that the Cx46 gap junctions and cytoskeletons synergistically support lens transparency by not only controlling the fiber cell morphogenesis during development but also regulating the exchanges of metabolites between fiber cells to maintain lens homeostasis. Using genetic manipulation and confocal imaging analysis, we have found that both B6‐CP49 and B6‐periaxin inhibit the severity of nuclear cataracts and that the 129‐periaxin proteins are predominantly colocalized with F‐actin and associated with abnormal protrusions on fiber cell surface. The degree of disrupted inner fiber cell surface topology is correlated with the severity of nuclear cataract formation. Metabolite profiling has revealed changes of antioxidants, glycolysis pathway intermediates and lipids compositions in Gja3 knockout (KO) lenses of different strain backgrounds. The Gja3 KO lenses have significant lower levels of glutathione (GSH) than wild‐type controls. Combined effects of gap junctions, CP49‐intermediate filament and periaxin‐mediated cytoskeleton synergistically regulate the interdigitation structures and metabolism of lens inner fiber cells, which modulate lens transparency, homeostasis and cataractogenesis.

Support or Funding Information

Supported by EY013849 grant from the National Eye Institute
The effects of eating a high fat or a ketogenic diet on sensitivity of female rats to methamphetamine
Authors: Elsey Madeline K , Beltran Nina M , Serafine Katherine M
Published: April 2020

Eating a high fat diet leads to negative health consequences such as obesity, type 2 diabetes, and dysfunction to dopamine systems. For example, eating a high fat laboratory chow enhances the sensitivity of rats to the behavioral effects of drugs of abuse (i.e., methamphetamine). Previous research has focused on diets that are high in fat and carbohydrates but low in protein. In contrast, a ketogenic diet is high in fat, low in carbohydrates, and low in protein. Ketogenic diets have been used in humans for the treatment of epilepsy and have been investigated for weight loss. Preclinical studies suggest that rats eating a ketogenic laboratory chow are less sensitive to the locomotor effects of cocaine (e.g., stereotypy). Based on this previous literature, it was hypothesized that eating high fat chow would enhance sensitivity of rats to methamphetamine‐induced locomotion and sensitization; however, rats eating ketogenetic chow would not differ from rats eating standard chow. To test this hypothesis rats eating standard chow (17% kcal from fat, 58% kcal from carbohydrate, 25% kcal from protein), high fat chow (60% kcal from fat, 21% kcal from carbohydrate, 18% kcal from protein), or ketogenic chow (90.5% kcal from fat, 0.3% kcal from carbohydrate, 9.2% kcal from protein) were tested once weekly with cumulative doses of methamphetamine (0.1–3.2 mg/kg; i.p.). After 4 weeks, rats eating high fat chow were more sensitive to the locomotor‐stimulating effects of smaller and intermediate doses of methamphetamine (e.g., 0.32, 1.0 mg/kg) than rats eating standard chow. Rats eating ketogenic chow were also more sensitive than rats eating standard chow to the locomotor‐stimulating effects of methamphetamine, but only at the largest dose tested (3.2 mg/kg). Future research will investigate the mechanisms underlying the effects of diet on sensitivity of rats to methamphetamine‐induced locomotion and sensitization, as well as examine the impact of these and other diets on sensitivity of rats to the rewarding and reinforcing effects of drugs of abuse.

Support or Funding Information

The authors have no conflicts to disclose. This work was supported by the National Institutes of Health, through the National Institute of General Medical Sciences under linked award numbers RL5GM118969, TL4GM118971, and UL1GM118970 and award number R25GM069621. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Elucidating the Mechanisms of Developmental Cell Death in the Retina Using Stem Cell‐derived Organoids
Authors: Brooks Tara , Park Yuna K. , Vielle Anne , Vergara Natalia
Published: April 2020

The development of stem cell technologies has opened up new opportunities for research and therapeutic development that, until recently, were unavailable. In particular, the development of human stem cell derived 3D tissues or “organoids” that recapitulate the native histoarchitecture, and to some degree the native functionality, have provided incredible advancements in our understanding of human physiology and disease. In this study we take advantage of retinal organoids as a new model to study the mechanisms of cell death affecting retinal ganglion cells during human development.

Human induced pluripotent stem cells (hiPSC) were directed to follow a neuronal lineage and were then further differentiated into 3D retinal tissue. Retinal organoids were collected at various time points during development and analyzed using Western blot and immunofluorescent staining for retinal ganglion cells and programmed cell death markers. Our results show the role of programmed cell death as a mechanism to control retinal ganglion cell numbers during development. This has important consequences for understanding not only basic developmental processes, but also the basis of congenital retinal abnormalities that can lead to diseases of vision. Moreover, this knowledge has potential impact for translational research using retinal organoid models.

Support or Funding Information

This work was supported, in part, by a Challenge Grant to the Department of Ophthalmology at the University of Colorado from Research to Prevent Blindness.
The role of Caspase‐1 in diet induced chronic liver injury and blood bile barrier regulation
Authors: Pradhan-Sundd Tirthadipa , Gudapati Shwetha , Brandon Sean
Published: April 2020

End‐stage chronic liver disease is a major cause of mortality worldwide. Irrespective of the underlying cause, most chronic liver diseases are characterized by fibrosis, hepatocellular necrosis, inflammation, ductular reactions and proliferation of the liver progenitor cells. Vast differences exist between experimental models that mimic these processes, and their identification is fundamental for translational research. However, the underlying differences in the molecular mechanism driving liver injury in different models remains largely unknown due to our inability to visualize the initiation and progression of liver injury in vivo in mice. Recently we have introduced quantitative Liver Intravital Microscopy (qLIM) that enables real‐time assessment of bile transport and blood‐bile barrier (BBlB) integrity in the intact liver of live mice (Pradhan‐Sundd et al., Gastroenterology, 2018) to visualize injury progression in two widely used experimental models of chronic liver injury ‐the choline‐deficient ethionine‐supplemented (CDE) or 3, 5‐diethoxycarbonyl 1, 4‐dihydrocollidine (DDC) diet, respectively. qLIM analysis revealed that chronic liver injury involves physical breach of BBlB, mixing of blood with bile and loss of bile transport across hepatocytes in both CDE and DDC diet fed mice. Mechanistically we showed an association of disruption of the BBlB and loss of various tight junctional proteins with sustained inflammation and apoptotic cell death (Pradhan‐Sundd et al., Gastroenterology, 2018). Thus, we hypothesized that blocking Caspase ‐1 induced inflammatory response can inhibit the loss of BBlB and thus postpone the chronic liver injury initiation in these diet fed mice. Indeed, mice with deletion of caspase‐1 displayed reduced liver injury, ductular reaction, fibrosis, inflammation, and apoptosis after two weeks of CDE and DDC diet. Remarkably, qLIM analysis showed intact blood biliary barrier at day 4 and 6 of CDE and DDC fed mice suggestive of delay in injury initiation. Furthermore, we demonstrate that the recovery from CDE and DDC diet induced liver injury is significantly accelerated in caspase‐1 KO mouse. We also found upregulation several tight junctional claudins in the liver upon caspase ‐1 knockdown. Current investigation is underway to analyse the regulation of tight junctional claudins by Caspase‐1. These findings may lead insight to disease progression and possible treatment of diet induced chronic liver injury.
Apo‐hemoglobin and Haptoglobin Limit Acellular Hemoglobin Negative Effects
Authors: Munoz Carlos Jose , Pires Ivan S. , Buehler Paul W. , Palmer Andre F. , Cabrales Pedro
Published: April 2020

Background

Haptoglobin (Hp), a scavenger protein for hemoglobin, and apo‐hemoglobin (apoHb), a chemically synthesized scavenger protein for free heme, present promise in preventing acellular hemoglobin toxicity. The purification process of both molecules is cost effective and can help a number of patients suffering from a septic shock, thalassemia, sickle cell and various other diseases.

Methods

Systematic parameters, microhemodynamics and pharmacokinetics were observed on Golden Syrian hamsters (n=32) using intravital microscopy on the dorsal skin fold. Hamsters were injected with apoHb, Hp, and a combination of apoHb and Hp via the carotid artery. This was followed by a single 10% top‐load of acellular Hb via the carotid artery

Results

A combined treatment with apoHb and Hp was found to reduce acellular hemoglobin toxicities. Systematic parameters and microhemodynamics of all sized arterioles and venules resulted in sustained values when compared to baseline and other treatments (apoHb and Hp alone). The pharmacokinetics data suggests strong scavenging functionality over 6 hours.

Conclusion

The results suggest a promising treatment against acellular hemoglobin and hemolytic anemia induced toxicity where the apoHb is stabilized in a molar excess of Hp preventing vasoconstriction and preserving microvascular blood velocity and blood flow. Ultimately, these results support the efficacy of using purified Hp and apoHb to control hemoglobin and heme induced toxicity.

Support or Funding Information

This work was supported by the NIH Heart Lung and Blood Institute under Grants T32‐HL105373, R01‐HL126945, and the DOD DMRDP under Grant W81XWH‐18‐1‐0059
Regulation of RpoS, the Stationary Phase Sigma Factor, by RbsD
Authors: Marte Esteban , Dorlean Shaina , Parsons Dylan , Peterson Celeste
Published: April 2020

RpoS is the master regulator of stationary phase in bacteria. In response to stress, the RpoS protein accumulates and activates the stationary phase response which makes the cells go dormant and provides protection. A screen for regulators of RpoS was carried out with an overexpression library and an RpoS’‐’LacZ fusion. Overexpression of the rbsD gene, which codes for a ribose pyranase necessary for efficient metabolism of ribose, was found to reduce RpoS levels. Using a beta‐galactosidase assay and Western blot analysis, we found that the effect of rbsD on RpoS is mostly at the post‐transcriptional level and the rbsD knock‐out does not affect RpoS. In addition, ribose also lowered RpoS levels. In summary, the identification of rbsD as a regulator or RpoS sheds light on how ribose affects RpoS levels.
Purification of Insoluble Protein Aggregates from Skeletal Muscle Using a Pre‐Clinical Model of Huntington’s Disease
Authors: Turner Kristen D. , Opatrny Lauren M. , Fuqua Jordan D. , Musetescu Andrei , Kronemberger Ana , Lira Vitor A.
Published: April 2020

Insoluble protein aggregates are distinctive attributes of several tissues affected by aging and are suggested to play a pathological role in the development of neuromuscular disorders (i.e., Huntington’s Disease). However, the influence of insoluble protein aggregates in skeletal muscle on muscle‐specific function is unclear. This is due, in part, to the absence of standardized protocols for isolating protein aggregates so they can be identified and further investigated in vivo. The overall aim of the present methodological study was to determine the most effective method for isolating insoluble protein aggregates from mouse skeletal muscle. This study was conducted using a pre‐clinical Huntington’s Disease model. Electroporation with plasmids encoding EGFP fused to exon 1 of human huntingtin containing 23 (EGFP‐Q23, control) or 74 polyglutamine repeats (EGFP‐Q74, aggregate‐prone) in contralateral TAs was performed (N = 9 muscles/group). Using three adapted protocols from the literature that combined mild detergents and centrifugation to purify insoluble and soluble fractions (Protocol 1 (1% Triton‐X‐100, pH 7.5), Protocol 2 (1% NP‐40, pH 7.4) and Protocol 3 (0.5% Triton‐X‐100, pH 8.0)), we hypothesized that the most efficient protocol would show aggregate‐prone proteins (i.e., EGFP‐Q74) accompanied by adaptors involved in the degradation of protein aggregates by autophagy (p62, NBR1), enriched in insoluble fractions. Approximately 6‐, 5‐ and 3‐fold increases in EGFP‐Q74 vs. EGFP‐Q23 were observed in the insoluble fractions of Protocols 1, 2 and 3, respectively (P < 0.05). 2.5‐fold increases in adaptor p62 in the same insoluble fractions from Protocols 1 and 2 were observed, while only a 2‐fold increase was observed with Protocol 3. Lastly, we observed trends for NBR1, ubiquitinated proteins and total proteins to be elevated using Protocol 1. These observations indicate that Protocol 1 and 2 might be more effective than Protocol 3 for isolating protein aggregates from skeletal muscle and these findings should be considered in future studies extracting insoluble proteins using this Huntington’s Disease model.

Support or Funding Information

This research is supported by an SDG grant from the American Heart Association (V.A.L.).
Anoxic Turtles May Be Blind, but Not Deaf: Responses of the In Vitro Pond Turtle Brain to Visual and Auditory Stimuli During Anoxia
Authors: Ariel Michael , Warren Daniel E.
Published: April 2020

Freshwater pond turtles are among the most anoxia‐tolerant vertebrates, an ability that allows some species to overwinter for months in ice‐locked ponds without oxygen. To determine if anoxic slider turtles can maintain the ability to detect visual and auditory stimuli, we monitored field potentials in an in vitro reduced brainstem while superfusing with either oxygenated or anoxic superfusate. Visual stimuli were recorded using glass recording pipettes placed medial to the optic tectum while flashing light on the eyes. For auditory stimuli, the pipette was placed medial to the eighth cranial nerve (the presumptive cochlear nucleus). In place of sound stimuli, a glass rod with a small polished tip was mounted to a piezo‐electric piston that moved onto the submerged tympanic membrane in brief inward and outward steps. Auditory responses of the brainstem occurred after a very brief latency to each step of the tympanic membrane, while there was a long latency to each light flash in the optic tectum
. An optical probe measured the concurrent oxygen tensions under control (oxygenated) and anoxic conditions. After switching from control to anoxic superfusate, there was no substantial change in the amplitude of the averaged auditory response to steps of the tympanic membrane. However, responses recorded in the optic tectum to light flashes while bathed in anoxic superfusate decreased strongly, but recovered upon return to the control superfusate. These results suggest that turtles submerged for long periods in anoxic ponds may retain sensory responses to auditory but not visual stimuli.

Support or Funding Information

National Science Foundation CAREER Award (1253939) to DEW.
Drosophila TRIM32 cooperates with glycolytic enzymes to promote cell growth
Authors: Bawa Simran , Brooks David S. , Neville Kathryn E. , Sagar Md Abdul Kader , Geisbrecht Brian V. , Tennessen Jason M. , Tipping Marla , Elicieri Kevin W. , Geisbrecht Erika R.
Published: April 2020

Control of tissue and organismal size requires the continual reprogramming of metabolic pathways to integrate biosynthetic and degradative signals. During cell growth and/or proliferation, one such mechanism that promotes the accumulation of cellular material is a switch from oxidative to glycolytic metabolism, whereby glycolytic intermediates are diverted towards anabolic pathways. How this switch is regulated in different tissues is not clear. Herein we identify a novel role for the tripartite motif (TRIM) family member, TRIM32, in the maintenance of glycolytic flux. Using a proteomics approach, we uncovered the glycolytic enzymes Aldolase (Ald) and Phosphoglycerate mutase 78 (Pglym) as TRIM32 interacting proteins. Loss of Drosophila TRIM32, encoded by the thin (tn) gene, showed a reduction in glycolytic activity and amino acid abundance. This altered metabolic profile caused a striking reduction in the overall size of two inherently glycolytic larval tissues – somatic muscles and the developing brain. Consistent with a role for metabolic intermediates in glycolysis‐driven biomass production, nutritional supplementation of amino acids in tn mutants restored muscle mass. Many tumors favor glycolytic metabolism to maximize substrate production for uncontrolled cell growth and proliferation. Remarkably, wing disc‐associated tumor growth is abolished upon loss of TRIM32. Our results reveal a novel connection between TRIM32 and the maintenance of glycolytic enzyme levels and upregulated pathway activity for the sustained growth of normal and cancerous tissue growth.

Support or Funding Information

This work was supported by a grant through the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) to E.R.G (R01AR060788). J.M.T. is supported by a MIRA award from NIGMS (R35GM119557).
Bringing Anatomy Back
Authors: Helgren Maureen , Giblin Jillian , Nankin Matthew , Grgurich Tania
Published: April 2020

The paradigm shift from the longstanding “2+2” medical school model to a contemporary curriculum aimed at competencies in clinical reasoning and decision making has challenged medical educators to consider pedagogical and content reform. The integrated curriculum model is an attempt at reform. To date, the outcome of the paradigm shift is uncertain. From a curriculum development view, there is no shortage of desired curricular content; there are time restraints. With the advent of open sources and virtual technologies specific to anatomy, costs of operating human anatomy laboratories, and curricular demands for truncated time dedicated to anatomical foundation, our institution has implemented a variety of curricular and co‐curricular initiatives for multiple learners to engage with anatomy in active learning settings. Most of the initiatives take advantage of collaborative learning between learners from different disciplines and all emphasize the value of understanding cadaveric anatomy from a functional approach to focus on diagnostic reasoning.

Examples of three successful curricular initiatives include: 1) Diagnostic imaging of our Silent Mentors. Diagnostic Imaging (DI) students who have completed a Human Anatomy course, prepare CT scans of Silent Mentors (cadaver donors) for students taking Human Anatomy courses in Occupational Therapy (OT), Pathology Assistant (PathA), Physical Therapy (PT), Physician Assistant (PA), and for 1st year medical students in system blocks of the undergraduate medical education (UME) curriculum. The scans are used for horizontal and vertical integration of sectional anatomy. 2) PathA students who have completed a Human Anatomy course return to the lab to partner with 1st year medical students on a 6‐month pathology project. 3) Students taking 4th year electives in medical education or pathology have the option to return to the human anatomy laboratory.

Examples of three successful co‐curricular initiatives include: 1) A series of interprofessional case‐based continuing medical education courses with a dissection component. Practicing clinicians and students from medicine, nursing, OT, PA, and PT review a patient case and share discipline‐specific perspectives. 2) IPE60. A peer learning strategy modeled after collegiate athlete “power hours”. Theme‐based series of one‐hour study groups of medical and PT students in the human anatomy lab. 3) Pipeline programs. Anatomy modules delivered by faculty and healthcare profession students interprofessionally or discipline‐specific to local high school and college students.

Assessment of curricular initiatives support their utility of meeting learning objectives with a high degree of student satisfaction.