bluuebunny/crossref_metadata_2025 huggingface dataset:
Stress‐Induced Cardiomyopathy Following Exertional Heat Stroke in Mice
Authors: Laitano Orlando , Garcia Christian K. , Mattingly Alex J. , Robinson Gerard P. , Murray Kevin O. , King Michelle , Ingram Brian , Ramamoorthy Sivapriya , Leon Lisa R. , Clanton Thomas L.
Published: April 2020

INTRODUCTION

Apparent return to homeostasis may not be the end of concerns for exertional heat stroke (EHS) victims. It is well known that patients with cardiovascular diseases are more prone to develop heat related illnesses due to impaired thermoregulation. Epidemiological data suggest EHS exposure is associated with a ~2.5 times higher risk of long‐sterm cardiovascular disease in humans. However, whether EHS results in cardiovascular disorders remains unknown.

OBJECTIVE

To determine the potential of EHS to contribute to development of a stress‐induced cardiomyopathy using a preclinical mouse model of EHS.

METHODS

Male and female C57bl/6J mice underwent forced wheel running for 1.5–3 h in a 37.5°C/40% relative humidity environmental chamber until symptom limitation, characterized by central nervous system dysfunction at peak core temperatures of 42.2 ± 0.3 °C. Sham exercise control mice ran for the same duration as EHS mice at 22.5°C. Myocardial and plasma samples (n = 8/group) were obtained between 30 min −14 d recovery, analyzed using metabolomics/lipidomics platforms and compared to sham exercise controls. Data are presented as fold‐change from control.

RESULTS

Females ran ~40% longer than males (121.16 ± 22 vs. 189.25 ± 35 min; P < 0.05), with a ~51% greater heat load (240 vs.160°C·min; P < 0.05). The immediate recovery period revealed an acute energy substrate crisis characterized by a ~0.5‐fold decrease in plasma lactate, pyruvate, glucose and fructose at 3 h post EHS (P < 0.05) from which both sexes recovered within 24 h. At 9–14 d post EHS, the myocardium of female mice developed ~0.5‐fold elevations (P < 0.05) in free fatty acids, ceramides, and diacylglycerols. Moreover, glycolysis shifted towards the pentose phosphate and glycerol‐3P dehydrogenase pathways. Tricarboxylic acid cycle metabolites revealed bottlenecks in substrate flow and build‐up of intermediate metabolites. These changes are consistent with cardiac metabolic dysfunction observed in chronic heart failure patients. Males exhibited only late stage reductions in acylcarnitines and elevations in acetylcarnitine.

CONCLUSIONS

We demonstrate that the myocardium of female mice is vulnerable to a slowly emerging metabolic disorder following EHS that may lead to long‐term cardiovascular complications. If these abnormalities persist over time, it raises the possibility that untoward outcomes of severe EHS exposure could emerge later in life. Lack of similar findings in males may reflect their reduced time of heat exposure.

Support or Funding Information

DOD Grant BA180078. The opinions or assertions contained herein are the private views of the author(s) and are not to be construed as official or reflecting the views of the Army or the DoD.
HNF4 paralogue gene expression and function in the kissing bug Rhodnius prolixus
Authors: de Oliveira Fernanda Almeida , Musselman Laura Palanker , Gondim Katia Calp , Majerowicz David
Published: April 2020

HNF4 is a transcription factor and member of the nuclear receptor superfamily, proteins that play important roles in development and metabolism. HNF4 is a key gene in the adaptation to fasting and regulates many genes involved in lipid oxidation and mobilization. Mammalian genomes contain two paralogues named HNF4 alpha and HNF4 gamma, but most insect genomes have only one HNF4 gene. After the publication of the Rhodnius prolixus genome, we noticed there were two paralogous HNF4 genes known as RpHNF4.1 and RpHNF4.2. Currently, little is known about HNF4 function in the kissing bug R. prolixus, so study of HNF4 paralogues may provide unique insight into the metabolism and physiology of this important vector for Chagas Disease. A qPCR analysis reveled different patterns of expression of these two genes in different R. prolixus organs, with RpHNF4.1 expressed mainly in ovaries while RpHNF4.2 is the main form expressed in fat body, midgut and flight muscle, suggesting they may have different functions. Drosophila melanogaster is a useful insect model organism with genetic tools that can be used to test the conserved functions of the HNF4 paralogues from R. prolixus. Because R. prolixus has a long life cycle and few genetic tools, we are constructing D. melanogaster transgenic flies to conditionally express each gene in wild‐type and dHNF4 null mutant backgrounds. Loss of dHNF4 in these flies produces adult lethality and these animals acumulate triacylglycerol in the fat body and have low expression of genes involved in the beta oxidation pathway as larvae. Using the GAL4‐UAS system, we aim to generate flies expressing only RpHNF4.1 or RpHNF4.2 without the interference of dHNF4. With these constructs, we will be able to evaluate alterations and possible rescue of the lifespan and lipid metabolism pathway independently for each R. prolixus HNF4 gene. This data may shed some light in their individual physiological roles and suggest new lines of investigation on the lipid metabolism of the Chagas desease vector. Ultimately, a better understanding of conserved features of HNF4 paralogues may provide new molecular targets for obesity treatment in other organisms.

Support or Funding Information

The first author is a Fulbright Scholar at Binghamton University. Special thanks to the Fulbright Comission Brazil. CAPES, CNPq and FAPERJ.
Evidence for a novel interaction between key enzymes in the lipid biosynthesis pathway in S. cerevisiae
Authors: Greenwood Brianna , Stuart David
Published: April 2020

Lipids are essential for energy storage and integrity of membranes in organisms ranging from humans to yeast. The biological properties of triglycerides and other lipids are determined by their composition and the order of assembly. Previous research has demonstrated that acyl‐CoA molecules are delivered to the enzymes involved in lipid biosynthesis in a specific order. It has been proposed that this ordered assembly is achieved by means of a membrane‐bound lipid synthesis complex in which the key enzymes interact through specific protein‐protein contacts. Preliminary data from yeast two‐hybrid and coimmunoprecipitation studies have revealed specific protein‐protein interactions between lipid synthesizing enzymes. Interactions were found between Ole1, the main desaturase in yeast, and Dga1, the terminal enzyme in the triglyceride synthesis pathway, as well as between Ole1 and Slc1, a protein that acylates lysophosphatidic acid. Future research aims to determine the other proteins in complex with Ole1 with a biotin ligase proximity assay and direct interactors via in vitro studies. This research is revealing the architecture of the enzyme complex and how it controls lipid synthesis and composition. Investigating the lipid biosynthesis complex will have implications for a wide variety of applied research, from treating lipid dysregulation in humans to biofuel production by microbes.

Support or Funding Information

NSERC, BioIndustrial Innovates Canada, Alberta Innovates Biofutures, University of Alberta Faculty of Medicine and Dentistry
Contribution of Kir4.1/Kir5.1 Channels to the Control of ENaC‐Mediated Apical Sodium Transport in the Cortical Collecting Duct
Authors: Isaeva Elena , Palygin Oleg , Shalygin Alexey , Bohovyk Ruslan , Fedoriuk Mykhailo , Klemens Christine , El-Meanawy Ashraf , Denton Jerod , Staruschenko Alexander
Published: April 2020

Recent studies using genetically modified animal models reveal that inwardly rectifying potassium channels Kir4.1 and Kir5.1, encoded by Kcnj10 and Kcnj16 genes, play an essential role in determining plasma potassium level and regulating blood pressure. Moreover, we have revealed recently that the deletion of Kcnj16 gene in Dahl salt‐sensitive (SS) rats has the profound effect on the blood pressure and renal damage. These data uncover a new potential target for the treatment of salt‐induced hypertension. Protein products of Kcnj10 and Kcnj16 are localized to the basolateral membrane of distal nephron segments forming functional homomeric (Kir4.1) and heteromeric (Kir4.1/Kir5.1) potassium channels that play an essential role in establishing a negative basolateral membrane potential and recycling K+ ions moved into the cytosol by the Na+/K+‐ATPase. In the present study we examined the effect of pharmacological inhibition of Kir4.1 and Kir4.1/Kir5.1 channels on sodium transport through the epithelial Na+ channel (ENaC) in the cortical collecting duct (CCD) cells. We have reported recently that Kir4.1 inhibitor nortriptyline (100 μM), but not fluoxetine (100 μM) inhibited whole‐cell potassium currents recorded from native principal CCD cells. First we examined whether fluoxetine and nortriptyline have different selectivity in targeting Kir4.1 and Kir4.1/Kir5.1 expressed in Chinese hamster ovary (CHO) cells. Single channel analysis revealed that application of 100 μM of amitriptyline produces a significant decrease in NPo of Kir4.1/Kir5.1 but does not affect Kir4.1 current activity, whereas fluoxetine selectively inhibits Kir4.1. Next we examined whether selective inhibition of Kir4.1 and Kir4.1/Kir5.1 affects an apical sodium transport via ENaC. We showed that amitriptyline, but not fluoxetine reduced (IC50=15.0 μM) the amiloride‐sensitive short‐circuit current (ISC) measured in polarized epithelial mCCDcl1 cells. In control experiments, application of amitriptyline did not directly affect ENaC activity overexpressed in CHO cells. Using different concentrations of recently developed Kir4.1 inhibitor, VU0134992, and patch‐clamp studies of single‐channel activity on mCCD cell line and native rodent and human CCD tubules, we confirmed that inhibition of Kir4.1/Kir5.1 channel but not Kir4.1 produces suppressive effect on ENaC activity. In summary, inhibition of Kir4.1/Kir5.1 channels in CCD cells indirectly reduces apical sodium conductance through ENaC, demonstrating the importance of basolateral potassium channels in the control of sodium reabsorption in distal nephron.

Support or Funding Information

National Institutes of Health grants R01 DK120821 (to J.D. and A.S.), R56 DK121750 (to O.P.), and R35 HL135749 (to A.S.)
A Torso Burn Injury Does Not Exacerbate Thermoregulatory Strain During Exercise‐Heat Stress While Wearing A Military Combat Uniform
Authors: Fischer Mads , Cramer Matthew N. , Huang Mu , Belval Luke N. , Watso Joseph C. , Cimino Frank A. , Secher Niels H. , Crandall Craig G.
Published: April 2020

The ability to dissipate heat during exercise is impaired in individuals with grafted skin over a large portion of their body. However, it is unknown whether a torso burn injury impairs thermoregulatory function in uniformed military personnel operating in a hot environment.

Purpose

To evaluate whether a torso burn injury exacerbates the rise in core temperature during exercise in a hot environment while wearing a military combat uniform.

Methods

Ten healthy individuals (8 males) performed 60 min of treadmill walking at 5.3 km/h with 3.7 ± 0.9% grade incline (altered to maintain a rate of metabolic heat generation of ~6.0 W • kg‐1) in a 40.0 ± 0.1 °C and 20.0 ± 0.6% relative humidity climate chamber. On separate, randomized occasions, participants wore a standard U.S. Army Combat Uniform and Outer Tactical Vest (Uniform) with or without a simulated burn injury imposed by placing absorbent vapor‐impermeable material across the torso, 20% body surface area (BSA), to hinder evaporative heat dissipation (Uniform + Burn). A third trial was conducted without the uniform or simulated burn with subjects wearing athletic shorts and sports bra for females (Control).

Rectal temperature and heart rate were monitored continuously, and metabolic heat production was calculated via indirect calorimetry. The change in outcome measures from baseline to the end of exercise were compared between trials with a two‐way (group × time) repeated‐measures ANOVA.

Results

At the end of exercise, no differences were revealed (P > 0.05) between the Uniform versus Uniform + Burn trials in all outcome variables: increase in rectal temperature (Uniform: Δ1.16 ± 0.3°C; Uniform + Burn: Δ1.21 ± 0.4°C), thermal sensation (Uniform: 6.0 ± 0.6; Uniform + Burn: 6.2 ± 0.8), rating of perceived exertion (Uniform: 14.2 ± 2.7; Uniform + Burn: 14.1 ± 3.0) and increase in heart rate (Uniform: Δ61 ± 13; Uniform + Burn: Δ56 ± 15 bpm). In contrast, the Control trial showed lower (P < 0.05): increases in rectal temperature (Δ0.93 ± 0.3°C), thermal sensation (4.8 ± 0.7), rating of perceived exertion (11.3 ± 2.2), and increases in heart rate (Δ44 ± 14 bpm) compared to both the Uniform and Uniform + Burn trials.

Conclusion

A 20% BSA simulated burn on the torso does not exacerbate thermoregulatory strain during exercise in a hot environment while wearing a military combat uniform in healthy young adults. These findings suggest that the risk of heat‐related illness in soldiers with torso burn injuries is no different than non‐injured soldiers when wearing a standard military combat uniform.

Support or Funding Information

This work was supported by awards from the Department of Defense (W81XWH‐15‐1‐0647 to CGC), National Institutes of Health (R01GM068865 to CGC), and a Natural Sciences and Engineering Research Council of Canada Postdoctoral Fellowship (to MNC).
Depletion of HIF‐1α‐mediated Mitochondrial Uncoupling Protein‐4 (UCP4) Expression Sensitizes Temozolomide (TMZ)‐Induced Apoptosis in Glioblastoma
Authors: Chen Jin , Li Zhaozhong , Kolpakova Jenya , Chan Sic L. , Cheng Zixi Jack
Published: April 2020

Introduction

Mitochondrial Uncoupling Protein‐4 (UCP4) UCP4 is exclusively expressed in the brain and has been shown to promote cell proliferation and enhance resistance to mitochondrial toxins by shifting cellular ATP synthesis from oxidative phosphorylation (OxPhos) toward glycolysis. Notably, a recent study demonstrated that the expression of cerebral UCP4 was upregulated when the animals were exposed to acute high‐altitude hypoxia. This upregulation may facilitate the adaptation of malignant glioma cells to their hypoxic microenvironment, and contribute to the subsequent mitochondria remodeling and metabolic reprogramming. UCP4 has become an interesting focus in the regulation of oxidative stress in age‐related neurodegenerative diseases. However, the function and mechanism of UCP4 upregulation under hypoxia remains poorly defined in gliomas.

Methods

The human glioblastoma cell line, U373MG was used for this study. UCP4 expression level under hypoxia was detected using Western Blot. Glioma cell proliferation, anchorage‐independent growth and invasion were evaluated using MTT assay, BrdU incorporation, Soft‐agar colonogenic assay, and Matrigel invasion assay. To silent UCP4, U373MG cells were infected with shRNA against UCP4. LDH release assay was employed to assess whether targeting UCP4 could sensitize the Temozolomine (TMZ) – induced the apoptosis in U373MG cells. The Human UCP4 base promoter region was cloned for Luciferase reporter assays.

Results

Here, we showed that 1) Hypoxia increased UCP4 which promoted tumor progression in gliomas. 2) Knockdown of UCP4 decreased tumor progression and increased the cell sensitivity to TMZ chemotherapy. 3) HIF‐1α regulated UCP4 expression via binding to the UCP4 upstream consensus sequence, and silencing HIF‐1α decreases UCP4 expression.

Conclusions

Our data suggested that hypoxia increased UCP4 expression which may contribute to cell adaptation to the hypoxic microenvironment. Targeting UCP4/HIF‐1α, or blocking HIF‐1α binding to UCP4 promoter in glioma cells may offer a new avenue for therapeutic intervention.

Support or Funding Information

Supported by the award Florida Center for Brain Tumor Research UF10262 (PI: Zixi Jack Cheng).
A versatile bioluminescent immunoassay approach to interrogate cellular signaling pathway regulation
Authors: Zegzouti Hicham , Hwang Byounghoon (Brian) , Engel Laurie , Alves Juliano , Goueli Said
Published: April 2020

Monitoring cellular signaling events can help better understand cell behavior in health and disease states. Traditional immunoassays to study proteins involved in signaling can be tedious, require multiple washing steps, and are not easily adaptable to high throughput screening (HTS) format. Here we describe a novel immunoassay approach based on bioluminescent enzyme subunit complementation technology. This bioluminescent immunoassay takes less than two hours to complete in a homogeneous “Add and Read” format and was successfully used to monitor the activation of multiple signaling pathways through specific nodes of phosphorylation (e.g pIkBα, pAKT, and pSTAT3) in unmodified cells. We also tested deactivation of these pathways with different small and large molecule inhibitors and obtained the expected pharmacology. A key advantage of this approach is that it does not require cell engineering. Therefore, the phosphorylation or total level of an endogenous protein is detected in any cell type. Our results demonstrate that this technology can be broadly adapted to streamline the analysis of signaling pathways of interest or the identification of pathway specific chemical or biologic inhibitors.
Twin enzymes, divergent control: The cholesterogenic enzymes DHCR14 and LBR are differentially regulated transcriptionally and post‐translationally
Authors: Capell-Hattam Isabelle Marie , Sharpe Laura J. , Qian Lydia , Hart-Smith Gene , Prabhu Anika V. , Brown Andrew J.
Published: April 2020

Cholesterol synthesis is a tightly regulated process, both transcriptionally and post‐translationally. Transcriptional control of cholesterol synthesis is relatively well understood. However, post‐translational regulation has only been examined for a few of the ~20 enzymes in cholesterol biosynthesis. Two of the four sterol reductases, DHCR14 and LBR, share evolutionary ties with a high level of sequence homology, and they uniquely share the same delta‐14 reductase activity in cholesterol biosynthesis. However, the post‐translational regulation of these twin enzymes is poorly understood. Using CHO‐7 cells stably expressing epitope tagged DHCR14 or LBR, we investigated the post‐translational regulation of these enzymes. We found that DHCR14 and LBR undergo differential post translational regulation, with DHCR14 being rapidly turned over, triggered by cholesterol and other sterol intermediates while LBR remained stable. DHCR14 is degraded via the ubiquitin‐proteasome system and via mass‐spectrometry we identified several E3 ligases that modulate basal DHCR14 levels. Significantly, we found that gene expression across an array of human tissues showed an inverse relationship between the C14‐sterol reductases; one enzyme or the other tends to be predominantly expressed in each tissue. Overall, our findings indicate that while LBR tends to be the constitutively active delta‐14 reductase, DHCR14 levels are tuneable, responding to the local cellular demands for cholesterol.

Support or Funding Information

This work was supported by Australian Research Council Grant DP170101178. PhD Candidates were funded by the Research Training Program from the Australian Government Department of Education.
Role of nitric oxide (NO) in the modulation of anti‐arthritic and anti‐inflammatory effects of Trigonella foenum‐graecum (fenugreek) ethanolic seed extract in rats
Authors: PAL RISHI , Kamal Parul , Nath Rajendra
Published: April 2020

Rheumatoid arthritis (RA) is an autoimmune disorder with a characteristic feature of swollen and painful joints. Trigonella foenum graecum (Fenugreek) has been used from ancient time as spice and also has medicinal value in different disease conditions like constipation, gastritis, inflammation, and diabetes. The current study was designed to evaluate protective role of ethanolic fenugreek seed extract (FSE) in CFA‐induced experimental arthritis and its comparison with methotrexate. The FSE was prepared by using standard procedures and tested over the male wistar rats (180–225 g) for anti‐arthritic and anti‐inflammatory effects. CFA (0.2 ml with sqaline, s.c.) was inoculated in right foot of rats on day “0” and evaluated daily for inflammation for 14 days. From day 14 to 28, FSE (200–400 mg/kg) and nitric oxide modulataors, L‐arginine (100 mg/kg) and L‐NAME (10 mg/kg) administered daily. FSE (200–400 mg/kg), shows significant protective role in arthritic and inflammatory parameters as well as oxidative stress markers in a dose dependent manner. The adjuvant‐induced changes in arthritic parameters like ankle diameter, paw volume, arthritic index, inflammatory cytokine, IL‐1β, TNF‐α & IL‐6 levels and oxidative stress markers, MDA & SOD were significantly reversed by FSE. These results are more or less comparable to the standard drug methotrexate. NO modulators further potentiated the protective effects of FSE. The protective effects of FSE was more prominent when ethanolic seed extract was given in combination with iNOS inhibitor, L‐NAME in comparison with FSE + L‐arginine. Our findings suggests that nitric oxide synthase inhibition leads to potentiated anti‐arthritic, anti‐inflammatory effects of ethanolic FSE and that may be mediated through pro‐inflammatory/anti‐inflammatory cytokines imbalance.
In Vitro Evaluation Of The Essential Oil Derived From Eucalyptus globulus Labill Against Rhipicephalus (Boophilus) annulatus Ticks
Authors: Adenubi Olubukola Tolulope , Abolaji Abiola Oluwasegun , Salihu Timothy , Akande Foluke Adedayo , Lawal Hassan
Published: April 2020

Ticks are of great economic importance to man and animals due to their role in disease transmission and other effects they have on their hosts. This impact warrants appropriate tick control strategies. The application of synthetic, chemical acaricides on the animal and/or the environment (the most commonly used tick control method globally) has led to the selection of tick populations that are resistant. Their adverse effects on ecology, human and animal health also, cannot be overemphasised. As a result of this, the search for alternatives that are natural and can overcome these adverse effects are strongly indicated. Using gas chromatography–mass spectrometry (GC‐MS) and adult immersion test (AIT), this study evaluated the chemical compositions and acaricidal activity respectively of E. globulus essential oil (EO) on R. (B.) annulatus ticks. The acaricidal activity was evaluated using different concentrations (1.25, 2.5, 5 and 10%) of E. globulus EO. Amitraz (0.1 and 0.2%) served as the positive control and 2% dimethylsulfoxide was the negative control. Three replicates of ten engorged female ticks each were immersed in the test samples for two minutes. The GC‐MS analysis showed the E. globulus EO has eucalyptol, cyclohexanone and menthol as major constituents. Eucalyptus globulus EO caused 97% acaricidal mortality at 10% concentrations. Lower concentrations (1.25, 2.5, 5%) reduced tick fecundity up to 90% in a dose‐dependent manner. This study provides support for ongoing possibility of plant EOs as alternative tick control strategy for humans and animals.
Implementing course‐embedded research projects in a large biochemistry lecture
Authors: Berndsen Christopher
Published: April 2020

The Biochemistry course at James Madison University is a one‐semester survey course covering everything from atoms to organisms. The students come from a variety of backgrounds and majors, thus it is challenging to make the content accessible to the students and cover the content in adequate depth to prepare students for their careers. I have taken this challenge as an opportunity to develop a series of lecture‐based projects that have students learn biochemistry content through research and experimentation. The large biochemistry lecture section is not an environment where course‐embedded research projects are often found. However, large lecture sections offer the opportunity to expose a large number of students with diverse backgrounds to an open, collaborative research experience. Over the semester long course, students use freely available databases and servers to explore biomolecule structure and function in projects which require students to apply their knowledge of chemistry to biological research questions in an authentic manner. Project topics include spectroscopy, protein purification, drug design, and biomolecule structure prediction and evaluation. In addition to learning the fundamentals of biochemistry, participants develop their skills in experimental design and improve their ability to communicate and disseminate science. To manage the projects on the scale of 50 to 100+ participants at a time, I use the Open Science Framework to store data and report out the findings. From post‐activity assessment, students report increased confidence in their ability to understand fundamental biochemical concepts while also showing increased professional skills. Students also can assemble their projects into portfolios of their work to show to future employers and admissions committees.

Support or Funding Information

Thomas and Kate F. Jeffress Memorial TrustAccelerating Creative Teaching grant from JMU Libraries
Comparison of Survivor Scores for Differentiation Therapy of Cancer to those for Check Point Inhibition.
Authors: Sell Stewart
Published: April 2020

Differentiation therapy (DT) is directed to the self‐renewing cancer stem cells, as well as their progeny transit amplifying cells, to force them to mature to terminal differentiation. Differentiation therapy is effective in treatment of neuroblastomas and myeloid leukemias. Check point inhibition (CPI) therapy removes blocks to cancer reactive T‐killer cells and allows them to react to malignant cells and limit growth of cancer. The percent of patients with a given cancer that respond to either therapy is less than hoped for and the duration of response is variable. Multiplying the response rate (% of patients responding to therapy) by the duration of response may be used to derive a survival score for patients treated with DT or CPI. By this criterion DT gives better survival scores than CPI. Yet, CPI is considered a great success, mostly because it may be applied to many different types of cancer and DT is considered relatively ineffective because it is limited to a few specific cancers. On the other hand, the cost of CPI treatment is 10 to 20 times more per patient than DT. Hopefully, future combined treatments and advances in both approaches will increase the effectiveness and decrease the cost of these cancer treatments. For example, resent results with combination therapy of melanoma with two check point inhibitors has substantially increased the survival score.

Support or Funding Information

No support
Design of Small Molecules to Modulate Transthyretin Aggregation
Authors: Fortin Jessica S. , Isa Nurhanis B. M. , Rashid Anisa M. , Thompson Thomas L. , O’Dell Malikah
Published: April 2020

Transthyretin (TTR) amyloid deposits have been causally implicated in familial amyloid polyneuropathy (FAP) and familial amyloid cardiomyopathy (FAC). An effective means to block formation and disaggregate transthyretin amyloid deposits of patients with FAP and FAC does not exist. We have designed and synthesized small molecules to stop formation of, as well as disaggregate these deposits. Our hypothesis is that TTR fibrils can be abrogated by TTR‐interactive compounds. The specific aim of this study is to define the activity of our novel small molecules on TTR aggregation and disaggregation with biophysical methods. Thioflavin T (ThT) fluorescence assay was used to monitor fibril formation with synthetic TTR truncated peptide (TTR81–127) treated with small molecules. At the end of a time‐course study, fibrils were directly detected by transmission electron microscopy. Preliminary results showed a few molecules that were able to abrogate fibril formation via the ThT fluorescence assay. The percentages of fibril formation measured at the plateau phase of the kinetics of fibril formation obtained with control and the most potent molecules were compared. One of our best compounds, JF‐19‐029, was ten time more potent than the positive control, resveratrol in inhibiting fibril formation. When TTR81–127 was incubated for 24h with JF‐19‐029, rarely fibrils were observed on the copper grid visualized with the electron microscope. This compound is also being studied to determine the potential for disaggregation of mature TTR81–127 fibrils. This project discovered novel small molecules that potentially reduce TTR fibril formation, with the eventual goal to improve the therapeutic arsenal for FAP and FAC.
Activation of Neutral Sphingomyelinase Contributes to Nitric Oxide‐Mediated Flow‐Induced Dilation in the Human Microvasculature
Authors: Schulz Mary K. , Gutterman David K. , Freed Julie K.
Published: April 2020

Flow induced dilation (FID) is a critical physiological mechanism to maintain tissue perfusion and modulate vascular homeostasis. Recently our laboratory has shown that sphingolipids can influence the endothelial‐derived mediator that is generated in response to flow. Exposure to elevated levels of ceramide, or, inhibition of ceramide metabolism, induces a transition in endothelial‐derived FID mediator from nitric oxide (NO) to hydrogen peroxide (H2O2), the same change in mechanism that occurs with presence of CAD. Interestingly, studies have suggested that endothelial nitric oxide synthase (eNOS) is regulated by ceramide metabolites, such as sphingosine‐1‐phosphate (S1P), suggesting that ceramide is necessary for NO formation due to shear stress. We therefore hypothesized that activation of the shear sensitive, ceramide‐producing enzyme, neutral sphingomyelinase (NSmase) is required for NO‐dependent FID. Resistance arterioles (100–200μm) were dissected from adipose tissue collected from healthy patients and were prepared for videomicroscopy. Following equilibration and preconstriction with endothelin‐1, changes in internal diameter to flow were recorded. L‐NAME (100mM) did not affect FID in healthy arterioles pre‐treated with the NSmase inhibitor, GW4869 (4mM, 16–20hrs), (69.1%±4.9 of maximal dilator capacity, n=4), however dilation was decreased in the presence of PEG‐Catalase (500 Units/ml) (15.2%±9.4, n=3). A similar effect was observed with acute inhibition (30 min) of NSmase. Vasodilation due to increased flow was maintained in the presence of L‐NAME (88.0%±3.5, n=3), however was impaired when exposed to PEG‐Catalase (−5.3%±1.3, n=3). GW4869 alone did not impair the overall magnitude of dilation due to flow (86.1%±3.9, n=2). These findings suggest that while elevated levels of ceramide promote H2O2‐dependent FID, NSmase‐derived products of ceramide are a key component of NO‐mediated signaling in the human microvasculature.

Support or Funding Information

K08HL141562
Imoxin Prevents Dexamethasone‐Induced Promotion of Muscle‐Specific E3 Ubiquitin Ligases and Stimulates Anabolic Signaling in C2C12 Myotubes
Authors: Eo Hyeyoon , Valentine Rudy J.
Published: April 2020

Muscle atrophy is the loss of skeletal muscle mass during several pathological conditions such as long‐term fasting, aging, cancer, diabetes, sepsis and immune disorders. Dexamethasone (DEX), a synthetic glucocorticoid, induces skeletal muscle atrophy by suppression of protein synthesis and promotion of protein degradation. Specifically, DEX increases the levels of muscle specific ubiquitin E3 ligases including MAFbx and MuRF1. The double‐stranded RNA (dsRNA)‐activated protein kinase R (PKR) plays a significant role in mediating inflammation and glucose homeostasis. However, pathological roles of PKR in muscle atrophy are not fully understood. The current study aimed to investigate the effect of imoxin, a PKR inhibitor, on DEX‐induced muscle atrophy in C2C12 myotubes. Myotubes were incubated with imoxin at different concentrations with or without 5 μM DEX for 24 h. Western blotting was performed to measure proteins involved in muscle protein synthesis as well as degradation. DEX significantly augmented the protein levels of MuRF1 and MAFbx (275 ± 5 % and 694 ± 36 %, respectively) compared with serum free control (CON). However, 5 μM imoxin treatment significantly reduced protein levels of MuRF1 by 88 ± 2 % and MAFbx by 99 ± 0 % compared with DEX, respectively. In addition, DEX suppressed FoxO4 phosphorylation (33 ± 9 %) compared to CON, but 5 μM imoxin treatment promoted FoxO4 phosphorylation (735 ± 44 %) compared to DEX. Additionally, DEX stimulated protein ubiquitination compared with control (124 ± 1 %) but 5 μM imoxin treatment reduced protein ubiquitination by 42 ± 4 % compared to DEX. At the same time, 5 μM imoxin treatment stimulated Akt phosphorylation (195 ± 5 %), mTOR phosphorylation (171 ± 21%) and p70S6K1 phosphorylation (314 ± 31 %) under DEX‐treated condition even though DEX treatment did not suppressed Akt/mTOR/p70S6K1 axis. These findings suggest that imoxin protects against DEX‐induced skeletal muscle atrophy by alleviating muscle specific E3 ubiquitin ligases and promoting protein synthesis via Akt/mTOR/S6K1 axis in muscle cells.

Support or Funding Information

None
Neuroinflammation and Age‐Dependent Salt‐Sensitive Hypertension
Authors: Nist Kayla M. , Wainford Richard D.
Published: April 2020

Aim

Normal aging and elevated dietary sodium intake have both been identified as contributors to hypertension (HTN). Further, recent data from multiple animal models suggest a role of central inflammation in HTN. Therefore, we speculate that central inflammation plays a role in age‐related salt‐sensitive HTN.

Methods

Male Sprague‐Dawley rats aged 3, 8, and 16 months (mo) old were randomly assigned to a normal salt (NS; 0.6% NaCl) or high salt (HS; 4% NaCl) diet for 21 days (n=4–6/group). In a subset of animals (n=6/group) MAP was assessed via femoral artery cannulation. Sympathetic tone was assessed via renal norepinephrine (NE) measured by ELISA on day 21 in a subset of animals (n=6/group). Additional groups of rats were sacrificed via transcardial perfusion using phosphate‐buffered saline and 4% paraformaldehyde (PFA). Brains were extracted and fixed in 4% PFA and 30% sucrose. Immunohistochemistry was performed for markers of microglial (CD11b/c) and astrocyte activation (GFAP) in the paraventricular nucleus (PVN) of the hypothalamus, and assessed using ImageJ software.

Results

When fed a NS diet, male rats exhibit age‐dependent HTN (MAP [mmHg] 3 mo 124 ± 2 vs. 8 mo 135 ± 4 vs 16 mo 149 ± 3, p<0.05). When fed a 21‐day HS diet, male rats develop salt‐sensitivite HTN (MAP [mmHg] 3 mo 126 ± 3 vs. 8 mo 143 ± 5 vs. 16 mo 169 ± 1, p<0.5). Aged rats display elevated levels of renal NE, with impaired HS‐evoked suppression of sympathetic tone (renal NE content [pg/mg] 3 mo NS 612 ± 36 vs HS 368 ± 32; 8 mo NS 835 ± 48 vs HS 722 ± 44, p<0.05; 16 mo HS 974 ± 13 vs NS 1019 ± 34, ns). There was no statistical difference between branching complexities on trend lines from Sholl analysis of microglia in all groups. On a NS diet, there is no change in astrocytic density in the PVN with age, (total astrocyte % tissue density: 3 mo 41.58 ± 6.00% vs. 8 mo 43.58 ± 3.65% vs. 16 mo 47.18 ± 3.76%, ns). In 3 mo and 8 mo rats, which do not get salt‐sensitive HTN, there is a significant increase in the activation of astrocytes in the PVN when fed a high salt diet (total astrocyte % tissue density: 3 mo NS 41.58 ± 6.00% vs. 3 mo HS 56.66 ± 4.51%, p<0.05; 8 mo NS 43.58 ± 3.65% vs. 8 mo HS 58.28 ± 2.63%, p<0.05). Sixteen month old rats, which become salt‐sensitive and hypertensive with age, exhibit a failure to increase PVN astrocyte levels on a high salt diet (total astrocyte % tissue density: NS 47.18 ± 3.76% vs. HS 50.66 ± 5.01%).

Conclusions

Male Sprague‐Dawley rats become hypertensive and salt‐sensitive with age. On a NS diet, inflammation does not play a role in age‐dependent HTN. However, inflammation plays a role in the salt‐sensitivity of blood pressure mediated by astrocyte reactivity. Our findings propose that with age, PVN astrocytes express a loss of a protective mechanism when challenged with a high salt diet.

Support or Funding Information

R01 AG062515, R01 HL139867, R01 HL141406, and R56 AG057687 to RDW
Effect of N‐Terminal Splice Variants and Post‐Translational Modification on Regulation and Activity of Human Cytosolic Malate Dehydrogenase
Authors: Cruz Melanie R. , Balagtas Aaliyah N. , Provost Joseph John
Published: April 2020

Post‐translational modification via phosphorylation is crucial in regulation of proteins, as well as in interactions between proteins. Human cytosolic malate dehydrogenase (hMDH1) is involved in multiple metabolic pathways including amino acid metabolism and gluconeogenesis, yet information on the impact of phosphorylation is very limited. There is however, growing evidence for regulation of hMDH1 in several disorders. The cytosolic hMDH1 is ubiquitously expressed, however several N‐terminal splice variants have been identified. Like regulation by phosphorylation, no work has been published on the effects of the splice variants on the structure or function of hMDH1. We first determined the impact of N‐terminal deletion for the three most common splice variants on kinetic values. We next analyzed the potential for phosphorylation using three phosphorylation prediction programs and performed meta‐analysis with existing mass spectrometry databases documenting phosphorylation. This bioinformatic analysis identified 24 residues with overlapping phosphorylation predictions between databases. Of these sites, the longest hMDH1 variant (hMDH1v3) has a unique phosphorylation site that is predicted in all databases and has documented phosphorylation in the N‐terminal extension. To examine the potential impact on MDH structure and function phosphomimetic mutations were generated. MDH S5E mutant of hMDH1v3, unique of all splice variants was created while additional mutations of the mid‐length, canonical variant of hMDH1 (hMDH1v1) at residues: S89, S188, S241, and T321 were also generated. These sites were selected for their potential effect on catalysis due to location within the catalytic or binding domain (S188 and S89, respectively), and the overlap of all phosphorylation prediction and mass spectrometry databases (S241 and T321). Our goal in creating phosphomimics is to determine the role of post‐translational modification (phosphorylation) on the regulation of hMDH1 in its stability, kinetic activity, effects on structural changes, and open the potential of phosphorylation of MDH on protein‐protein interactions.

Support or Funding Information

NSF EHR‐IUSE Project support NSF‐1726932
Activation of Sirtuin 1 (SIRT1) Signaling by Resveratrol Increases Human Beta‐Defensins‐2 and ‐3 (HBD2, HBD3) Production in Response to Lipopolysaccharide (LPS) in Human Amniotic Epithelial Cells (HAEC): Pregnancy Complicated by Diabetes (PCD) vs. Normoglycemic Pregnancy (NP)
Authors: Szukiewicz Dariusz , Szewczyk Grzegorz , Stangret Aleksandra , Watroba Mateusz
Published: April 2020

Human beta defensins (HBDs) are small cationic peptides with chemoattractant activities. HBD2 and HBD3 are generally expressed at low levels in physiologic conditions and are induced in response to microbial challenge (e.g. LPS), whereas HBD1 low expression is constitutive, with little regulation in response to infection or other stimuli. HBDs produced by HAEC co‐create an innate antimicrobial immune response in the materno‐placento‐fetal unit. PCD increases the risk of infection. According to recent studies, PCD activates the innate immune response through TLR5 or TLR1/2 on neonatal monocyte, but the data about HBDs are still lacking. The aim of this study was to examine comparatively the effect of resveratrol on SIRT1 level and HBD2 and HBD3 production in HAEC isolated after PCD (White’s class C) vs. NP. HAEC were isolated from the amnion immediately after cesarean section delivery terminating PCD (N = 24, group I) and NP (N = 24, group II) using trypsin‐based method and cultured in vitro under normoxia at 37 degrees C. Upon reaching confluence, HAEC were cultured in media supplemented with 0.5 μM resveratrol and SIRT1 levels were determined in HAEC culture lysates obtained once a day for 7 consecutive days. After pretreatment with LPS (1 μg/mL), HBD2 and HBD3 concentrations in the HAEC culture supernatant, obtained in the days 1,3 and 7 of the culture with resveratrol, were assessed in both goups using ELISA. Nonparametric Wilcoxon rank‐sum test was used and the differences between the groups were considered statistically significant if p < 0.05. In both groups resveratrol increased significantly SIRT1 levels in consecutive days. The response was stronger in PCD as compared to NP. Despite of this, observed increase in both HBD2 and HBD3 concentrations were significantly lower in PCD, amounting to 32.8 / 37.7, 45.5 / 42.9 and 55.3% / 49.5% (HBD2 / HBD3 days 1,3 and 7, respectively) of the values obtained in group II (NP). Importantly, the mean viability of cells in both groups did not differ significantly at the consecutive time points. Thus, susceptibility to infections in diabetes may be to some extent caused by compromised HBDs‐dependent antimicrobial immune respose. The nature of impaired SIRT1 – HBDs interaction is still under investigation.

Support or Funding Information

WUM grant: 2M2‐W2‐18/2M2‐W1‐19
5‐HT2 and 5‐HT2B Receptor Antagonism Regulates Fibrotic Potential of Peritoneal Fibroblasts in CAPD by Targeting TGF‐β1‐Src‐ERK1/2‐STAT3 Pathway
Authors: Chaturvedi Saurabh , Agarwal Vikas , Prasad Narayan
Published: April 2020

Peritoneal fibrosis (PF) results in ultrafiltration failure in continuous ambulatory peritoneal dialysis (CAPD) patients. 5‐hydroxytryptamine (5‐HT; serotonin) induces extracellular matrix (ECM) synthesis in peritoneal fibroblasts in a transforming growth factor beta 1 (TGF‐β1) dependent manner. We evaluated anti‐fibrotic role of inhibitors of 5‐HT2 (Terguride) and 5‐HT2B (SB204741) in human peritoneal fibroblasts (HPFB) of CAPD patients. Peritoneal fibroblasts isolated fromCAPD patients (n=6) and controls (n=8), were incubated with 5‐HT (1μM)/TGF‐β1 (10ng/ml) for 1 hour and later with 5‐HT (1μM)/TGF‐β1 (10ng/ml) and terguride or SB204741 (1μM, each) for 24 hours (Post‐treatment strategy). In pre‐treatment strategy, cells were pre‐treated with terguride or SB204741 (1μM, each) for 1 hour and later with only 5‐HT (1μM)/TGF‐β1 (10ng/ml) for 24 hours. Real time PCR for pro‐fibrotic TGFB1, Type 1 collagen (COL1A1), Type 2 collagen (COL1A2), Actin alpha 2 (ACTA2), Connective tissue Growth factor (CTGF) and Fibronectin 1 (FN1) and anti‐fibrotic genes Matrix mettaloproteinase‐2/Tissue inhibitor of metalloproteinase (MMP2/TIMP1) expression was performed. Type I collagen and α‐SMA, phosphorylation status of Smad‐3, ERK1/2, Src and STAT‐3 was examined by western blotting.

In 5‐HT/TGF‐β1stimulated HPFB, upregulated pro‐fibrotic gene expressionwas observed, which significantly reduced on co‐culture with 5‐HT2/5‐HT2B inhibitors, with no effect on anti‐fibrotic genes mRNA expression. In 5‐HT stimulated HPFB, treatment with both 5‐HT inhibitors decreased type 1 collagen and α‐SMA with reduced ERK1/2 phosphorylation (figure 1), however, Smad‐3 phosphorylation remain unaltered (figure 2). In 5‐HT/TGF‐β1 simulated HPFB, 5‐HT inhibitors decreased STAT3 phosphorylation, without affecting Src phosphorylation. 5‐HT receptor antagonists attenuate 5‐HT/TGF‐β1 mediated pro‐fibrotic potential of HPFB by suppressingTGF‐β1‐Src‐ERK1/2‐STAT3 pathway.

Support or Funding Information

This project received funding from intramural grant(Grant No ‐ 2017‐1‐IMP‐95) of Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India.
Indaziflam herbicidal action: a potent auxin signaling inhibitor
Authors: Behnami Sara , Bonetta Dario
Published: April 2020

Cellulose biosynthesis is a common feature of land plants. Therefore, cellulose biosynthesis inhibitors (CBIs) are useful tools in decoding fundamental aspects of cellulose biosynthesis. Here, we characterize the herbicide indaziflam, which has a unique mode of action for resistance management and appears to prevent plant growth by inhibiting cellulose biosynthesis and causes radial swelling and ectopic lignification; typical responses to treatment with CBIs. Previous work has demonstrated that Arabidopsis thaliana mutants that are indaziflam resistant can be identified through forward genetic screening. Since indaziflam is also active in the moss Physcomitrella patens, we took advantage of this system to attempt to understand the mode of action of indaziflam.

Using UV‐induced mutagenesis, we generated mutants in Physcomitrella patens that were resistant to 30μM of indaziflam (izr), interestingly among the izr mutants; some were cross‐resistant to synthetic auxin 2,4‐D and NAA, which is predicted that indaziflam affects multiple pathways required for plant growth and development, since auxin affects the production and remodeling of plant cell wall either directly or indirectly. The izr mutants displayed a phenotype similar to that of auxin treated wild type plants, with small, undifferentiated, and leafless filaments, so we tested their responses to exogenous auxin, and it appears mutants were insensitive to synthetic auxin and started differentiation of chloronema to caulonemal filaments and gametophore development. To positionally clone the causative mutations, we have generated segregating populations by crossing the izr mutants with the Villersexel (Vx) wild type using a protoplast fusion technique.

Our screen was designed to select mutant lines with indaziflam and auxin resistance; as a result, it establishes how critical elements within the indaziflam mode of action can refine auxin repressor activity, and have the long‐term developmental outcome. As a herbicide, indaziflam could potentially be used for weed management and eliminate the effects of these invasive grasses.

Support or Funding Information

NSERC (The Natural Sciences and Engineering Research Council of Canada)
Mapping of the Neural Circuits that Control Intrinsic Lung Function
Authors: Sun Xin , Verheyden Jamie , Su Yujuan , Barr Justinn , Xu Albert
Published: April 2020

The lung is commonly known as the gas‐exchange organ. However, given its vast surface area and direct exposure to the atmospheric environment, it is also an under‐recognized major sensory organ. The lung is exposed to signals such as oxygen, carbon dioxide, allergens, pollutants, pathogens, and subjected to mechanical stretch. Little is known on how these stimuli are sensed, processed and translated into distinct physiological outputs, including immune cell infiltration, airway tone and pulmonary blood pressure, all responses at the core of lung diseases. The lung is richly innervated. Multiple lines of evidence support that neural innervation controls many aspects of lung function. In this NIH SPARC funded research, we have used an array of cutting‐edge technologies to map the pattern determine the specific role of the neurons in modulating lung responses to inputs. Knowledge on the anatomical architecture as well as molecular code of the lung innervating neurons will set the foundation for modulating these circuits with precision.

Support or Funding Information

NIH 1OT2OD023857‐01S3