bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of Nociceptive C‐fiber Subtypes in Mouse Skin by Bee Venoms
Authors: Jurcakova Danica , Undem Bradley J.
Published: April 2020

Bee stings are extremely painful and in allergic subjects a cause of life‐threatening anaphylaxis. We have previously developed a dorsal thoracic skin‐nerve preparation to define sub‐groups of afferent C‐fibers innervating mouse skin. These comprise low mechanical threshold C‐fibers (C‐LTMs) which failed to directly respond to inflammatory mediators, and high mechanical threshold C‐fibers (C‐HTMs). The C‐HTMs could be further subdivided into two subgroups, one responding strongly to pruritogens such as histamine, chloroquine (CQ), BAM8‐22, and mast cells activation; the other failing to respond to these pruritogens, but respond strongly to 5‐HT and ATP (Mol Pharmacol. 2018, 94:1047–1056). Both types of C‐HTMs are activated by capsaicin. Here we evaluated the response of the two subgroups of C‐HTMs to venoms applied directly to the receptive fields in isolate skin. We evaluated the venom of honey bees, wasps and yellow jackets. Venom from each activated both types of C‐HTMs. Based on peak Hz of C‐fiber discharge, the relative strength of activation was: yellow jacket > honey bee ≈ wasp. CQ‐sensitive C‐fibers were activated by venoms to a greater extent than CQ‐insensitive C‐fibers. For example, bee venom evoked 6 ± 2 Hz (n=3) and 2 ± 0.4 Hz (n=7) in CQ‐sensitive vs CQ‐insensitive C‐fibers, respectively. Melittin, an active ingredient in bee venom mimicked the venoms in strongly activating C‐HTMs, with CQ‐sensitive being more strongly stimulated than CQ‐insensitive (9 ± 4 Hz (n=3) vs 1 ± 0.2 Hz (n=3), respectively). We hypothesize that CQsensitive C‐fibers are more strongly activated because unlike the CQ‐insensitive C‐HTMs, they can be stimulated as a consequence of mast cell activation (Neuroscience. 2019; 410:55–58). Indeed, melittin (10 μg/ml) caused mast cell activation (histamine release) in isolated skin fragments (76 ± 19 ng/g, above baseline, n=4); for comparison, compound 48/80 (30 μg/ml) evoked a net 203 ± 46 ng/g of histamine. The degree of melittin‐induced histamine release was not different between wildtype and MrgprB2 knockout mice, whereas compound 48/80 evoked histamine release only from the skin of wildtype mice. The data show that bee venom directly and strongly activates the terminals of virtually all high threshold nociceptive C‐fibers in mouse skin. The prurigentic subtype of C‐fibers are the most strongly activated, and this may be due to the additional indirect effect of mast cell activation, that occurs independently of MrgprB2 receptors.

Support or Funding Information

This work was supported by Department of Education, Slovakia (VEGA 1/0306/18).
PNUTS and CDK12 enhance KSHV gene expression independently of RNA quality control pathways
Authors: Rosado-González Gabriela Tatiana , Devlin Anne , Conrad Nicholas
Published: April 2020

Nuclear RNA quality control (QC) pathways degrade potentially deleterious transcripts arising from cryptic transcription. These QC pathways also target RNAs that are generated by improper mRNA capping, splicing, 3′ end formation or export. Kaposi’s sarcoma‐associated herpesvirus (KSHV) is a double‐stranded DNA virus whose transcripts avoid many of host RNA QC pathways, and it is used as a model to study mammalian cells RNA decay systems. One of the ways KSHV ensures this is by encoding ORF57 to protect viral transcripts from RNA decay systems. Previous pulse‐chase assay experiments with an unstable nuclear KSHV RNA demonstrated higher steady state KSHV RNA in presence of ORF57. Moreover, we recently found that the host proteins PPP1R10 (PNUTS) and CDK12 play important roles in controlling the steady‐state viral RNA levels. However, it was unknown if these processes work in the same pathway. The purpose of this study was to determine a possible synergistic or independent association between ORF57, PNUTS & CDK12 proteins in RNA stability. To accomplish this, PNUTS or CDK12 were knocked down with siRNAs in 293A‐TOA cells, followed by transient transfection of an ORF57‐responsive ORF59‐GFP reporter. The ORF59‐GFP reporter was co‐transfected with ORF57 or an empty vector control. We assayed RNA levels of the ORF59‐GFP reporter by northern blotting after PNUTS or CDK12 knockdown in the presence or absence of ORF57. Western blot verified that PNUTS and CDK12 were efficiently knocked down in cells treated with siRNA. Depletion of PNUTS or CDK12 resulted in higher steady‐state levels of ORF59 compared to controls. However, depletion of PNUTS or CDK12 in the presence of ORF57 resulted in higher steady‐state levels of ORF59. Our results suggest that PNUTS and CDK12 regulates RNA by a distinct mechanism than ORF57. Because ORF57 protects ORF59 from RNA QC pathways, we conclude that CDK12 and PNUTS do not affect RNA stability, but likely affect transcription or RNA processing.
Induction of Free Fatty Acid‐mediated Angiopoietin‐Like Protein‐4 Gene Expression is Independent of PPARδ Activity in Skeletal Muscle Cells In Vitro
Authors: Son Yura , Paton Chad
Published: April 2020

Angiopoietin‐like protein‐4 (ANGPTL4) inhibits lipoprotein lipase activity in skeletal muscle. Free fatty acids (FFAs) activate ANGPTL4 expression, yet the method by which FFAs induce ANGPTL4 in skeletal muscle is not well‐known. The purpose of this study was to examine the effect of FFAs on peroxisome proliferator‐activated receptor δ (PPARδ)‐mediated ANGPTL4 expression in C2C12 cells. Chronic exposure of mono‐ (MUFA) and poly‐unsaturated fatty acids (PUFA), but not saturated fatty acids (SFA), significantly induced ANGPTL4 mRNA expression in C2C12 cells (MUFA: 4.5±1.1, p=0.02; PUFA: 5.6±1.6, p=0.02; SFA: 0.4±0.1, p=0.4 respectively). To analyze the mechanism of FFA‐induced PPARδ activation of ANGPTL4, mRNA expression of PPARδ, which is believed to induce ANGPTL4 activity, was measured. However, there was no significant difference in PPARδ expression after FFA treatments. Next, we used the PPARδ agonist (GW0742) and antagonist (GSK0660) to modulate PPARδ activity independent of FFA treatment and examined ANGPTL4 expression. GW0742 significantly increased ANGPTL4 in undifferentiated and differentiated C2C12 cells (12.6±3.9, p=0.01 and 2.8±0.2, p<0.001 respectively), while GSK0660 significantly suppressed basal ANGPTL4 expression (0.2±0.1, p=0.05 and 0.2±0.03, p<0.001 respectively). Yet, in the presence of FFA and GSK0660, ANGPTL4 gene expression significantly increased despite the absence of PPARδ activity in both undifferentiated and differentiated C2C12 cells (7.1±2.5, p=0.03 and 3.8±0.4, p<0.001). Taken together, these results indicate that FFAs may activate ANGPTL4 in a PPARδ independent manner, and a revised mechanism of FFA‐induced skeletal muscle ANGPTL4 expression may be warranted.
Synergistic effect of sorafenib and resveratrol in human breast cancer MDA‐MB‐231 cells
Authors: lo bennett lunawati
Published: April 2020

The incidence of breast cancer mortality is one of the major challenges in the scientific community. Despite advances in diagnosis and treatment options, death among women worldwide are still high.

The desired outcome of the cancer therapies is to increase cellular apoptosis, to decrease the proliferative activity of the cancer cells, to minimize drug resistance, and to decrease the toxicity of the anticancer drugs. Sorafenib (SF), a component of ERK1/2 pathway, is a multi‐kinase inhibitor that target VEGFR‐1, VEGFR‐3, PDGFR‐h, and others. SF has been shown in the previous preclinical studies to slow cancer progression in human hepatocellular, renal, colon, pancreatic, thyroid, ovarian, lung, and breast cancer. The use of natural dietary antioxidants such as curcumin, resveratrol (RSV) in cancer treatment has gained popularity due to their minimal cytotoxic effects with no evidence of causing damage to normal cellular tissue. RSV is a natural polyphenol compound found in red grapes, berries, peanuts and other fruits with anti‐oxidant and chemo‐preventive properties. Several studies mentioned that RSV possesses potent anti‐cancer activity against human breast cancer by inhibiting cancer initiation, promotion, and progression.

In the present study, RSV was studied as a chemo‐enhancing potential agent in combination with low dose of SF for the treatment of breast cancer using MDA‐MB‐231 cells. Cells viability measured by MTT assay (3‐[4, 5‐dimethylthiazol‐2‐yl] ‐2, 5‐diphenyl‐tetrazolium bromide), Hoechst33342, H2DCFDA, and Rhodamine123 staining using fluorescent microscope were conducted to determine cell viability, chromatin inducing apoptosis, intracellular generation of ROS, and mitochondrial membrane potential, respectively. Western Blot to detect different signaling protein expressions such as Casp‐3, Casp‐9, Casp‐8, P53, Bax, Bcl2, Apaf‐1, Smac/Diablo, MMP2, BRCA1, BRCA2, Bim, and Map kinases were conducted to test the hypothesis that RSV has efficacy with low dose of SF to decrease cancer cell proliferation and progression. The concentration of SF used was 3 μM and RSV was 50 μM. This finding suggests RSV can potentiate SF as anticancer drug in breast cancer treatment.

Support or Funding Information

Union University College of Pharmacy
Local Heating Mediated Vasodilation Increased 48 Hours After Remote Ischemic Preconditioning
Authors: Hinde Jurnie F. , Kim Jahyun , Barlow Sydney E. , Lang James A.
Published: April 2020

Introduction

Remote ischemic preconditioning (RIPC), induced by brief periods of blood flow occlusion to a limb followed by reperfusion, protects distant tissues from ischemia‐reperfusion (IR) injury. A single bout of RIPC confers a delayed window of protection that begins 24 hrs after RIPC and last for 2–3 days; however, whether this timeline also applies to the microvasculature is unclear. We hypothesize that the hyperemic response to local heating will increase after a single bout of RIPC.

Methods

Sixteen healthy, young adults (7M, 9F) underwent a single session of RIPC, which consisted of four periods of inflating an arm cuff to 200mmHg for five min interspersed with five min of reperfusion. Laser speckle contrast imaging (LSCI) was used to measure the hyperemia response to local skin heating (Tsk = 42°C) at five time points; before treatment, and 24, 48, 72, and 168 hrs post‐RIPC. LSCI data are expressed as cutaneous vascular conductance (CVC = LSCI flux/mean arterial pressure).

Results

Compared to pre‐RIPC (1.58 ± 0.09 flux · mmHg−1), the vasodilation response to local heating was increased only at the 48‐hr post‐RIPC time point (24 hrs: 1.59 ± 0.10, 48 hrs: 1.69 ± 0.10, 72 hrs: 1.54 ± 0.13, 160 hrs: 1.53 ± 0.11 flux · mmHg−1).

Conclusion

These results indicate that the modest increase in local‐mediation dilation exhibited a delayed response that peaked 48 hrs following a single RIPC bout. This suggests that a single bout of RIPC may have a protective effect on the microvasculature.

Support or Funding Information

NASA – Iowa Space Consortium Grant
Particulate Matter Activates SIRT1‐SREBP1‐PIR Pathway to Promote Pulmonary Fibroblast Inflammation and Enhance Lung Fibrosis
Authors: Tien Chia-Ping , Chang Yu-Chan , Hung Shih-Chieh , Hsiao Michael
Published: April 2020

Particulate matter (PM) is infamous for its strong connection with a variety of human disorders and diseases, including bronchitis, asthma, chronic obstructive pulmonary disease (COPD), cancer, allergy, stroke, Alzheimer’s disease, anxiety, cardiovascular disease, productive dysfunction, anemia, and diabetes. While mechanisms driving PM‐induced pathogenesis are poorly understood, it’s believed associated with PM’s unique character, small size with large surface area, allowing PM easily enters the body with a great amount of toxin attached. In an attempt to identify the microenvironment induced by PM, we treated human pulmonary fibroblasts (HPF) with different doses of PM, and our in vitro results showed that PM activated sterol regulatory element‐binding protein 1 (SREBP1) and its downstream PIRIN (PIR) and Nod‐like receptor protein 3 (NLRP3) inflammasome through downregulating Sirtuin1 (SIRT1). Our further in silico analysis revealed that overexpression of PIR was related to tobacco and consistent with early COPD, while our IHC analysis reported the correlation of PIR with PM from lung tissues of PM‐fed mouse models. Taken together, we concluded that PM caused lung fibrosis and COPD via SIRT1‐SREBP1‐PIR/ NLRP3 inflammasome axis, which may provide several plausible therapeutic targets for PM‐related adverse sequelae.

Support or Funding Information

This work was supported by grants from Academia Sinica and Ministry of Science and Technology [MOST 104‐0210‐01‐09‐02, MOST 105‐0210‐01‐13‐01, and MOST 106‐0210‐01‐15‐02] to Michael Hsiao.
Identification of a novel player in insulin‐mediated lipolysis inhibition
Authors: Zadoorian Armella , Stoeckli Jacqueline , James David Ernest
Published: April 2020

Lipolysis is a tightly regulated catabolic process essential for maintaining whole‐body energy homeostasis. It exists in a dynamic balance between activation by β‐adrenergic stimulation during starvation or prolonged exercise, and inhibition by insulin following nutrient intake. Dysregulation of lipolysis can lead to ectopic fat accumulation in the blood and insulin‐sensitive tissues, encouraging the development of metabolic disorders such as type 2 diabetes and non‐alcoholic fatty liver disease. Despite this, the mechanism by which insulin regulates lipolysis remains controversial and incomplete. The prevailing model hinges on the enzymatic function of the Akt substrate, phosphodiesterase 3B (PDE3B) in explaining insulin’s antilipolytic action. However, several studies have challenged this and suggested that whilst PDE3B is essential, its function alone cannot explain this regulation. Instead, we implicate another protein, α/β‐hydrolase domain‐containing protein 15 (ABHD15) as a novel player in insulin‐mediated lipolysis inhibition. Through stable knockdown or knockout of ABHD15 in 3T3‐L1, brown or fat explant adipocytes, we demonstrate a significant defect in insulin’s inhibition of lipolysis that is rescued upon ABHD15 re‐expression. Moreover, through the characterisation of ABHD15 and PDE3B mutants lacking novel insulin‐regulated phosphorylation sites previously identified by our lab, we have explored a putative mechanism by which these proteins facilitate insulin’s inhibition of lipolysis. In summary, we have identified a novel regulator of lipolysis that will likely shed light on the mechanism of this important process.

Support or Funding Information

Australian Government Research Training Program Scholarship, The Chen Family Research Scholarship.
Investigating the mechanism by which Naproxen and Prometryn causes Cardiovascular dysfunction
Authors: Sule Rasheed , Gomes Aldrin
Published: April 2020

Background and Objective

Cardiovascular disease (CVD) is the leading cause of death and a major cause of disability worldwide. Previous studies have shown that the long‐term and chronic use of nonsteroidal anti‐inflammatory drugs (NSAIDs) increases the risk of developing stroke and cardiovascular diseases. Naproxen is an FDA‐approved NSAID that is currently used to treat several medical conditions such as pain, osteoarthritis, rheumatoid arthritis, polyarticular juvenile idiopathic arthritis, tendonitis, etc. Acetaminophen, the active ingredient in Tylenol, is not an NSAID but it has been associated with increased risk of liver toxicity. Similarly, the long‐term exposures to pesticides such as Prometryn amongst others have been established to be dangerous to human lives and capable to disrupt the physiology of different organs in the body, including the cardiovascular system. Prometryn, a diamino‐1,3,5‐triazine, is used as a selective pre‐ and post‐emergence herbicide. It is used to control annual grasses and broadleaf weeds in cotton and celery. This study aimed to investigate the effects of Naproxen, Acetaminophen, and Prometryn in cardiomyocytes and the underlying mechanisms by which it causes cardiovascular dysfunction.

Methods

The embryonic cardiomyocyte cell line H9c2 is commonly used in numerous in vitro studies because morphological parameters of their cells resemble immature embryonic cardiomyocytes. It has been previously shown that H9c2 cell line and primary neonatal cardiomyocyte cells show similar hypertrophic responses in vitro. H9c2 cardiomyocytes were plated in a 96 well plate at a concentration of 4000 cells/well. The cells were then treated with different concentrations of Naproxen, Acetaminophen, and Prometryn for 24h at 37°C and 5% CO2. The cell viability, reactive oxygen species (ROS) formation, and mitochondrial membrane potential (ΔΨm) were examined.

Results

H9c2 cells treated with 100μM Acetaminophen and 20μM Prometryn showed significant decreases (> 30%) in cell viability. Hydrogen peroxide (H2O2), which was used as a positive control, also resulted in significant decreases in cell viability at 100μM. ROS formation was significantly increased in the presence of 200–400μM Naproxen. Acetaminophen showed significant amount of ROS formation at a higher concentration of 200μM while Prometryn also showed a significant increase in ROS formation at a higher concentration of 30μM. The effect of varying concentrations of Naproxen showed that ΔΨm was decreased by ~30% at 100μM. Prometryn decreased ΔΨm by ~20% at 10–20μM while Acetaminophen also decreased ΔΨm by ~20% at 100–200μM.

Conclusion

These results suggest that both concentration and exposure time are important for NSAID‐induced cardiotoxicity. Acetaminophen, which is not an NSAID, increased ROS generation and decreased ΔΨm at higher concentration. This experimental data suggests that Naproxen, Acetaminophen, and Prometryn causes cardiomyocyte dysfunction at varying concentrations.
Microglial Involvement in Stress‐induced Prolonged Sympathetic Nervous Excitation in Rats
Authors: Yoshizawa Masashi , Fukushi Isato , Takeda Kotaro , Kono Yousuke , Hasebe Yohei , Koizumi Keiichi , Toda Takako , Okada Yasumasa
Published: April 2020

Hypertension is one of the most important risk factors causing cardiovascular diseases. It has been assumed that hypertension is caused by continuous over‐excitation of the sympathetic nervous system, which is often accompanied by psychological stress. We have previously reported that glial cells, especially astrocytes, are importantly involved in blood pressure elevation during stress and post‐stress recovery period in air jet stress loaded rats. However, it has not been well clarified whether another glial cell, microglia, is also involved in stress‐induced sympathetic nervous excitation. In the present study, we aimed to address this issue using adult rats (n=9, 23–26 weeks age, body weight 300–467g). A blood pressure telemeter was implanted intraperitoneally with a pressure transducer tip in the abdominal aorta under anesthesia. After at least one week recovery period, blood pressure and pulse rate responses to psychological stress were measured in an unanesthetized freely moving condition by blood pressure telemetry system (Kaha Sciences, Auckland, New Zealand). Measurement was first conducted without pretreatment with minocycline, which is an inhibitor of microglial activation. Then, in more than one week after this control measurement, the animal was pretreated with minocycline (25mg/kg/day) for three consecutive days, and blood pressure and pulse rate responses to stress were again measured under the suppression of microglial activation. Air jet stress was loaded to the rat by blowing compressed air to the face of the animal (10L/min, 15min). We recorded blood pressure and pulse rate before, during and after the stress loading, and compared these data between the conditions before and after minocycline pretreatment. Pretreatment with minocycline did not affect either blood pressure or pulse rate in a pre‐stress resting condition. However, after pretreatment with minocycline, stress‐induced augmentation of both blood pressure and pulse rate were attenuated during and after air jet stress loading with statistical significance (paired t‐test). These findings indicate that activated microglia is involved in the augmentation of sympathetic nervous activity during and after stress loading.

Support or Funding Information

This work was supported by JSPS KAKENHI Grant Numbers 17K08559, 17H05540, 18K17783, 19K17386 and 19K17620 and a Grant‐in‐aid of the Cardiovascular Research Fund, Tokyo, Japan.
Deletion of the Microtubule‐associated protein tau (Mapt−/−) results in diastolic heart failure and altered skeletal muscle function in vivo
Authors: Rajalingam Sahith , Kambrath Anuradha Valiya , Vidal Ruben , Willis Monte S.
Published: April 2020

According to 2019 statistics, Alzheimer’s affects 5.8 million Americans. It is an undertreated disease with increased prevalence. The microtubule‐associated protein tau (Mapt) gene encodes the Tau protein, which plays a crucial role in the pathology and progression of Alzheimer’s disease in the brain. The Tau protein interacts with microtubules and plays a role in neuronal microtubule stability. Surprisingly, Tau is equally expressed in the heart, skeletal muscle, kidney, adipose, and soft tissue at the protein level, and its function is mostly unknown. Since microtubule stability plays an essential role in cardiac function, we hypothesized that Mapt−/− mice would develop cardiac dysfunction and skeletal muscle function in vivo. In the present study, we investigated both cardiac function and skeletal muscle phenotypes at 12 months of age. Conscious echocardiography (VisualSonics Vevo 2100) and ECG analysis (ECGenie) was performed in male and female Mapt−/−. Mice and compared to age‐ and strain‐matched wildtype controls. Echocardiographic analysis identified an “apparent” increase in Mapt−/− systolic dysfunction compared to wildtype mice (EF%: 83.4±1.9 vs. 79.1±1.5, FS%: 51.5±2.0 vs. 46.9±1.5%, N=6, 7, respectively) at 12 months of age, although both systolic and diastolic LV volumes were significantly decreased in the Mapt−/− mice. Parallel Doppler studies of the mitral valve E/A ratio (1.5±0.4 vs. 2.3±0.5) identified a significant diastolic (relaxation) dysfunction, which may be driving the “apparent” systolic dysfunction and consistent with the clinical parameters of heart failure with preserved ejection fraction (HFpEF). Electrophysiologic analysis of Mapt−/− mice at 12 months of age identified a significantly decreased heart rate (and corresponding increased RR interval), with evidence of an increased heart rate variability (e.g., CV%) indicative of autonomic changes in the heart. No other changes in ECG intervals (i.e., PQ, PR, QRS, QT, ST, QTC ) or ECG amplitudes (SR, R) were identified. These studies demonstrate the Tau protein’s role for the first time in cardiac and skeletal muscle function phenocopying clinically relevant heart failure (HFpEF), characteristic of patients with Alzheimer Disease in recent small studies. Furthermore, these studies indicate that anti‐Tau therapies for Alzheimer Disease currently in development for primary brain disease could potentially have unexpected off‐target effects that should be considered, particularly since heart failure and skeletal muscle weakness in the AD patients is associated common co‐morbidities and mortality.

Support or Funding Information

Lilly Foundation/Indiana Center for Musculoskeletal Health/ IU School of Medicine, Physician Scientist Initiative, Scientific Research Initiative
Multilayer regulatory mechanisms of ERK1/2 signals transmitted through the Shoc2 scaffolding complex
Authors: Galperin Emilia , Jang HyeIn , Jang Eun Ryoung , Chawla Udeep , Wilson Pat
Published: April 2020

Signaling scaffolds guide the flow of information and the spatial organization of the enzymes within the ERK1/2 signaling pathway. However, mechanisms that control assembly and dynamics within scaffolding complexes as well as mechanisms regulating the cellular distribution of these complexes remain largely unknown. Our studies unravel a novel, multi‐level paradigm in which allosteric modifications alter the ability of the scaffold protein Shoc2 to actively accelerate transmission of ERK1/2 signals. Shoc2 accelerates ERK1/2 signaling by tethering to close proximity essential proteins of the pathway, Ras and RAF‐1. Germ‐line mutations in Shoc2 affect spatial distribution of Shoc2 or its ability to assemble the signaling complex and cause Noonan‐like syndrome, a congenital disorder that presents with a wide spectrum of developmental abnormalities.

Our lab studies mechanisms that fine‐tune ERK1/2 signals transmitted through the Shoc2 complex. We found that Shoc2 assembles an elegant multi‐protein complex that incorporates a number of proteins of the ubiquitin system. To fine‐tune amplitude of ERK1/2 signal transmitted via the complex, Shoc2 tethers the E3 ligase HUWE1, the (AAA+) ATPases, PSMC5 and VCP/p97 as well as the deubiquitinating enzyme, USP7. All of these enzymes are integral to the intricate feedback mechanism by which ubiquitination controls amplitude of the Shoc2‐ERK1/2 signals.

Our studies show that HUWE1 ubiquitinates Shoc2 and RAF‐1, while ATPases modulate ubiquitination of Shoc2 and RAF‐1 through remodeling of the complex. We used fluorescence microscopy and biochemical analysis to demonstrate that PSMC5 and VCP/p97 are involved in recruitment of the Shoc2 scaffolding complexes to endosomes for complex remodeling. Importantly, we found that in the context of the Shoc2 complex, USP7 functions as a “molecular switch” that uses its enzymatic activity to trigger the “turn‐off” of the ERK1/2 signals transmitted via the scaffolding complex. We found that congenital Shoc2 mutations affect mechanisms controlling Shoc2 ubiquitination leading to aberrant signal transmission.

In summary, our studies are the first to demonstrate that the Shoc2 scaffold employs multi‐protein enzymatic machinery to govern the amplitude of Shoc2‐ERK1/2 signals. We also show that mislocalization of the Shoc2 complexes affects remodeling of the scaffolding module and may lead to the developmental pathology. Overall, these studies significantly advance our understanding of the mechanisms by which non‐enzymatic scaffolds regulate specificity and dynamics of the ERK1/2 signaling networks.

Support or Funding Information

National Cancer Institute (R00CA126161 to EG), National Institute of General Medical Sciences (GM113087 to EG), American Cancer Society (RSG‐14‐172‐01‐CSM to EG)
Computational Studies of Glycosylated Photosensitizers with Plasma Proteins
Authors: Guevara Johnny , Zahran Mai , Samaroo Diana
Published: April 2020

Photodynamic therapy (PDT) is a promising treatment against cancer, it utilizes photosensitizers that when excited by light undergo photochemical reactions that can kill abnormal cancer cells. However, in order for photosensitizers to reach their target, they must effectively bind serum albumin, the most abundant plasma protein circulated in an organism’s blood stream. In our previous study, we synthesized three glycosylated photosensitizers (PGlu, CGlu and IGlu) and performed in vitro spectroscopy studies that revealed each glycosylated photosensitizer effectively binding bovine serum albumin and human serum albumin (BSA and HSA). The aim of this work was to implement an in silico approach to study how PGlu, CGlu, and IGlu are binding onto BSA and HSA. We performed molecular docking simulations to predict a favorable mode of binding between each of our glycosylated photosensitizers to BSA and HSA. Further, we performed a 5 ns molecular dynamics (MD) simulation for each of our predicted systems; consequently, we calculated the RMSD of the systems to attest for stability, performed binding free energy calculations to correlate with experimental binding constant results and analyzed residue interactions critical to each binding event. We observed PGlu, CGlu and IGlu favorably binding the hemin site located in subdomain IB of BSA, forming strong interactions with the Trp134 residue, as well as favorably binding Sudlow Site 1 located in subdomain IIA of HSA, forming strong interactions to Trp214. RMSD studies revealed the BSA‐IB and HSA‐IIA systems plateauing after a short time indicating stable binding events; in addition, a Pearson correlation coefficient of R=0.87 between the experimental binding constants and computational binding free energies showed a strong correlation between in vitro and in silico results. Overall, our computational results corroborated our experimental results, revealing that our glycosylated photosensitizers can bind serum albumin effectively through critical tryptophan residue interactions and can potentially aid in the progression of drug development for PDT.

Support or Funding Information

This research used resources of the National Energy Research Scientific Computing Center (NERSC), a U.S. Department of Energy Office of Science User Facility operated under Contract No. DE‐AC02‐05CH11231.
“If It Looks Like Windows ‘98, I’m Not Using It”: Factors Influencing Students Use of Technology‐Enhanced Learning (TEL) Resources in Anatomy
Authors: Clunie Lauren , Morris Neil P. , Hallam Jennifer L. , Pickering James D.
Published: April 2020

The use of technology‐enhanced learning (TEL) resources is now ubiquitous within anatomy education. Anatomy educators and commercial partners are constantly pushing the boundaries of technological innovation in education, and students now have access to a wide variety of TEL resources from both their own institution and other online sources. This study aimed to identify factors that may influence a student’s decision to utilise TEL resources when studying anatomy.

This study, approved by the university ethics committee, was undertaken with Year 2 medical students at a UK university. The study employed surveys and focus groups in an exploratory sequential mixed methods approach. Firstly, pilot Likert scale items were developed following analysis of existing surveys from both anatomy TEL evaluation literature (identified from a previously published systematic literature review) and broader education literature. The resultant pilot survey responses (α=0.891; n=131) were analysed using principal components analysis (PCA). Secondly, the results of the pilot survey were explored in greater depth using focus groups with survey respondents (n=12). Focus groups were audio recorded, transcribed verbatim and analysed using thematic analysis. Finally, triangulation of the data collected from both the pilot survey and focus groups informed the development of a refined survey scale. The consecutive Year 2 medical cohort completed the survey (n=129). PCA was utilised to validate the adapted survey scale (α=0.893), the results of which revealed four emergent factors. Further statistical tests revealed no significant difference in responses when comparing gender, resource preference (i.e. TEL or non‐TEL) and academic achievement.

Using a robust mixed method approach, this study developed the validated 23‐item Anatomy TEL Utility Scale with the following emergent factors: (1) Attitude Towards TEL; (2) Perceived Effectiveness; (3) Visual Appeal; (4) Personal and Social Influence. The results from this study revealed that, in addition to affective attitude towards TEL, students’ use of TEL resources for studying anatomy may be influenced by their perceptions of resource usefulness, ease of use and visual appeal. Furthermore, students revealed they are most likely to seek resources that are aligned to their curriculum by using resources provided by their institution, or resources validated by their instructor or peers. These findings highlight the responsibility held by anatomy instructors in ensuring they recommend appropriate TEL resources to students and, if developing their own resources, emphasizes the importance of evidence‐informed instructional design.
MicroRNA‐378a Loss Enhances Mitochondrial Bioenergetics and Lessens Cardiac Contractile Dysfunction in the Type 2 Diabetic Heart
Authors: Durr Andrya J. , Hathaway Quincy A. , Kunovac Amina , Taylor Andrew D. , Pinti Mark V. , McLaughlin Sarah L. , Shepherd Danielle L. , Singh Atul K. , Hollander John M.
Published: April 2020

During type 2 diabetes mellitus (T2DM), microRNA (miR) function can diverge and become pathological. Decreases in mitochondrial bioenergetic function contribute to the development of cardiovascular contractile dysfunction, making the mitochondrion and pathologically inclined miRs, a potential target for therapeutic intervention. MiR‐378a interacts with the mitochondrial genome, and during T2DM, is associated with a decrease in the mitochondrial genome‐encoded protein, ATP6. Loss of ATP6, a key component of the ATP synthase complex, results in decreased ATP synthase activity and ATP generating capacity of the electron transport chain (ETC). The objective of this study was to determine if genetic loss of miR‐378a could ameliorate the decrements in cardiac function observed in the type 2 diabetic heart, through restoration of ATP6 protein content, improved ATP generating capacity, and improved cardiac contractile dysfunction. A miRNA‐378a knockout (KO) mouse model was bred into a Db/Db background to produce miR‐378aKO/Db/Db (KO/Db/Db) mice. At 25 weeks of age, M‐mode echocardiography‐based measures (ejection fraction and fractional shortening (p < 0.05)), indicated preservation of cardiac contractile function in KO/Db/Db mice which was complemented by a reversal of long‐axis diastolic radial and longitudinal strain patterns (p < 0.05 for both). Mitochondrial oxygen consumption was evaluated in KO/Db/Db mice using glutamate/malate (GM) and palmitoylcarnitine (PM) substrates. A higher GM‐dependent respiratory control ratio (RCR) was observed in KO/Db/Db mice when compared to Db/Db mice (p < 0.05). Further, KO/Db/Db mice displayed significantly lower PM‐dependent state 3 and state 4 respiration rates when compared to Db/Db counterparts (p < 0.05), and a higher RCR in both diabetic groups when compared to wild‐type controls (p < 0.05). ETC complex activity analyses revealed increased ATP synthase activity in KO/Db/Db mice (p < 0.05), that was supported by increases in mt‐ATP6 and mt‐ATP8 protein content in KO/Db/Db mice (p < 0.05). In summary, loss of miRNA‐378a content in the type 2 diabetic heart preserved cardiac contractile function and lead to a reduced dependence on fatty acid metabolism as suggested by reduced ability to utilize PM substrate and increased GM‐dependent RCR. This data provides evidence for the efficacy of miRNA targeted mitochondrial therapeutics in a mouse model of T2DM.

Support or Funding Information

This work was supported by: The National Heart, Lung, and Blood Institute [R01 HL‐128485] (JMH), American Heart Association [AHA‐17PRE33660333] (QAH), West Virginia IDeA Network of Biomedical Research WV‐INBRE support by National Institute of Health Grant [P20GM103434], and the Community Foundation for the Ohio Valley Whipkey Trust.
The connector between the kinase and RNase domains of IRE1α differentially controls the activities of this stress sensor
Authors: Ricci Daniela , Tutton Stephen , Chomistek Steven , Marrocco Ilaria , Ying Mingjie , Blumental Daniel , Eletto Daniela , Vargas Jade , Boyle Sarah , Fazelinia Hossein , Paton James , Paton Adrienne , Tang Anthony , Hu Chih-Chi Andrew , Gidalevitz Tali , Argon Yair
Published: April 2020

Inositol requiring enzyme 1 (IRE1α) is a sensor that responds to endoplasmic reticulum stress with self‐phosphorylation, oligomerization and RNase activity. As a result, IRE1α activates the critical transcription factor XBP1, while reducing the expression of select other transcripts, to cope with the level of ER stress. In response to stress IRE1α molecules also cluster transiently, with similar time frame to the enzymatic activity.

The response of IRE1α is inhibited by mutations in the kinase and RNase active sites of IRE1α‐, but not by most mutations with no direct catalytic role. Here we describe amino acid substitutions in the segment connecting the kinase and RNase domains which affect IRE1α clustering, its RNase activities or both. The more detrimental substitutions involve Pro mutations and they do not support RNase activity, fail to auto‐phosphorylate in response to stress and do not oligomerize. The substitution L827P is dominant‐negative in vivo: it dimerizes with full‐length WT IRE1α and consequently inhibits its activation in lymphoma and multiple myeloma cells. Expression of the IRE1α L827P mutant also increases the sensitivity of lymphoma cells to ER stress and inhibits their growth. Less drastic substitutions, such as L827F have intact RNase and kinase activities but fails to oligomerize and displays altered self‐phosphorylation, in particular on Ser729. Higher sensitivity to partial proteolysis indicates directly that the conformations of L827P, L827F and WT IRE1α are distinct.

Furthermore, our data show that clustering of IRE1α depends on sensing ER stress but not on its RNase activity. There are conditions that initiate IRE1α RNase activity without inducing clustering, and conversely ‐ RNase‐impaired molecules can cluster. We hypothesize that depending on which residues are phosphorylated, IRE1α assumes different conformations, only some of which are permissive for clustering. Therefore, this work shows that the kinase‐RNase connector is important for attaining the stress‐responsive, clustering‐capable conformation of IRE1α and that co‐expression of variant IRE1α sensors profoundly inhibits the normal signaling.

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T32 HL 7954‐19 (R.D.)
A TGFβ1‐BIGH3‐Apoptosis Axis Comprising Peptidase and Integrin α3β1 Promotes Renal Cell Death
Authors: LeBaron Richard G. , Perez Louis , Randolf Lois , Phelix Clyde F.
Published: April 2020

Transforming growth factor‐β1 (TGFβ1) helps contribute to diabetes complications in various organs including the renal system. TGFβ1 signaling activates genes encoding various extracellular matrix (ECM) molecules that accumulate and promote renal cell injury, increasing the probability of end stage renal disease (ESRD). The mechanisms underlying development of ESRD are complex and not known completely. We have shown previously that TGFβ1 strongly stimulates renal proximal tubule epithelial (PTE) cells to express the gene that encodes an ECM protein called BIGH3. This newly‐synthesized endogenous BIGH3 protein increased PTE cell apoptosis. Treating PTE cells with exogenous recombinant BIGH3 protein increased apoptosis. Anti‐BIGH3 antibody blocked endogenous and exogenous BIGH3‐mediated apoptosis. These findings signify a TGFβ1‐BIGH3‐Apoptosis axis that augments kidney disease progression. BIGH3’s unstructured C‐terminus encodes integrin‐binding sequences EPDIM and RGD. In the kidney, BIGH3 C‐terminal proteolysis (CTP) provides bioactive C‐terminal fragments (BCTF) that comprise EPDIM and RGD and account for PTE cell apoptosis. The EPDIM motif in BIGH3 induced two‐fold greater apoptosis of PTE cells when compared to apoptosis induced by the RGD motif. In order to provide evidence that BIGH3 CTP and integrin interaction with EPDIM is required for PTE cell apoptosis, we used recombinant BIGH3, PTE cells, BIGH3 mutagenesis, protease inhibitors, TUNEL, immunoblots, integrin α3 antibody and siRNA. Homology modeling and molecular docking software Raptor X, ZDOCK and AutoDock Vina were used to generate monomeric and heterodimer models of α3β1 integrin; followed by in silico analysis of EPDIM interaction with α‐integrin subunit. Results show that a serine peptidase accomplishes BIGH3 CTP, which we regard as mechanistic in the proposed axis. A 21% chemical inhibition of BIGH3 CTP resulted in a 43% decrease in PTE cell apoptosis. In silico analysis indicates that the BCTF encoding EPDIM, bound to α3 subunit, prevents full activation and disrupts integrin‐mediated pro‐survival signals. We conclude that BIGH3 mediates at least some of the destructive arm of TGFβ1 signaling in diabetic nephropathy. BIGH3 CTP provides local mediator BCTFs that act through paracrine or autocrine pathways to block α3β1 pro‐survival signaling leading to PTE cell apoptosis. The data indicate that molecules of the axis pathway, including BIGH3, BIGH3 peptidase, and BIGH3 BCTFs are potential therapeutic targets to treat diabetic nephropathy. In a broader perspective, we have evidence that a similar mechanism is active in retinal endothelial and retinal pericyte apoptosis in diabetic complications of the ocular system.
Downregulation of Fis1 in Endothelial Cells Does Not Impact Normal Endothelial Cell Mitochondrial Bioenergetics, Metabolism, or Mitochondrial Protein Content
Authors: kakarla mamatha , Wang Jingli , Waters Heather , Puppala V. krishna , Tyagi Sudhi , Hofeld Benjamin , Ying Rong , Anger Amberly , Harwig Megan Cleland , Egner John , Hill Ronald Blake , Widlansky Michael E.
Published: April 2020

Our prior data suggest that knockdown of Fis1 protein improves vascular endothelial health by reversing endothelial dysfunction associated with dysglycemic conditions and reducing endothelial inflammation and permeability of the endothelium. Fis1 is an outer mitochondrial membrane protein that triggers mitochondrial fission when bound by dynamin‐related protein‐1. Fis1 is important to mitochondrial fission process specifically under pathophysiological conditions making Fis1 an attractive therapeutic target. We hypothesize that reducing Fis1 expression will not alter other mitochondrial proteins and mitochondrial bioenergetics under different glycemic conditions. We measured mitochondrial proteins, and mitochondrial bioenergetics in wild type immortalized human microvascular endothelial cells (HMEC‐1) and HMEC‐1 cells that were transfected with Fis1 siRNA (20 nM) or scrambled siRNA (20 nM). Upon transfection, cells were exposed to different glycemic conditions: normal (5mM), high (30mM for six hours), and low (2mM for two hours) glucose levels. Mitochondrial proteins expression in Fis1 siRNA HMEC‐1 cell lysates and scrambled control cell lysates were assessed under different glycemic conditions by high throughput immunodetection WES system. The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in transfected cells under different glycemic conditions were measured by Seahorse mitochondrial and glycolytic stress tests. There were no significant differences in mitochondrial bioenergetics (OCR and ECAR; n=5, p>0.05), and expression of other mitochondrial proteins (MFF, MFN1, MFN2, Drp1, OPA1, Cyt‐c, TOMM20, MFF, pAMPKa, POLG, P62, NDUFB8, GABARAP) between siFis1 cells and scrambled controls in any of the glycemic conditions (n=4, p>0.05). There is significant increase in expression of other mitochondrial proteins (MFF, MFN1, Cyt‐c, TOMM20, MFF, pAMPKa, POLG, P62, NDUFB8, GABARAP) under low glucose conditions (n=4, p<0.05) compared to normal and high glucose conditions. Reducing the expression of Fis1 did not alter other mitochondrial proteins and mitochondrial bioenergetics that are critical for normal physiological processes. Therapeutic potential of Fis1 knockdown is enhanced by a lack of impact on endothelial cell metabolism and other mitochondrial protein expression.

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This project was supported by the National Institutes of Health, through Grant Number
R01‐HL128240 and
HL125409.
Hypertension and Exaggerated Sympatho‐excitatory Reflexes are Reduced by Low Salt Diet in Male Obese Zucker Rats with no Effect in Lean Zucker Rats
Authors: Sun Fen , Das-Earl Paromita , Schreihofer Ann M.
Published: April 2020

Adult male obese Zucker rats (OZRs) develop sympathetically‐driven hypertension and exaggerated sympatho‐excitatory reflexes compared to age‐matched male lean Zucker rats (LZRs). Compared to LZRs, male OZRs on a standard rodent diet (0.5–1% NaCl) display attributes seen in salt sensitive rats consuming a high salt diet, such as suppressed plasma renin activity and exaggerated sympatho‐excitatory reflexes. The present study examined whether these attributes would be reduced in male OZRs on a low salt diet (0.1% NaCl). Rats were placed on diets with 0.5–0.85% NaCl (control diet; 10 OZRs and 8 LZRs) or a diet with 0.1% NaCl (low salt diet; 12 OZRs and 10 LZRs) for 14–28 days. After consumption of a low salt diet, baseline mean arterial pressure (MAP) was reduced in urethane‐anesthetized OZRs (125.7±4.4 vs 111.8±4.1 mmHg, P <0.05) but not in LZRs (116.3±4.6 vs. 113.1±5.6 mmHg). Stimulation of sciatic nerve (1 msec pulses for 5 sec at 20 Hz, 100–800 μA) evoked intensity‐dependent increases in MAP and splanchnic sympathetic nerve activity (SNA) in OZRs and LZRs on both diets. Consumption of a low salt diet reduced the sciatic stimulation‐induced rises in SNA in OZRs (800 μA increased SNA 96±7.6% with control diet and 75.3±4.7% with low salt diet, P <0.05) with no effect of diet in LZRs (800 μA increased SNA 87.4±4.4% with control diet and 83.0±8.8% in with low salt diet, n.s.). In contrast, low salt diet reduced the sciatic stimulation‐induced rises in MAP in OZRs and LZRs. These data suggest that consuming a diet containing 0.5–1.0% NaCl contributes to elevated baseline MAP in OZRs and exaggerated sympatho‐excitatory reflexes compared to male LZRs. However, both OZRs and LZRs show reduced sympathetically‐mediated rises in MAP on low salt diet. This study suggests that in male OZRs consumption of a standard rodent diet with 0.5–1.0% NaCl promotes some physiological effects observed in salt‐sensitive strains of rats on a high salt diet.

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Mechanisms for Impaired Short‐Term Control of Blood Pressure with Obesity (R01HL132568, Dr. Ann M. Schreihofer)
Trafficking of the Prorenin Receptor in Endothelial Cells
Authors: Harris Nicholas R. , Ogola Benard O. , Visniauskas Bruna , Katakam Prasad V. , Meadows Stryder M. , Prieto Minolfa C. , Lindsey Sarah H.
Published: April 2020

Diabetic patients are at higher risk for hypertension and cardiovascular mortality than their normotensive, nondiabetic counterparts. Diabetic patients also have increased circulating prorenin which can bind to its receptor (PRR) to cause activation of the renin‐angiotensin system and fibrosis. We hypothesized that diabetes induces dysregulation of the endothelial PRR to exacerbate cardiovascular damage. Mouse pancreatic endothelial cells (MS1) were cultured and exposed to high glucose or low calcium (Ca2+) conditions for one hour. Cells were fixed for immunocytochemistry or fractionated and immunoblotted for PRR. Treatment with 25 mM glucose or 25 mM mannitol for one hour did not impact full length PRR expression or localization in the total membrane fraction. When Ca2+ was removed from the media, full length PRR increased in the cytosolic fraction by 3.6‐fold (P=0.022) compared to control. Restoration of physiological Ca2+ returned PRR expression to control levels in the cytosolic fraction, while neither sPRR nor full length PRR were altered in the total membrane fraction. Confocal imaging of fixed cells suggested that PRR in the total membrane fraction may be encapsulated in vesicles. These data indicate that Ca2+ may be an important regulator of endothelial PRR trafficking, possibly via a Ca2+‐dependent PRR cleavage pathway. Ongoing studies will establish the relationship between Ca2+ and sPRR/PRR and determine the impact of PRR trafficking on endothelial dysfunction. These studies will determine whether targeting of PRR can lower the risk of cardiovascular disease in patients with diabetes.

Support or Funding Information

Tulane Center for Engaged Learning and Technology, NIH HL133619, DK104375