bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 InformationNational 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 InformationThis 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 InformationThis 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.Support or Funding InformationT32 HL 7954‐19 (R.D.)
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.Support or Funding InformationThis 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.Support or Funding InformationMechanisms 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 InformationTulane Center for Engaged Learning and Technology, NIH HL133619, DK104375