bluuebunny/crossref_metadata_2025 huggingface dataset:
Nucleic‐Acid Mediated Expression of Cardiopoietic Markers in Human Umbilical Cord‐Derived Mesenchymal Stem Cells
Authors: Arocha Sinibaldo Rafael Romero , Li Mark , Singh Raman Deep , Terzic Andre , Behfar Atta
Published: April 2020

The management of patients with heart failure focuses on halting the progression of disease. To date, no therapeutic approach has been able to restore cardiac function to its original baseline state. Cell therapies, such as cardiopoietic (CP) stem cells, have shown to be promising platforms to restore cardiac function. The CP platform enhances the cardioregenerative potency of mesenchymal stem cells (MSCs). CP cells are characterized by the robust expression and nuclear translocation of cardiac transcription factors such Mesoderm Posterior Basic Helix‐Loop‐Helix Transcription Factor 1 (MESP1), early cardiac transcription factor Nkx2.5, myocyte enhancer factor 2C (Mef2C), and zinc finger‐containing transcription factor GATA4. Clinical trials using CP cells showed signs of safety and efficacy in improving cardiac function in patients with heart failure. However, the costs, time, and complexities associated with the manufacturing process have precluded the adoption of the therapy as a large‐scale clinical practice. In response, we adopted a microencapsulated‐modified‐messenger RNA (M3RNA) platform as a cost‐effective and time‐saving alternative to induce high expression of cardiopoietic markers. We hypothesized that the transfection of human umbilical cord‐derived mesenchymal stem cells (hUC‐MSCs) with M3RNA encoding mesodermal and/or cardiac transcription factor(s) will induce the expression of cardiopoietic markers (MESP1, Nkx2.5, Mef2C, and GATA4). hUC‐MSCs were transfected with M3RNA encoding Brachyury (this protein plays a key role in cardiomyocyte differentiation) and MESP1, Nkx2.5, Mef2C, and GATA4 using the transfection reagent Lipofectamine STEM. Protein expression was measured and quantified using fluorescence microscopy and flow cytometry. hUC‐MSCs transfected with M3RNA encoding mesodermal and/or cardiac transcription factor(s) demonstrated rapid protein expression at 4 hours post‐transfection that reached peak levels between 24 hours and 48 hours post‐transfection. Immunocytochemistry demonstrated similar morphology between transfected and non‐transfected cells. Thus, hUC‐MSCs can be transfected with M3RNA with high efficiency to produce rapid and predictable protein expression in vitro. We here demonstrate a simple, cost‐effective, and time‐saving approach to induce the expression of cardiopoietic markers in UC‐MSCs using M3RNA, which can serve as a highly translatable platform to circumvent many burdens associated with current strategies and development of cell‐based therapies for future clinical trials and practice.

Support or Funding Information

Mayo Clinic Center for Regenerative Medicine, Van Cleve Cardiac Regenerative Medicine Program, Marriott Foundation, Michael S. and Mary Sue Shannon Family, NIH NHLBI HL134664, Mayo Clinic Regenerative Sciences Post‐Baccalaureate Training Program
Ripk2 dictates blood insulin and glucose responses to cancer drug and microbial xenobiotics
Authors: Schertzer Jonathan , Duggan Brittany , Cavallari Joseph , Foley Kevin , Barra Nicole
Published: April 2020

Xenobiotics, including therapeutic drugs, and components of microbes can promote inflammation that contributes to insulin resistance and dysglycemia. Tyrosine kinase inhibitors (TKIs) used in cancer have been considered as a therapy in diabetes and its complications. It is not clear which molecular target of TKIs impact blood glucose or insulin during obesity.

One objective here was to define the role of the innate immune adapter protein, Ripk2 in TKI‐mediated changes in blood glucose and insulin during obesity. We tested gefitinib, which inhibits Ripk2 and imatinib, which does not inhibit Ripk2 as TKI interventions in diet‐induced obese mice. We also tested whether Ripk2 and these TKIs prevented the insulin sensitizing properties of bacterial components that we already know lower glucose and insulin resistance via Nod2‐Ripk2. We previously found specific bacterial cell components that lower blood glucose and insulin resistance. Now we found that crude bacterial extracts from the upper gut also lower blood glucose. Hence, the other objective was to determine if Ripk2 propagated insulin sensitizing properties of extracts from commensal bacteria.

We found gefitinib lowered blood glucose during a glucose tolerance test (GTT) in WT, Nod1‐null, Nod2‐null and Ripk2‐null mice that were obese. Gefitinib lowered glucose stimulated blood insulin levels only in obese Ripk2‐null mice. Hence, deletion of Ripk2 in mice promoted the insulin sensitizing potential of gefitinib. Gefitinib, but not imatinib, prevented improvements in glucose control caused by a bacterial postbiotic that improves insulin sensitivity. Further, Ripk2 was required for improvements in glucose control when mice were injected bacterial extracts from the upper gut of obese or lean mice.

We conclude that multiple TKIs, including gefitinib and imatinib lower blood glucose in obese mice, independent of Ripk2. However, Ripk2 participated in TKI‐induced lowering of blood insulin in response to an oral glucose load. The effects of TKIs on Ripk2‐mediated metabolism should be measured if this drug class is considered in treatment of diabetes or its complications. Also, the role of Ripk2 in propagating bacterial‐induced changes in blood glucose and insulin warrants further investigation.

Support or Funding Information

Supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) discovery grant. BD was supported by Ontario Graduate Scholarships. JC was supported by a Frederick Banting and Charles Best Canada Graduate Scholarship. KF was supported by a NSERC fellowship. JS holds a Canada Research Chair in Metabolic Inflammation.
Sex Differences in Baseline and Reactive Cerebrovascular Compliance
Authors: Moir M. Erin , Klassen Stephen A. , Zamir Mair , Shoemaker J. Kevin
Published: April 2020

Adjustments in cerebrovascular resistance represent the primary characterization of cerebral autoregulation to date. However, recent studies have demonstrated that cerebrovascular compliance contributes to the relationship between BP and cerebral blood velocity (BV) during dynamic changes in BP. Therefore, the present study tested the hypotheses that i) hypotension‐induced autoregulatory processes involve increases in cerebrovascular compliance and ii) sex differences exist in cerebrovascular compliance. Twenty young healthy adults (24 ± 3 years, 10 females) completed a sit‐to‐stand protocol involving three minutes of sitting and three minutes of standing while brachial artery BP (Finapres Finometer) and middle cerebral artery BV (Multigon Neurovision) were collected. Individual BP (with temporal shift) and corresponding BV waveforms were input into a four‐element lumped parameter Windkessel model to calculate indices of cerebrovascular compliance (Ci) and resistance (Ri). Beginning 20 beats prior to standing and continuing 40 beats following the stand, every other heartbeat was extracted so that a total of 30 beats were analyzed (i.e., 10 beats during sitting, 20 beats during standing). Seated baseline Ci was calculated as the average of the 10 beats of sitting. Peak Ci minus seated baseline Ci determined the change in (Δ) Ci. Females demonstrated higher Ci than males during seated baseline (1.1e−3 ± 4.4e−4 vs. 7.1e−4 ± 2.1e−4 cm·s−1·mmHg−1; P = 0.04, d = 1.3). Upon standing, large and rapid increases in Ci were observed in both groups but females exhibited a lesser increase when compared with males (114 ± 48 vs. 202 ± 50 %; P < 0.01, d = 1.8). No differences were observed in peak Ci with standing (females: 2.1e−3 ± 6.7e−4, males: 2.1e−3 ± 5.5e−4 cm·s−1·mmHg−1; P = 0.89). While the drop in mean BP with standing was attenuated in females compared with males (−14 ± 5 vs. −25 ± 9 mmHg; P < 0.01, d = 1.5), no relationship existed between Δ Ci and Δ mean BP (b = −7.0e−7 cm·s−1·mmHg−2; r2 = 3.0e−4; b P = 0.94). Importantly, the increase in Ci preceded reductions in Ri (2 ± 2 vs. 11 ± 4 beats following standing; P < 0.01, d = 2.8). Therefore, large and rapid increases in Ci occur with standing‐induced hypotension that contribute to dynamic cerebral autoregulation. Further, females demonstrate attenuated Ci responses to standing despite exhibiting higher seated baseline Ci compared with males.

Support or Funding Information

Supported by the Natural Sciences and Engineering Research Council of Canada.
The Impact of Spatial Ability on Learning with a 3D Stereoscopic Model of the Mediastinum
Authors: Labranche Leah , Terrell Mark , Wilson Tim D. , Carty Nancy , Kulesza Randy J.
Published: April 2020

Three‐dimensional (3D) stereoscopic anatomy models have shown success in demonstrating complex spatial anatomical relationships. However, there is conflicting evidence as to whether or not such models are beneficial for students with low spatial visualization ability (Vz). A previous study suggests that students with low Vz may be hindered by the complex spatial cues provided by a 3D stereoscopic resource compared to students with high Vz. This study aimed to investigate the educational effectiveness of a digital 3D stereoscopic model of the human mediastinum, compared to a cadaveric specimen, for novice students in a biomedical masters‐level graduate program. In addition, this study aimed to further examine the effect of Vz on learning with a 3D stereoscopic anatomy model. We hypothesized that participants learning with the 3D stereoscopic model of the mediastinum would improve performance overall on an anatomy knowledge post‐test more than participants learning with a cadaveric specimen, and that high Vz participants would benefit more from the use of the 3D stereoscopic model compared to their low Vz counterparts. In addition, we hypothesized that participants learning with the 3D model of the mediastinum would rate the model more favorably on a post‐session survey compared to those learning with a cadaveric specimen. Participants were recruited from the Master of Medical Science (MMS) program at Lake Erie College of Osteopathic Medicine (LECOM), prior to the start of their Gross Anatomy course.

Participants completed a demographic survey and Mental Rotations Test (MRT) pretest to determine their level of Vz. Participants were then assigned to either a 3D Group or a Cadaver Group, normalized for MRT score and sex. Each group was invited to a brief learning session using either a 3D stereoscopic model or cadaveric specimen of the mediastinum respectively. The 3D model was developed at LECOM using Amira® software, and projected stereoscopically using a dual projection system. All participants completed a pre‐ and post‐test in each session, consisting of spatially‐oriented multiple choice questions pertaining to anatomy of the mediastinum. Student satisfaction with either resource was also examined using a qualitative survey. Data collection and analysis will be completed in Spring 2020. Results of this study should be considered when developing and implementing anatomical resources for health professional education, to maximize learning for students of varying Vz.

Support or Funding Information

Funded by the Lake Erie Consortium for Osteopathic Medical Training and the LECOM Internal Seed Grant
Caffeine Abolishes TrkB Receptor‐Dependent Respiratory‐Related Spinal Motor Facilitation in Isolated Neonatal Rat Brainstem‐Spinal Cords In Vitro
Authors: Johnson Stephen M. , Vasdev Ranveer M.S. , Miller Mckayla M. , Baker Tracy L. , Watters Jyoti J.
Published: April 2020

Caffeine is the main treatment for apnea of prematurity because it stimulates breathing, reduces duration of respiratory support, and increases infant survival. Caffeine has multiple neuronal effects, but it is a well‐established adenosine A2a receptor (A2aR) antagonist. In adult rats, A2aR blockade increases the magnitude of intermittent hypoxia‐induced, TrkB receptor‐dependent, long‐term facilitation of phrenic motor output. In neonates, it is not known whether caffeine blocks A2aRs and enhances TrkB receptor‐induced facilitation of respiratory motor output. To test this question, neonatal (P0‐P3) rat brainstem‐spinal cords were isolated and respiratory‐related motor output recorded from C4‐C5 ventral spinal roots. Bath‐applied dihydroxyflavone (DHF, TrkB receptor agonist, 5–10 μM, 9‐min, n=22) increased respiratory motor burst amplitude by 13 ± 3% relative to baseline at 75 min following drug application, whereas vehicle dimethylsulfoxide‐treated time controls decreased burst amplitude by 11 ± 4% relative to baseline (n=15; p<0.001). A background caffeine application (50–250 μM; 15 min) that overlapped a DHF application (5–10 μM, 9 min) abolished DHF‐induced spinal motor facilitation (burst amplitude was decreased by 7 ± 4% relative to baseline at 75 min). The lack of DHF‐induced spinal motor facilitation with caffeine pretreatment could be due to decreased endogenous signaling in neonatal rat A2aRs (i.e., little or no crosstalk inhibition), or other caffeine effects, such as phosphodiesterase inhibition. If TrkB‐dependent spinal motor facilitation is beneficial in neonates, then caffeine administration may compromise one compensatory mechanism during apnea of prematurity.

Support or Funding Information

Supported by: NS085226 (JJW), HL105511 (TLB)
HIV‐1 Capsid Binding Host Factor CPSF6 is Post Transcriptionally Regulated by the Cellular MicroRNA miR‐125b
Authors: Chaudhuri Evan , Dash Sabyasachi , Balasubramaniam Muthukumar , Holland Ocey L , Sowd Gregory , Villalta Fernando , Engelman Alan , Pandhare Jui , Dash Chandravanu
Published: April 2020

The cleavage and polyadenylation specificity factor 6 (CPSF6) is a cellular protein involved in RNA cleavage and polyadenylation. Emerging evidence also suggest that CPSF6 plays a key role during HIV‐1 infection by guiding integration of the viral DNA into gene‐dense regions of the host genome. Since viral DNA integration is a critical step of HIV‐1 infection, the role of CPSF6 in the virus lifecycle is being intensely investigated. Surprisingly, the cellular mechanisms that regulate CPSF6 expression are largely unknown. In this study, we report a post‐transcriptional mechanism of regulation of CPSF6. Our initial bioinformatics analysis revealed that the 3′ untranslated region (3′UTR) of Cpsf6 contains a binding site for the cellular miRNA miR‐125b that is strikingly conserved across different mammalian species. Since miRNAs negatively regulate protein expression, we carried out knock‐down and over‐expression studies of miR‐125b. Results from these experiments revealed that miR‐125b expression is negatively associated with CPSF6 protein levels. Interestingly, HIV‐1 infection resulted in the down‐regulation of miR‐125b concurrent with induction of CPSF6. To probe that CPSF6 expression is post‐transcriptionally regulated by miR‐125b, we cloned the 3′UTR regions of Cpsf6 mRNA into a luciferase reporter. Results from luciferase assay provide evidence that miR‐125b expression negatively regulates Cpsf6 3′UTR activity. Accordingly, mutations in the miR‐125b seed sequences abrogated the regulatory effect of the miRNA on Cpsf6 3′UTR. Pull‐down studies demonstrated that miR‐125b physically interacts with the Cpsf6 mRNA. Continuing studies probe the necessity of productive HIV‐1 infection for negatively regulating miR‐125b expression as well as to elucidate the pathway through which HIV‐1 infection can knockdown miR‐125b expression. Collectively, these findings establish a post‐transcriptional mechanism of CPSF6 expression and describe a novel function of miR‐125b in virus‐host interaction.

Support or Funding Information

This work is partly supported by grants DA024558, DA30896, DA033892 and DA021471 from NIDA/NIH to CD. We also acknowledge the RCMI Grant G12MD007586, the Vanderbilt CTSA grant UL1RR024975, the Meharry Translational Research Center (MeTRC) CTSA grant (U54 RR026140 from NCRR/NIH, the U54 grant MD007593 from NIMHD/NIH, and Tennessee CFAR grant (P30 AI110527).
The F‐box Protein FBXL16 Upregulates the Stability of c‐myc Oncoprotein by Antagonizing FBW7 Function
Authors: Morel Marion , Shah Krushangi , Long Weiwen
Published: April 2020

F‐box proteins are essential components of the SCF (SKP1‐CUL1‐F‐box) E3 ubiquitin ligases as they are responsible for substrate recognition. F‐box proteins bind to SKP1 through the F‐box motif and bring the substrate to the E3 ligase complex for ubiquitination. So far, 69 F‐box proteins have been identified in humans, and they fall into 3 families depending on their substrate recognition domains: FBXLs (Leucine‐Rich Repeats or LRR), FBXWs (WD repeats) and FBXOs (Other domains). FBXL16 is a poorly studied F‐box protein, which was shown to be a transcriptional target of E2F1 (Sato et al, 2010). As it doesn’t interact with the scaffold protein Cul1, FBXL16 might not form a functional SCF‐E3 ligase complex (Honarpour et al, 2014). In our present study, we found by data mining that FBXL16 is highly upregulated in many cancers, and its upregulation is often associated with poor patient survival, indicating that FBXL16 may play important roles in cancers. To decipher the role of FBXL16 in oncogenesis, we first analyzed its biochemical function in regulating protein levels of different SCF‐E3 substrates. Unexpectedly, we found that FBXL16 upregulates oncogenic proteins targeted by SCF‐E3 ligases, such as Steroid Receptor Coactivator 3 (SRC‐3) and c‐myc. Here, we have focused on c‐myc, a well‐known oncoprotein overexpressed in most human cancers, and found that FBXL16 interacts with and stabilizes c‐myc protein. This stabilization is dependent on c‐myc phosphorylation on both Threonine 58 and Serine 62, the phosphodegron recognized by FBW7 for its subsequent ubiquitination. Further, we found that FBXL16 antagonizes FBW7‐E3 ligase activity, leading to reduced c‐myc ubiquitination. In line with its role in upregulating the levels of oncoproteins, we found that FBXL16 promotes cancer cell growth and migration. Taken together, our findings reveal FBXL16 as a unique F‐Box protein antagonizing the activity of another F‐box protein FBW7 and as a new positive regulator of c‐myc. Further investigation is being carried out to determine the oncogenic roles of FBXL16 in vivo and may unravel FBXL16 as a new therapeutic target of cancers.

Support or Funding Information

This research was supported by NCI of the National Institutes of Health under award number R01CA193264.
The therapeutic effects of masitinib in complex regional pain syndrome in a mouse model of tourniquet‐induced extremity ischemia‐reperfusion
Authors: Qian Junliang , Tu Huiyin , Zhang Dongzhe , Wadman Michael C. , Li Yu-Long
Published: April 2020

Tourniquet, as a life‐saving device, is widely used to stop extremity hemorrhage in civilian medicine and on the battlefield. However, tourniquet use and subsequent release can induce a long‐term increase in pain sensitivity called complex regional pain syndrome. Complex regional pain syndrome is a form of chronic pain triggered by inflammation and nerve dysfunction, with a great resistance to different treatments for relieving pain. Increasing evidence showed a pivotal role of neuroinflammation in the formation of hyperalgesia and allodynia. In our study, we investigated the therapeutic effects of masitinib, an anti‐neuroinflammatory drug, on ischemia‐reperfusion (IR) increased pain sensitivity in a mouse model of tourniquet‐induced hindpaw IR. Unilateral hindpaw of C57/BL6 mice were subjected to 3 hours of ischemia by placing a rubber band at ankle joint and followed 4 weeks of reperfusion. Masitinib treatment (28 mg/kg/day, i.p.) began on the day of IR induction and last for 4 weeks. Tourniquet‐induced IR caused nerve damage in the skin and abolished the paw sensation to 2 g of mechanical stimulation before the 3rd day of reperfusion. In the mice with tourniquet‐induced IR, mechanical allodynia appeared at day 7 (0.54 ± 0.10 g, compared to 0.83 g ± 0.10 in sham mice; p<0.05), reached to the maximum reaction at 2 weeks (0.19 g ± 0.04 g), and lasted 4 weeks. In the mice with masitinib plus tourniquet‐induced IR, 33% (4/12) of mice at day 1 and 40% (6/15) of mice at day 3 responded to 2 g of stimulation. Treatment with masitinib significantly alleviated IR‐induced mechanical allodynia from 2 weeks to 4 weeks. Additionally, paw thickness, a marker for hindpaw edema, was observed. Treatment with masitinib also attenuated IR‐induced expansion of the hindpaw. Our data provide the evidence that treatment with masitinib effectively alleviates complex regional pain syndrome in tourniquet‐induced hindpaw IR.
Large and Small Substrate Binding to DesD via ITC Assay Development
Authors: Weaver Madison , Hoffmann Katherine
Published: April 2020

Desferrioxamines (“dfo”s) are iron‐chelating compounds which have been used medicinally in cases of iron overload. The synthesis of dfoE involves an enzyme called DesD which is a dimer that has an active site on each chain, but the mechanism of it binding both large or small substrates is unknown. We are studying DesD as a model enzyme for the non‐ribosomal peptide synthetase independent siderophore (NIS) pathway and as a potential new drug target. The purpose of this research is to analyze the co‐factor specificity and large substrate verse small substrate specificity of DesD by comparing substrate dissociation constants to analog dissociation constants (KD, where a smaller number correlates to tighter binding). Future drugs will have to out‐compete the substrates in binding, so we will use these studies to determine which substrate components contribute to maximum binding. Techniques in analytical chemistry will be used to regenerate deferoxamines that were bound to cofactors in previous studies with the DesD enzyme. The binding properties of DesD were explored in a 25% glycerol Isothermal Titration Calorimetry (ITC) buffer. Prior experimentation with this protein was conducted in a buffer that resulted in precipitation of DesD out of solution, thus prompting the need for repeated binding assays in a buffer that maintains synthase solubility. GraphPad Prism 8.0 will be used to analyze the KD and occupancy of DesD. High Performance Liquid Chromatography (HPLC) will be used to separate deferoxamines from substrates they were bound to. An Ocean Optics Spectrometer will be used to quantify the deferoxamines by pairing these substrates to a metal compound that will allow us to identify its absorbance and determine if isolation was effective.

We confirmed previous results that the KD of DesD binding to ATP is in the M range, compared to non‐ATP cofactors which result in a KD in the M range. Future studies will compare dfoG (substrate) binding to DesD compared to analog dfoB or product dfoE, and also explore smaller substrates, hydroxysuccinylcadaverine (HSC), vs. analogs. Troubleshooting runs have indicated that isolation of the deferoxamines is feasible. It is expected that substrates like dfoG will be separated from ATP and ADP cofactors. We have confirmed that DesD has a strong binding preference for ATP over non‐ATP cofactors in a buffer that maximizes solubility. This data indicates that the phosphates are essential to tight binding in any future competitive inhibitors. Regeneration of each deferoxamine appeared to be a cost‐effective assay that will allow for these compounds to be used in later experimentation. Future work will expand our understanding of critical regions in the binding site, and aid in developing new inhibitors that could block the NIS pathways and be lead drugs for a new class of antibiotics.
Estradiol signaling interacts with Sirt1 in the regulation of autophagy and adipogenesis
Authors: Cheng Zhiyong , Tao Zhipeng , Shi Limin , Zheng Louise
Published: April 2020

Adiposity is regulated by estradiol signaling and the nutrient sensor Sirt1. It has been recognized that autophagy plays a key role in adipogenesis and metabolism. However, it remains largely unknown whether and how estradiol signaling interacts with Sirt1 in the regulation of autophagy. Here we showed that activation and overexpression of Sirt1 suppressed autophagy in 3T3L1 cells, which was associated with suppressed adipocyte differentiation. Treatment of preadipocytes with estradiol induced Sirt1 and dampened autophagy, thereby suppressing adipogenesis. Consistently, knockdown of Sirt1 induced autophagy and adipocyte differentiation, which was recapitulated by estrogen receptor inhibition or knockout. In addition, knockdown of Sirt1 abolished the effects of estradiol on autophagy and adipogenesis. The modulation of autophagy and adipogenesis by estradiol and Sirt1 was ascribed at least in part to mTOR‐ULK1 cascade. Protein analysis did not show significant changes in the acetylation levels of autophagy regulators (e.g., Atg5, Atg7, or LC3) during Sirt1 modulation. Taken together, our data reveals an estradiol‐Sirt1‐autophagy axis that regulates adipogenesis. Future study will be of interest and significance to determine the potential role of the estradiol‐Sirt1‐autophagy axis in sex‐dependent fat distribution.

Support or Funding Information

American Heart Association Grant 18TPA34230082 (Z.C.) and USDA National Institute of Food and Agriculture Hatch Project 1020373 (Z.C.).
Decoding Glycoproteome Remodeling in Sepsis through Integrative Multi‐Omics Analysis and Parts‐Based Data Representation
Authors: Sorrentino James T. , Toledo Alejandro Gomez , Golden Gregory , Diaz-Peña Ramon , Campos Alexandre Rosa , Nizet Victor , Malmstrom Johan , Smith Jeffrey W. , Lewis Nathan E. , Esko Jeffrey D.
Published: April 2020

Sepsis is a major clinical challenge accounting for unacceptably high‐rates of morbidity and mortality in intensive care units around the world. After many years of extensive research, reliable markers for diagnosis and treatment monitoring remain desperately needed. The plasma glycoproteome offers a suitable readout to monitor systemic alterations in the body. Thus, tracking structural changes in plasma glycoproteins may uncover new molecular windows to target bacterial sepsis. Unfortunately, the analytical challenge posed by the large heterogeneity of plasma carbohydrate and protein components has severely limited advancements in this area. Here we show that Staphylococcus aureus bacteremia induces a dramatic shift of the plasma glycoproteome in a murine model of monobacterial sepsis. Significant changes in the levels of plasma glycoproteins in circulation, as well as their associations with vascular surfaces were revealed through an integrative glycomics, chemical proteomics, and glycoproteomics approach. Additionally, the global and site‐specific glycosylation patterns of the plasma N‐ and O‐linked glycoproteome were significantly altered during sepsis. Deconvolution of these changes through non‐negative matrix factorization methods uncovered the co‐existence of discrete patterns of glycoproteome remodeling associated with specific subgroups of glycoproteins. This included changes in the levels of N‐glycan core‐fucosylation, terminal sialylation, and increased protein deglycosylation, including complete glycan truncations. Furthermore, computational analysis demonstrates that these molecular patterns are sufficient to robustly stratify the samples based on their infection status. In short, we show that multi‐Omics integration has the power to dissect local alterations in the plasma glycoproteome during monobacterial sepsis, possibly reflecting the contribution of discrete biological processes of cellular glycosylation.

Support or Funding Information

This work was supported by grant P01HL131474 to J.D.E. and J.W.S. J.T.S. acknowledges support from the National Institutes of Health, USA (NIH grant T32GM008806) J.W.S. was supported by R01 GM107523. G.G. was supported by Microbial Sciences Graduate Research Fellowship Awards 1‐F17GG and 1‐F18GG.
Polymorphisms in RocA contribute to the molecular pathogenesis of serotype M28 group A streptococcus
Authors: Bernard Paul Edward , Kachroo Priyanka , Eraso Jesus M. , Zhu Luchang , Madry Jessica E. , Duarte Amey , Linson Sarah E. , Saavedra Matthew Ojeda , Cantu Concepcion , Musser James M. , Olsen Randall J.
Published: April 2020

Two‐component systems (TCSs) are signal transduction proteins that enable bacteria to respond to external stimuli by altering the global transcriptome. Accessory proteins interact with TCSs to fine‐tune their activity. In group A streptococcus (GAS), RocA (regulator of Cov) is an accessory protein that functions with the CovRS (control of virulence regulator/sensor) TCS. Previous data suggest that the N‐terminal predicted transmembrane domains are important for RocA function and interaction with the CovRS TCS. Whole‐genome sequencing analysis of serotype M28 GAS strains collected from invasive infections in humans identified a higher number of missense (amino acid altering) and nonsense (protein truncating) polymorphisms in rocA than expected. We hypothesized that polymorphisms in RocA alter the global transcriptome and virulence of serotype M28 GAS. We used naturally‐occurring clinical isolates with rocA polymorphisms, an isogenic rocA deletion mutant strain, and isogenic rocA polymorphism mutant strains to perform genome‐wide transcript analysis (RNA‐seq), in vitro virulence factor assays, and mouse and nonhuman primate pathogenesis studies to test this hypothesis. Results demonstrated that polymorphisms in rocA result in either a subtle transcriptome change causing a wild‐type‐like virulence phenotype, or a substantial transcriptome change leading to a significantly increased virulence phenotype. Each polymorphism had a unique effect on the global GAS transcriptome. Additionally, in contrast to previously published data on functional domains of RocA, polymorphisms in both the N‐terminal transmembrane domains and the C‐terminal predicted non‐functional histidine kinase ATPase domain result in an altered virulence phenotype suggestive of loss of RocA activity. Taken together, our data show that naturally‐occurring polymorphisms in one gene encoding an accessory protein can significantly alter the global transcriptome and virulence phenotype of GAS, an important human pathogen.

Support or Funding Information

Fondren Foundation, Houston Methodist Hospital and Research Institute (to JMM and PEB)American Heart Association Predoctoral Fellowship award 19PRE34380820 (to PEB)NIH grants R21AI139369‐01A1 and R21AI146771‐01 (to JMM)
Regulation of Coronary Blood Flow by the Carotid Body Chemoreceptors in Ovine Heart Failure
Authors: PACHEN MRIDULA , Abukar Yonis , Lever Nigel , Ramchandra Rohit
Published: April 2020

Background

The carotid body (CB) is an arterial chemoreceptor organ located in the carotid bifurcation, which carries out essential functions in cardiorespiratory control. In response to hypoxia, carotid chemosensory discharge produces respiratory, autonomic, and cardiovascular adjustments critical for body homeostasis. Previous studies have shown that chemoreflex sensitivity is augmented animal models of heart failure (HF). However, the role of the chemoreflex in modulating coronary blood flow during HF is not clear. We hypothesized that carotid chemoreceptor stimulation would increase coronary blood flow in both normal sheep and sheep with HF.

Methods

Heart failure was induced by the microembolization technique and instrumentation was performed when ejection fraction was below 40%. Both groups of sheep were implanted with a coronary artery flow probe and an arterial pressure catheter. The responses of mean arterial pressure (MAP) and coronary blood flow (CoBF) were determined in conscious sheep following CB stimulation with intracarotid potassium cyanide (KCN; 30μg/kg). To determine the cardiac vagal contribution to the CoBF response, CB activation was repeated following atropine infusion (8 mg bolus followed by 24 mg/h infusion for 30 minutes).

Results

In control animals, activation of the CB increased MAP (114±1%) and CoBF (116±4%) but coronary vascular conductance (CVC) was unaltered. In the HF group, CB stimulation increased MAP (108±1%), CoBF (126±3%), and CVC (114±2%). The MAP increase was significantly attenuated after atropine infusion in both animal groups, however, the CoBF and CVC responses to KCN were unchanged. These responses persisted when the heart was paced at a constant rate to eliminate the role of altered metabolic demand.

Discussion

Our data indicate that in an ovine model of HF, carotid body mediated increases in MAP are attenuated compared to control animals. There was a significant increase in coronary vascular conductance in the HF animals following CB activation which was absent in the normal animals. Overall, our data suggest that the hyperactive carotid body chemoreflex may serve to increase coronary blood flow in the HF condition.

Support or Funding Information

Health Research Council of New Zealand, National Heart Foundation of New Zealand
Generating Phosphorylation Site Antibodies to Study α2A‐Adrenergic Receptor Phosphorylation by G Protein‐coupled Receptor Kinase 2 in Intact Cells
Authors: Barnett Jessica , Hebert Aaron , Weldin Erica , Farina Zachary , Varney Matthew , Guirguis Jeska , Sterne-Marr Rachel
Published: April 2020

G protein‐coupled receptors (GPCRs) are transmembrane proteins which are activated by the binding of agonist to create a cell signal response. GPCRs are regulated through phosphorylation by G protein‐coupled receptor kinases. We focus on G protein‐coupled receptor kinase 2 (GRK2) and have previously characterized phosphorylation of the β2‐adrenergic receptor (β2‐AR) by this kinase. Currently, we are trying to determine if the same mutations shown to effect β2‐AR phosphorylation are also important for phosphorylation of the α2A‐adrenergic receptor (α2A‐AR). The α2A‐AR plays a role in regulating insulin, neurotransmitter secretion, and is a pharmaceutical target for pain relief. It differs from β2‐AR in that α2A‐AR has a larger third intracellular loop, containing four consecutive serine residues. These residues can be phosphorylated by GRK2 in the presence of agonist, causing a desensitization response. One way to detect receptor phosphorylation is to use antibodies that specifically recognize phosphorylated receptor. Since there is no commercially available antibody that can detect phosphorylation of α2A‐AR, we developed an antibody, RB617, which detects the phosphorylation of two serine residues. Using this antibody, we have developed methodologies to study GRK2 phosphorylation of α2A‐AR in intact COS‐7 cells. We tested the dose‐response of α2A‐AR to epinephrine and determined the EC50 for GRK2‐dependent phosphorylation to be 35 nM. In the future, we plan to assess the effect of GRK2 mutations previously shown to affect β2‐AR phosphorylation to better understand the role of GRK2 in α2A‐AR desensitization.

Support or Funding Information

This work was funded by NSF MCB0744739 and Siena College Center for Undergraduate Research and Creative Activity (CURCA).
Down‐regulation of Circulating miRNAs in Canines with Chronic Kidney Disease may be Indicative of Renal Fibrosis
Authors: Tavener Selena , Panickar Kiran S. , Jewell Dennis E.
Published: April 2020

Mature microRNAs (miRNAs) are single‐stranded RNAs approximately 22 nucleotides in length. They are generated from short hairpin RNAs that are produced by the ribonucleases, Drosha and Dicer. miRNAs act as post‐transcriptional regulators by base‐pairing with complementary sequences to mRNAs, generally leading to the silencing of mRNA through either degradation or less efficient translation of their target mRNAs. Circulating miRNAs may also serve as biomarkers for renal dysfunction. One characteristic of chronic kidney disease (CKD) is renal fibrosis which results in a progressive loss of renal function, subsequently leading to renal failure. Mechanisms underlying renal fibrosis, generally characterized by excessive extracellular matrix synthesis and deposition, are not clear but deregulation of miRNAs has been intricately linked to renal fibrosis. We assessed the circulating levels of miRNAs using Qiagen’s Canine mScript miRNA PCR array, in the blood of dogs that were clinically diagnosed as having CKD, post‐mortem. Further, end‐of‐life pathology reports indicated interstitial inflammation, fibrosis, and thickening of the Bowman’s capsule. Blood samples were collected at necropsy from dogs with CKD (n=10; 2–17 yr) and control dogs (n=10; 5–13.5 yr). There was a significant decline in the levels of miRNAs cfa‐let‐7a, let‐7c, let‐7f, and let‐7g in dogs with CKD when compared to controls (p<0.05). Down‐regulation of certain let‐7 miRNAs (let‐7a, let‐7b) has been associated with experimental models of induction of renal fibrogenesis. There was also a >2‐fold reduction (NS) in levels of cfa‐miR‐93, cfa‐miR‐122, cfa‐miR‐200a, and cfa‐miR‐204, in CKD when compared to controls. These microRNAs have been demonstrated to have anti‐fibrogenesis effect, and are also down‐regulated in rodent models and in vitro mechanistic models of renal fibrosis leading to increased fibrosis. There was also a down‐regulation of cfa‐miR‐16, which is consistent with its reported role in attenuating kidney injury independent of fibrosis. Taken together with the literature, down‐regulation of these miRNAs may be indicative of a reduction in their role in attenuating renal fibrogenesis and injury. Importantly, the circulating miRNAs may serve as non‐invasive biomarkers for renal fibrosis in CKD and also as nutritional targets for slowing the progression of fibrosis in kidney disease.

Support or Funding Information

This study was funded by Hill’s Pet Nutrition, Inc
Increase in Interleukin‐16, a Pro‐inflammatory Cytokine, and Other Distinct Markers of Lymphocytic Activation, in Kidney Tissue of Cats with Renal Dysfunction
Authors: Panickar Kiran S. , Tavener Selena , Hollar Regina L. , Jewell Dennis E.
Published: April 2020

Inflammation plays an important role in the initiation and subsequent progression of chronic kidney disease. An increase in inflammatory markers including cytokines as well as markers of oxidative stress is associated with reduced kidney function. Inflammatory markers and the underlying mechanisms that contribute to the pathophysiology of kidney disease are not clear and likely are multifactorial. Another feature associated with renal dysfunction and inflammatory response is the formation of calcium oxalate stones formed from the crystal deposits in the tubular epithelial cells. The aim of the present study was to investigate changes in gene expression in the renal cortex obtained from cats with kidney disease or calcium oxalate stone formers (CaOx) at necropsy in order to identify novel inflammatory biomarkers associated with renal dysfunction. At the time of death the circulating levels of creatinine as well as symmetric dimethyl arginine (SDMA), both markers of kidney decline in cats, were significantly higher in cats with renal disease (n=11) or stone‐forming cats (CaOx, n=12) when compared to controls (n=19). Using RNAseq in kidney tissue, we found a significant increase in the expression of IL‐16, a T‐cell chemoattractant, in both cats with kidney disease (6.72 fold) and stone formers (7.03 fold) compared to controls (both p<0.0001). There was also a significant increase in caspase recruitment domain family member 11 (CARD11), an activator of T and B‐lymphocytes and NFkB, in cats with kidney disease (9.2 fold) and stone formers (10 fold) compared to controls (p<0001). However, some differences between the kidney disease and CaOx groups were also observed. In cats with kidney disease there was an increased expression of LY9 (CD299), found in T and B‐cells and thymocytes, IL2Rg, a marker of lymphocyte activation, and SPP1 (Osteopontin), expressed in activated T and B‐cells (all >6‐fold) when compared to controls (p<0.0001) but such increased expression was not seen in CaOx cats. In summary, while IL‐16 was increased in both groups, our results indicate important molecular differences in the pathogenesis of kidney dysfunction in cats with kidney disease and stone formers, and that distinct lymphocyte markers may be involved in the persistent decline of kidney function. Our studies suggest that the optimal nutritional therapy to slow the progression of kidney dysfunction may be different for cats with kidney disease or stone formers.

Support or Funding Information

This study was funded by Hill’s Pet Nutrition, Inc
Loss of Peptidyl‐Arginine Deiminase 1‐mediated Citrullination Drives Esophageal Epithelial Barrier Impairment in Allergic Inflammation
Authors: Mehta Rishi S. , Rochman Mark , Rothenberg Marc E.
Published: April 2020

Background

Citrullination or deimination is a post‐translational modification in which neutral arginine residues are converted to positive citrulline by members of the peptidyl‐arginine deiminase (PADI) family. PADI1 is an esophageal enriched gene that is significantly downregulated in Eosinophilic Esophagitis (EoE), an eosinophilic‐driven chronic allergic inflammatory disorder characterized by reduced tissue differentiation and impaired esophageal epithelial barrier formation. While the role of PADI1 in deiminating structural proteins has been implicated in epithelial cell populations, including the skin, the functional significance and extent of PADI1‐mediated citrullination in allergic inflammation remains understudied.

Hypothesis

Herein we hypothesize that the loss of PADI1‐mediated citrullination drives esophageal epithelial barrier impairment in allergic inflammation.

Methods

PADI1 WT and KO EPC2 immortalized human esophageal epithelial cells generated by CRISPR‐Cas9 were grown in either monolayer or at Air‐Liquid Interface (ALI) to mimic esophageal differentiation. Transepithelial Electrical Resistance (TEER) measurements of EPC2 cells grown at ALI were measured as a function of barrier integrity. Transcriptional, protein and epigenetic analyses were conducted on EPC2 cells grown at ALI treated with or without IL‐13, a T‐cell derived cytokine known to induce responses involved in EoE. Immunofluorescence (IF) was used to visualize structural proteins and assess overall level of citrullination.

Results

PADI1 protein was found to be most highly expressed in the Esophageal Mucosa as compared to all other tissue types, specifically in the suprabasal layer of differentiated esophageal cells. PADI1 RNA expression was decreased eight‐fold (P < 0.0001) in esophageal biopsies of patients with EoE as compared to control patients. Treatment of EPC2 cells with IL‐13 (100 ng/ml) at confluence reduced PADI1 mRNA by three‐fold (P < 0.01). PADI1 KO cells demonstrate altered differentiation as assessed by decreased levels of citrullinated proteins and structural epithelial proteins, including filaggrin, involucrin, desmoglein‐1, E‐cadherin, and cornulin compared with wild‐type control cells.

Conclusion

Our data suggest that loss of PADI1‐mediated citrullination may drive esophageal epithelial barrier impairment in allergic inflammation.

Support or Funding Information

This work is supported by PHS Grant R01‐AI124355 and the Ohio Physiological Society.
Inflammation‐induced Extracellular Vesicles Cause Endothelial Barrier Injury
Authors: Chatterjee Victor , Yang Xiaoyuan , Cha Byeong , Creasy Alexandria , Zheng Ethan , Ma Yonggang , Yuan Sarah Y.
Published: April 2020

Extracellular vesicles (EVs) are a heterogenous population of sub‐micron sized vesicles released by cells during physiological or pathological stimulation. Circulating vesicles not only serve as important diagnostic/prognostic biomarkers, but also interact with blood or vascular cells and regulate multiple cellular processes by transferring bioactive cargo. In this study, we characterized EV production and cargo signature in the blood of healthy human subjects and burn patients, as well as in cultured human endothelial cells (ECs) after inflammatory stimulation. We also examined the effects of EVs on endothelial barrier structure and function. The results showed that the plasma level of EVs was increased during sterile inflammation in burn patients, with a significant portion of them derived from leukocytes and the endothelium. Interestingly, the EVs from burn patients expressed higher levels of c‐Src, protein kinase C and focal adhesion kinase, compared to EVs from healthy blood donors. In cultured ECs treated with tumor necrosis factor alpha, there was an increased production of EVs into the culture media. Furthermore, treating endothelial cells with EVs induced myosin light chain and VE‐cadherin phosphorylation and actin stress fiber formation in concomitance with adherens junction discontinuity. Functionally, EV treatment caused an increase in albumin flux across endothelial monolayers in a concentration‐dependent manner. EVs derived from c‐Src knockdown parent ECs displayed an attenuated effect on barrier protein phosphorylation and permeability. Likewise, pharmacological inhibition of Src activity reduced EV‐induced protein phosphorylation and barrier dysfunction in endothelial cells. Together, the data suggest that EVs produced during inflammation carry protein kinases capable of damaging the endothelial barrier.

Support or Funding Information

NIH grants HL126646, HL070752 (to SYY), and GM097270. The human blood study was approved by the University of South Florida IRB and supported by the institutional research funds.
Ras Structure, Dynamics and Conformational States Assessed by MD Simulations and Solution Wide angle X‐ray Scattering
Authors: Volmar Alicia Yvette , Guterres Hugo , Zhou Hao , Mattos Carla , Makowski Lee
Published: April 2020

Ras is a small GTPase that is involved in up to 30% of cancers. A wealth of evidence suggests that despite high sequence similarity of the Ras isoforms, H‐Ras, K‐Ras, and N‐Ras, there are distinct differences in signaling, localization, mutation position, and outcomes in cancer. Even in the absence of the C‐terminal hypervariable region (HVR), there are differences between the isoforms in the rates of GTP hydrolysis and in the details of Ras structure and dynamics. Here we present accelerated molecular dynamics simulations paired with solution x‐ray scattering that reveal differences in the conformational sampling of H‐Ras, K‐Ras4B, and N‐Ras. Specific interactions within water‐mediated allosteric networks are altered by the presence of isoform specific residues that impact accessibility to the catalytic state. Thus, the isoforms sample the same main three conformational states, but the population of each one is shifted by changes driven by isoform‐specific residues that may be associated with fine‐tuning preferences for signaling through distinct pathways and susceptibility to specific mutations observed in cancers.

Support or Funding Information

NSF MCB‐1517295