bluuebunny/crossref_metadata_2025 huggingface dataset:
Contribution of ASICs to Cardiac Remodeling and Dysautonomia Associated with Heart Failure
Authors: Monaghan Karley , El‐Geneidy Maram , Sabharwal Rasna , Yang Liping , Gibbons David , Harding Anne M. S. , Ward Chad , Benson Chris
Published: May 2022

INTRODUCTION

The American Heart Association projects that by 2030 more than 8 million American adults will have heart failure (HF). HF is a condition in which the heart muscle is unable to sufficiently pump blood to meet the needs of the body. A characteristic of HF, and a major driver of the disease process, is an imbalance in the autonomic nervous system. Our lab and others have shown that the Acid Sensing Ion Channel subunit 3 (ASIC3) is highly expressed in the sensory nerves innervating the heart. Recent studies showed that chemically ablating these nerves attenuates cardiac remodeling after myocardial infarction (MI).

METHODS

Wild type (WT) and ASIC3‐/‐ mice underwent either left anterior descending coronary artery ligation or a sham surgery. Echocardiograms were done before surgery as well as two days and three weeks after surgery to evaluate the size and function of the heart. Radio telemeters were implanted one week after surgery to record hemodynamic changes to autonomic nervous system altering drugs. In a separate cohort a catheter was inserted into the left ventricle and isoproterenol was administered to evaluate left ventricular (LV) contractile reserve. Finally, histological staining was done on heart slices to quantify the level of fibrosis in each of the groups.

RESULTS

We have demonstrated that knocking out ASIC3 prevents disadvantageous remodeling after MI in mice. Two days after MI surgery both WT and ASIC3‐/‐ mice have a decrease in their LV ejection fraction (LVEF), a measure for how well the left ventricle is able to pump blood to the rest of the body. However, three weeks after MI the ASIC3‐/‐ mice have a significant increase in their LVEF compared to their WT counterparts. However, preliminary autonomic studies found no differences between WT and ASIC3‐/‐. Also, there wasn’t a difference between WT Sham and WT MI, indicating that mice don’t undergo the same changes in autonomic tone that we see in other animals such as humans or rats. Although not statistically significant, isoproterenol infusion shows a trend towards a higher contractile reserve in ASIC3‐/‐ MI mice compared to WT MI mice.

CONCLUSION

Our echo data shows that ASIC3 contributes to the remodeling that occurs after MI. Along with that, our data from the isoproterenol study indicates that knocking out ASIC3 in mice after MI may help to conserve cardiac contractile reserve. The preliminary work done for our autonomic studies indicate that the dysregulation of the autonomic nervous system in mice is different than what is expected from studies done in humans and rats.
Dissecting the Component Parts of the Excitation‐Contraction Coupling Machinery in Drosophila melanogaster
Authors: Ormerod Kiel
Published: May 2022

The Drosophila neuromuscular system is widely used to characterize synaptic development and function. However, little is known about how specific synaptic and muscular alterations effect neuromuscular transduction and muscle contractility that ultimately dictate behavioral output. Here a system developed to characterize excitation‐contraction coupling at Drosophila larval neuromuscular junctions (NMJs) was exploited to examining how specific neuronal, synaptic, and muscle manipulations disrupt muscle contractility and locomotion. Increases in motor neuron stimulation frequency and duration increased muscle force in a predictable fashion, but show considerable plasticity between 5–40 Hz and saturating above 50 Hz. Fictive contraction recordings from larvae with CNS intact revealed motor neuron burst frequencies of 20‐30 Hz, consistent with the endogenous machinery operating within the range of contractility of greatest plasticity. Experimental parameters such as saline composition (e.g. calcium and magnesium) temperature, larval size and orientation, were explored to examine their impacts on muscle force production. Subsequently, genetic and pharmacological approaches were taken to dissect several molecular pathways which contribute to muscle contraction and modulation of force. First, well‐established calcium regulation pathways were manipulated via application of ryanodine, caffeine, thapsigargin, and felodipine to demonstrate the role of the ryanodine receptors, SERCA pump, and postsynaptic calcium channels in modulating the magnitude and time‐course of muscle contractions. Presynaptic calcium channels and ryanodine receptor expression were genetically altered to further examine their role in muscle force production. Next using both null and knock‐down (Gal4/UAS system) approaches combined with tissue‐specific expression of drivers, screens for motor, synaptic, and muscle proteins contributing to excitation‐contraction coupling (ECC) were conducted. Altering the expression of the motor proteins kinesin, dynein or dynactin all resulted in a significant reduction in muscle force production. Critical synaptic proteins like synaptotagmin, complexin, cacophony, gbb (homolog of BMP), significantly impacted ECC in Drosophila larvae. Several critical muscle‐specific proteins alter the magnitude and time‐course of muscle contractions when altering their expression in a sub‐set of muscle fibers. Lastly a screen for modulators of muscle contractility led to identification and characterization of the molecular and cellular pathway by which the FMRFa peptide, TPAEDFMRFa, increases muscle performance. These findings indicate Drosophila NMJs provide a robust system to correlate synaptic dysfunction, regulation, and modulation, to alterations in excitation‐contraction coupling.
High‐Fat Diet and Environmentally Relevant Secondhand Smoke Co‐exposure and Cardiovascular Regulation in Mice
Authors: Pan Shiyue , Karey Emma , Pinkerton Kent E. , Chen Chao‐Yin
Published: May 2022

BACKGROUND

Over the past few decades, cardiovascular disease incidence significantly decreased by 27%. Paradoxically, the number of deaths from cardiovascular diseases increased by 42% over the same period, owing to population growth and longevity. Consequently, cardiovascular diseases remain the leading cause of global death. Cardiovascular diseases are complex, multifactorial diseases that cannot be ascribed to a single factor. High‐fat diet (HFD) and secondhand smoke (SHS) are two preventable risk factors for the development of cardiovascular diseases. Studies have shown that SHS exposure is associated with poor dietary habits, suggesting the importance of potential confounding diet effects on health effects of SHS exposure. We previously showed that, in mice, environmentally relevant SHS exposure resulted in 10% decrease in baroreflex sensitivity (BRS) and 13% decrease in measures of heart rate variability (HRV). Here, we sought to characterize how HFD interplay with SHS in inducing cardiovascular dysfunction.

METHODS

Male C57BL/6J mice (11 wk) implanted with BP/ECG telemetry devices were fed with either normal diet (ND, 13.6 kcal% fat) or HFD (60 kcal% fat) and exposed to either filtered air (FA) or SHS (3 mg/m3, 6 hr/d, 5 d/wk) for 11 weeks. 36‐hr continuous BP/ECG recordings were performed. Data were divided into three 12‐hour periods: the first dark cycle (dark 1) immediately after the last day of exposure, and the following light and dark (dark 2) cycles. Spontaneous baroreflex sensitivity (BRS) was determined with the sequence method. Standard time domain heart rate variability (HRV) parameters were obtained from normal‐to‐normal R‐R intervals.

RESULTS

HFD‐fed mice had significantly greater weight gain than ND‐fed group (+15.3 g and +2.8 g over 11 weeks, HFD and ND, respectively). SHS exposure had no effect on body weight. Compared to ND mice, HFD mice had significantly higher BP (~6.5 mmHg), HR (~98 bpm), and lower BRS (~15% decrease) in the light cycle. Furthermore, HFD significantly reduced overall and short‐term HRV (36% and 47% reduction for SDNN and rMSSD, respectively, p < 0.05) across all three light cycles. The reduction in HRV is unlikely due to a higher HR in HFD mice because normalizing the HRV measures to baseline R‐R intervals did not change the results. SHS exposure did not pose additional effects on these HFD‐induced changes. While HFD alone did not affect BP variability, co‐exposure of SHS and HFD significantly increased BP variability during the first dark cycle (16.8%, 15.6%, and 17.3% higher than HFD+FA group, for systolic, diastolic, and mean BP variability, respectively, p < 0.05), suggesting a co‐exposure‐induced BP dysregulation.

CONCLUSIONS

This study emphasizes the importance of accounting for differences in diet when studying the cardiovascular effects of SHS.
Anti‐CD3 mAb Induced Necrotizing Enterocolitis in Formula‐Fed Neonatal Mice through a Gut Microbiome Colonization Dependent Mechanism
Authors: Subramanian Saravanan , Geng Hua , Du Chao , Chou Pauline M. , Bu Heng‐Fu , Wang Xiao , De Plaen Isabelle G. , Tan Xiao‐Di
Published: May 2022

Necrotizing enterocolitis (NEC) is a serious disease that affects the small intestine of premature infants. Previous studies showed that formula feeding is a risk factor for NEC. Furthermore, gut microbiome and T cells are revealed to play a critical role in the pathogenesis of NEC. It has been reported that anti‐CD3 mAb induces T cell activation and small intestinal injury in adult mice. However, how the small intestine of neonatal mice responses to anti‐CD3 mAb treatment is uncertain. In the present study, we aimed to study the susceptibility of dam‐fed and formula‐fed neonatal mice to the anti‐CD3 mAb challenge. For this purpose, mouse pups (P0) were nursed by dam feeding (DF) and formula feeding (FF) respectively. On postnatal day 2, pups were subjected to subcutaneous treatment with 10 mg/kg anti‐CD3ε mAb. They were processed to measure intestinal permeability to FITC‐dextran and euthanized at 24 h after administration of anti‐CD3 mAb. Histology examination showed that FF but not DF pups developed NEC‐like injury in the small intestine after anti‐CD3 treatment. FF Pups were noted to display an increase in mortality, intestinal permeability, and inflammatory mediators upon anti‐CD3 mAb challenge compared to DF pups. Even though the inflammatory markers were increased, the intestinal mucosa in DF pups remains intact after being challenged with anti‐CD3. Remarkably, disruption of postnatal colonization by gavaging streptomycin attenuated NEC incidence in anti‐CD3 mAb‐treated FF pups, suggesting that bacterial colonization plays a role in predisposition of mouse pups by formula feeding to anti‐CD3 mAb‐induced intestinal injury. Taken together, this study provided the first evidence that anti‐CD3 mAb induces NEC‐like injury in neonatal mice receiving FF but not in DF via a microbiome colonization dependent manner. The underlying mechanisms regarding this pathophysiological event are to be studied in the future.
6‐O‐sulfation of endothelial glycocalyx heparan sulfates regulates systemic coagulation
Authors: Oshima Kaori , Davizon‐Castillo Pavel , Hernandez Giovanny , Baird Christine , Manco‐Johnson Marilyn , Schmidt Eric
Published: May 2022

Objective

The endothelial glycocalyx is a glycosaminoglycan‐rich layer that lines the surface of all vessels and is critical for vascular homeostasis, including the maintenance of an anticoagulant vascular surface. Heparan sulfate (HS), a linear polysaccharide composed of repeating disaccharide units of glucosamine and iduronic acid, is the major glycosaminoglycan comprising the endothelial glycocalyx. The mechanism by which glycocalyx HS regulates coagulation is unknown. As the biological function of HS derives from its unique pattern of sulfation (2‐O‐, 6‐O‐, and/or N‐sulfation) within constituent disaccharides, we hypothesized that the degree of heparan sulfate sulfation critically regulates systemic hemostasis.

Methods

We performed thromboelastography on whole blood collected from tamoxifen‐treated VE‐Cadherin‐Cre‐ERT2‐Sulf1/2 knockout mice, which have loss of endothelial sulf‐1 (a 6‐O specific HS sulfatase) and thus increased heparan sulfate 6‐O‐sulfation at the endothelial surface. Tamoxifen‐treated floxed mice (no Cre recombinase) served as controls. To determine the effects of HS in clot initiation and propagation, separate from platelet contributions, we treated platelet‐poor plasma from healthy donors (pooled and deidentified) with highly‐sulfated HS (HS09) and lower‐sulfated HS (HS01) and measured endogenous thrombin potential with calibrated automated thrombography. To explore the potential mechanism by which HS functions as anticoagulant, we repeated calibrated automated thrombography with antithrombin‐III‐, heparin cofactor‐II depleted plasma.

Results

Mice lacking endothelial sulf‐1 demonstrated increased R‐time, decreased angle, and increased K‐time compared to tamoxifen‐treated controls, suggesting that increased endothelial‐surface 6‐O HS sulfation decreases coagulation. In platelet poor plasma, highly‐sulfated HS09 attenuated thrombin generation in dose‐dependent manner while such effect was not observed with the same dose of less‐sulfated HS01. Repeated analysis using platelet poor plasma depleted of antithrombin‐III and of heparin cofactor‐II showed dose‐dependent decrease of thrombin generation with HS09, suggesting that HS exerts this anticoagulant effect independently of heparin‐like activation of antithrombin‐III.

Conclusion

The anticoagulant effect of endothelial‐surface HS is dependent on 6‐O sulfation of its constituent disaccharides, and this effect is mediated by the mechanisms distinct from well‐known heparin anticoagulation mechanisms.
Characterizing the Real‐Time Endogenous Methane Release in a Rat for 26‐days: Effect of Diets, Fasting, and Ethanol Consumption Across Different Time Scales
Authors: McKenna Kenneth Z. , Lighton John R. , McCue Marshall D.
Published: May 2022

Introduction

Methane is a byproduct of methanogenic archaea that oxidize H2 gas produced by other microflora in the gut. This methane gas is readily absorbed into the into systemic circulation and excreted in the breath down its concentration gradient (similar to CO2) or may be acutely expelled as flatus. While ruminant animals can generate over 100 liters of methane per day, technological limitations have precluded detailed measurements of methane production and release in humans and other non‐ruminants.

Methods

We paired a Promethion metabolic measurement system with a high‐precision (<1ppb) methane analyzer capable of real‐time methane measurements in an individual mouse or rat. We then subjected a rat to multiple diet switches, 48h of fasting, and ethanol treatments and continuously measured food/water intake, metabolic rate, and methane release over 26 days.

Results

VCO2 responses were unaffected by diet changes and only decreased slightly during the fasting treatment. Methane release was diminished by some diet treatments and could be restored by others. Ethanol had no observable effects. We discovered that methane production generally followed a circadian cycle whereby total rates were ~50% higher and large flatus events were more frequent during the scotophase. Methane release during flatus varied by >1,000‐fold ranging from as little as 0.1 up to 60μl/min. Integrating the continuous data stream (at 1 Hz) we confirmed that the vast majority of methane release occurred in the breath and not in the flatus as is often repeated in the literature. Finally, we provide evidence that the constitutive rate of methane release in the breath is positively correlated with rates of CO2 release, the latter of which is indicative of overall activity levels of the rat.

Conclusion

Given the growing number of molecular‐based studies investigating how gut microbiota can alter physiological function at higher levels of biological organization the approach of using real‐time methane measurements promises to provide a powerful new tool to establish causal linkages between microbiome dynamics and whole‐animal responses.
Proteins with Known Structure but Previously Unknown Function Identified as Hydrolases
Authors: Roberts Rebecca , Wilner Samantha
Published: May 2022

The Protein Data Bank (PDB) contains the structures of over 4000 proteins of unknown function. In an attempt to assign function to some of these structures we combined in silico and in vitro analyses focusing on those most likely to be of the hydrolase class. We used bioinformatics tools including active site‐alignment software, BLAST, Pfam, and Dali to predict putative protein functions. We expressed and purified the proteins and carried out in vitro activity assays, informed by the in silico‐predicted enzyme function. We present data confirming the in vitro hydrolase function of several proteins of known structure but previously unidentified function. These studies used the Biochemistry Authentic Scientific Inquiry Lab (BASIL) modules as a framework and were carried out as part of an undergraduate course‐based research experience (CURE).
Skeletal Muscle Mitochondrial Capacity in Patients with Statin‐Associated Muscle Symptoms (SAMS)
Authors: Mangone Laura , Parker Beth A. , Schmelzer Robert , Thompson Paul D. , White Michael , Kwon Oh Sung
Published: May 2022

Background

Mitochondrial dysfunction may be a key mechanism underlying SAMS. Near infrared spectroscopy (NIRS) is a non‐invasive technique of assessing tissue oxygenation and mitochondrial function and has never been evaluated as a diagnostic tool for SAMS.

Methods and Results

SAMS was confirmed using an 8‐week randomized, double‐blind crossover trial of simvastatin 20 mg / day and placebo separated by a 4‐week washout phase in 39 adults with self‐reported SAMS (Age: 65 ± 6 y; 25 female, 14 male). Tissue oxygenation was measured using NIRS during a handgrip exercise protocol at increasing intensities of maximal voluntary contraction (MVC) before and after statin or placebo treatment. Patients were considered to have SAMS only if they had muscle symptoms on simvastatin and not placebo. Similar to our previous trials, 17/39 (44%) patients were confirmed as having SAMS. There was a significant interaction (p < 0.01) between placebo vs. statin, pre‐ vs. post‐treatment, and SAMS/not SAMS. Tissue oxygenation for average (20‐40%) MVC was significantly lower with placebo when symptoms were present compared with when participants did not have symptoms on a statin (15.66% ± 28.21% vs. 2.35% ± 19.13%, respectively) (p = 0.02). Neither group changed tissue oxygenation with statin therapy (both p > 0.22; SAMS: 28.0 ± 17.6% vs. 22.6 ± 11.9%; No SAMS: 30.5 ± 18.8% vs. 31.0 ± 16.4%, respectively), but the decrease was numerically greater in SAMS patients. There were no significant differences in MVC change score based on symptoms however, patients with SAMS experienced more variable MVC change score responses compared with patients who did not have symptoms.

Conclusions

These results indicate that NIRS may be effective for differentiating patients with SAMS from those with non‐specific muscle pain, however this must be verified with a larger sample.
Characterization of Weight Gain and Motivation for Palatable Food in MAGEL2‐Deficient Rats
Authors: Mietlicki‐Baase Elizabeth G. , Kern Katherine A. , DiBrog Adrianne M. , Williams Abigail
Published: May 2022

Disrupting expression of MAGEL2, part of the MAGE family, is associated with disorders such as Prader‐Willi syndrome (PWS). PWS involves changes to energy balance including hyperphagia and weight gain, but the neurohormonal mechanisms underlying these changes have not been fully elucidated. As energy balance is controlled by the central nervous system, rodent models represent an important tool in understanding the neurobiology of PWS and how gene expression changes such as MAGEL2 disruption may impact feeding, weight gain, and motivation for food. Here, we used a novel MAGEL2‐deficient rat to examine how disruption of MAGEL2 expression impacts weight gain and motivation for palatable food. We hypothesized that MAGEL2‐deficient rats would have increased body weight post‐weaning and would exhibit increased motivation to obtain palatable food. Ongoing studies suggest that, contrary to our hypothesis, MAGEL2‐deficient rats weigh less than wild type controls in the 6 weeks after weaning (p<0.05). When wild type and MAGEL2‐deficient rats were trained to lever press for a palatable sucrose solution or fat solution on a progressive ratio (PR) schedule of reinforcement, no significant genotype differences were observed for active lever responses, reinforcers earned, or breakpoint for sucrose self‐administration. For fat self‐administration, no significant effects of genotype were observed on these measures in the first day of PR testing; on a second day of PR testing, MAGEL2‐deficient rats had a lower breakpoint than did wild type counterparts (p<0.05), suggesting that they do not exhibit greater motivation to work for fat solution and may in fact be less motivated to obtain this palatable food compared to wild type rats. Interestingly, in ad libitum feeding studies, more MAGEL2‐deficient rats exhibited food hoarding than did wild type controls (p<0.05). Other studies in our lab are continuing to investigate meal patterns and palatable fluid intake in the MAGEL2‐deficient rat model. Collectively, these findings suggest that the MAGEL2‐deficient rat may be useful for assessing mechanisms of hoarding behavior in PWS, but may be less useful for examining the underpinnings of PWS‐associated weight gain as the MAGEL2‐deficient rat does not exhibit increased body weight like humans with PWS.
Tryptophan Fluorescence as a Probe for Conformational Dynamics in Zinc Solute Binding Proteins
Authors: Serrano Fred A. , Yukl Erik
Published: May 2022

Bacterial ATP binding cassette (ABC) transporters are important for the uptake of various nutrients, including essential metals such as zinc. These systems require an extracellular solute binding protein (SBP) to bind the substrate with high affinity and specificity and deliver it to the membrane permease for transport. Generally, substrate binding elicits a change from an open to closed conformation that is likely important for permease recognition and transport. However, crystal structures for the metal‐bound and metal‐free forms of several zinc SBPs show relatively subtle or even negligible conformational changes that accompany metal binding. Thus, solution studies are needed for a thorough understanding of the role of conformational dynamics in substrate binding and transport. Here we investigate conformational states and dynamics in a group of zinc SBPs using tryptophan fluorescence intensity and lifetime as a sensitive reporter for the environment of this residue. Useful changes were observed in homologues of the protein AztC from Paracoccus denitrificans and the human pathogen Citrobacter koseri, whereas fluorescence from Citrobacter koseri ZnuA showed no change in fluorescence behavior upon zinc binding. These data combined with isothermal titration calorimetry (ITC) suggest a dynamic equilibrium of at least two conformational states in the apo form and compensatory changes in the holo form that provide for a significant entropic contribution to zinc binding. Correlation with available crystal structures suggests that the formation of a Trp‐Phe pi‐stacking interaction in AztC that is absent in ZnuA may mediate the observed changes in fluorescence. The conformational dynamics identified here highlight diversity in binding mechanisms for zinc SBPs with relevance to the exploitation of these proteins as potential antibiotic drug targets.
Sexually Dimorphic Effects of Nociceptor LKB1 on Mitochondrial Metabolism and Mechanical Allodynia After Fasting
Authors: Garner Katherine M. , Burton Michael
Published: May 2022

Background

Pain induces metabolic stress in pain sensing neurons (nociceptors); alleviating this stress represents an avenue for treating pain. Liver kinase B1 (LKB1) is involved in maintaining energy homeostasis and is a key mediator of the cellular response to metabolic stress. If cellular energy modulation and pain relief is LKB1‐dependent, then its removal could lead to metabolic imbalance and innocuous stimuli becoming painful (allodynia).

Hypothesis

In a novel mouse transgenic model where LKB1 is removed from Nav1.8+ nociceptors (Nav1.8LKB1‐), we predict nociceptors will have reduced abilities to cope with metabolic stress, leading to energy imbalance and subsequent allodynia.

Methods

After baselining for mechanical allodynia, animals were fasted for 24hr to induce metabolic stress. A subset of animals had their primary dorsal root ganglia nociceptors cultured for 6 hours for the Seahorse MitoStress assay. The remainder of animals were tested for mechanical allodynia once a day for 5 days after food was returned.

Results

As expected, a 24hr fast induced mechanical allodynia in a genotype dependent manner, with Nav1.8LKB1‐ animals responding to the fast in a sexually dimorphic manner. While fasting initially caused allodynia in males of both genotypes, Nav1.8LKB1‐ animals took longer to recover. However, in females, only Nav1.8LKB1‐animals exhibited allodynia after fasting. A sexually dimorphic and genotype dependent effect was also seen in neuronal mitochondrial function. In control females, fasting increased neuronal oxidative metabolism, suggesting the LKB1 pathway is necessary to mitigate fasting‐induced energy depletion. Neither fasting nor genotype changed neuronal mitochondrial function in males, suggesting an alternate mechanism was responsible for their initial allodynia.

Conclusions

Although Nav1.8LKB1‐ animals of both sexes developed genotype‐dependent allodynia in response to a metabolic stressor (24hr fast), only females exhibited a difference in mitochondrial function in response to this fast. These data support existing literature that suggests neuronal processes are responsible for the development of allodynia in females but not males and suggests that these differences are metabolic in nature. However, additional studies are needed to determine how other sex‐ and cell‐specific metabolic processes can contribute to pain development. This includes how the metabolism of immune cells, such as microglia and macrophages, which have been shown contribute to pain in males but not females, can be affected in an LKB1‐dependent manner to reduce a painful phenotype. Acknowledgements: We would like to thank Luz Barron and Han Jeong for their contributions.
Inhibitory Effect of Podocyte‐Specific Silencing of Acid Sphingomyelinase Gene on NLRP3 Inflammasome Activation and Glomerular Injury During Hyperhomocysteinemia
Authors: Huang Dandan , Li Guangbi , Zou Yao , Li Ningjun , Ritter Joseph K. , Li Pin‐Lan
Published: May 2022

Acid Sphingomyelinase (ASM) has been reported to increase tissue ceramide and thereby mediate hyperhomocysteinemia (hHcy)‐induced NLRP3 inflammasome activation in podocytes and glomerular sclerosis. The present study was designed to test whether somatic podocyte‐specific silencing of ASM gene (its mouse code is Smpd1) attenuates hHcy‐induced NLRP3 inflammasome activation and associated exosome release in podocytes and thereby suppresses glomerular inflammatory response and injury. We first constructed a floxed Smpd1 shRNA construct (Vigene Bioscience, Cat# IS100001) that has both RFP and GFP as reporter genes, where RFP is expressed if there is no cre excision. If cre excision occurs, GFP is expressed. This floxed Smpd1 shRNA was transfected into podocytes isolated from WT/Podocre mice, which decreased ASM mRNA and protein levels by 90% and 60%, respectively. This suggests efficient gene silencing in podocytes with cre gene. When this floxed Smpd1 shRNA was transfected into mouse kidney by intravenous infusion and ultrasound‐microbubble directed introduction, ASM expression decreased by 65% in WT/Podocre mice and 51% in Smpd1trg/Podocre mice after 8 weeks of shRNA transfection. It was also found that transfection of floxed Smpd1 shRNA substantially attenuated hHcy‐induced increase in NLRP3 inflammasome product, IL‐1β. By nanoparticle tracking analysis, floxed Smpd1 shRNA transfection was shown to significantly block hHcy‐induced elevation of urinary exosome excretion, an indicative of NLRP3 inflammasome product release from the cells. In Smpd1trg/Podocre mice, hHcy was found to induce exaggerated NLRP3 inflammasome activation, exosome excretion and glomerular inflammatory response such as immune cell infiltration compared to WT/WT mice. This exaggerated glomerular pathological response to hHcy was also significantly suppressed by podocyte‐specific Smpd1 gene silencing. Besides, podocyte‐specific Smpd1 gene deletion substantially attenuated proteinuria and albuminuria. Taken together, our results suggest that ASM in podocytes plays a crucial role in the control of NLRP3 inflammasome activation and associated exosome release and that podocyte‐specific silencing of ASM gene may be an important therapeutic strategy for treatment of glomerular injury and ultimate sclerosis during hHcy.
Single Molecule Analysis Reveals Dimer Formation of the Norepinephrine Transporter at the Plasma Membrane of Live Cells
Authors: Luethi Dino , Schütz Gerhard J. , Sitte Harald H.
Published: May 2022

Background

Monoamine transporters are the principal regulators of extracellular monoamine levels in the brain. At the plasma membrane, monoamine transporters form oligomers, which may play an important role in transporter trafficking, drug‐induced monoamine efflux, and drug tolerance development. Using single molecule microscopy, we previously reported that the serotonin transporter (SERT) co‐exists as monomers, dimers, and higher‐order oligomers, while the dopamine transporter (DAT) forms monomers and dimers only. Furthermore, whereas SERT oligomers are kinetically trapped at the plasma membrane by the phospholipid phosphatidylinositol (4,5)‐bisphosphate (PIP2), DAT dimerization is PIP2 independent. The subunit stoichiometry of the norepinephrine transporter (NET) is currently unknown. Based on a high degree of sequence homology between DAT and NET, we aimed to test the hypothesis that NET oligomerization resembles the one of DAT.

Methods

We fused monomeric green fluorescent protein (mGFP) to the N‐terminus of human NET and expressed the construct in Chinese hamster ovary cells. We assessed NET oligomerization using single molecule total internal reflection fluorescence (TIRF) microscopy in live cells. In order to determine the role of cell membrane lipids in NET oligomerization, we pharmacologically depleted the cell membrane of cholesterol and PIP2 before imaging.

Results

Single molecule brightness analysis revealed that NET co‐exists as monomers (~60%) and dimers (~40%) at the plasma membrane. NET oligomerization displayed signs of transporter density dependence and subunit exchange. Cholesterol depletion did not affect NET dimerization, whereas PIP2 depletion resulted in a small but significant decrease in the average oligomeric state (Student's t‐test, P<0.05).

Conclusion

NET forms monomers and dimers but no higher‐order oligomers, thereby resembling the oligomerization of DAT. However, unlike DAT, NET dimers exchange subunits over time and dimerization is affected by the transporter density and PIP2 concentrations. This study therefore demonstrates that the oligomerization behavior of each monoamine transporters has unique features.
Modulating Protein Photophysical Properties with Unnatural Amino Acids
Authors: Holod Chloe G. , Camilo Angelica , Harris Darcy , Brewer Scott , Phillips‐Piro Christine
Published: May 2022

Superfolder green fluorescent protein (sfGFP) is a 244 residue, ß‐barrel protein that has been used in a wide range of applications including as a biosensor for subcellular imaging of organelles and protein complexes. The photophysical properties of sfGFP are primarily the result of a cyclized tripeptide chromophore inside the ß‐barrel, consisting of residues T65, Y66, and G67. The protonation state of the central tyrosine 66 residue (Y66) is directly related to the photophysical properties of the proteins ‐ specifically the electronic absorption and emission spectral properties. Here we have replaced this tyrosine residue with several unnatural amino acids (UAAs) that were selected to vary the pKa of the side‐chain hydroxyl group to alter the photophysical properties of the protein. The UAAs were site‐specifically incorporated with high efficiency and fidelity using the Amber codon suppression methodology. The successful incorporation of these UAAs was verified through time‐of‐flight mass spectrometry. The altered photophysical properties of these sfGFP constructs in addition to the experimentally determined pKa of the tyrosine hydroxyl group in the protein will be presented in addition to initial efforts to determine the X‐ray crystal structures of these constructs.
RAS‐mediated nitric oxide signaling in podocytes
Authors: Semenikhina Marharyta , Bohovyk Ruslan , Klemens Christine A. , Russell Dayvia , Oates Jim C. , Staruschenko Alexander , Palygin Oleg
Published: May 2022

In the kidney, glomerular cells can release nitric oxide (NO) and regulate microvascular hemodynamics in concert with the renin‐angiotensin system (RAS). It has been shown that inducible nitric oxide synthase (iNOS) expression plays a pathophysiological role in glomerulonephritis, and NO deficiency in the glomeruli leads to podocyte damage and oxidative stress in glomerular cells. However, the functional effects of NO in podocytes and whether they express other NOS subunits are not well‐established. Our results suggest that NO signaling in podocytes is triggered by Ang II stimulation via Angiotensin II type 2 receptor (AT2R) since AT2R antagonist (PD123319) inhibited Ang II‐mediated NO production. To detect how different components of the RAS may modify NO production and intracellular Ca2+ signaling, we treated a conditionally immortalized human podocyte cell line with a variety of angiotensin metabolite peptides and measured changes in their NO production, intracellular Ca2+ flux, and cell volume. Understanding these fundamental pathways in podocytes is essential for discovering new therapeutical approaches to treat diseases of the glomerular filtration barrier (GFB). Our experiments determined that of the Ang metabolites tested, Ang III had the most pronounced effect on NO production. In contrast, Ang 1‐7 and Ang 1‐9 produce only small increases in NO, while Ang IV (and other peptides mainly targeting AT1R or the β‐arrestin pathway) do not produce any NO response. We also examined changes in cell volume using Scanning Ion Conductance Microscopy and found that NO production is associated with increase in podocyte cell volume. In contrast, Ca2+ influx without NO generation results in podocyte cell shrinkage. Lastly, we repeated these experiments with nNOS and iNOS specific inhibitors (Nω‐Propyl‐L‐arginine hydrochloride and L‐NIL, respectively), and determined that in normal podocyte cells, nNOS signaling is predominant compared to iNOS (70% and 30% of total response respectively, p<0.0001). In summary, these results demonstrate that nNOS‐ and iNOS‐mediated biosynthesis can be triggered by AT2R activation. This NO signaling can modulate podocyte cell motility, potentially affecting the integrity of the GFB. Additional experiments are needed to determine whether overproduction of NO and iNOS‐mediated pathway activation can cause pathophysiological changes in podocytes.
High Mobility Group Box 1 (HMGB1) is Central to the Cytomix‐Induced Pro‐Inflammatory Response in Human Pulmonary Microvascular Endothelial Cells
Authors: Nelin Leif D. , Jin Yi , Liu Yusen
Published: May 2022

Endothelial cell death is an early event in acute lung injury (ALI) leading to acute respiratory distress syndrome (ARDS) and bronchopulmonary dysplasia (BPD). We have previously shown that cytomix (lipopolysaccharide (LPS), interleukin (IL)‐1β, interferon‐γ, and tumor necrosis factor‐α), but not LPS alone, results in robust cell death in human pulmonary microvascular endothelial cells (hPMVEC). We hypothesized that the inflammatory cascade associated with cytomix treatment in hPMVEC would result in the release of HMGB1, a damage‐associated molecular pattern (DAMP), which would enhance the pro‐inflammatory response. Treatment of hPMVEC with cytomix for 24 hours resulted in a robust (25‐fold) induction of HMGB1 protein levels in the media compared to controls. Cytomix treatment of hPMVEC resulted in nearly 5‐fold greater cleaved caspase‐1 protein levels and nearly 60‐fold greater cleaved IL‐1β protein levels than in control hPMVEC. Viable hPMVEC numbers were significantly reduced after cytomix treatment. To assess the role of TLR4 in the inflammatory response of hPMVEC following cytomix treatment, we utilized TAK‐242, a TLR4 antagonist. The cytomix‐induced increase in media HMGB1 levels was significantly attenuated by TAK‐242. TAK‐242 also significantly attenuated the cytomix‐induced increases in cleaved caspase‐1 and cleaved IL‐1β. To assess the role of caspase 1 in the inflammatory response of hPMVEC following cytomix treatment, we utilized Ac‐YVAD‐CMK, a caspase‐1 antagonist. Treatment with Ac‐YVAD‐CMK attenuated the cytomix‐induced increase in media HMGB1 and cleaved IL‐1β; and partially restored viable cell numbers following cytomix treatment. To further understand the role of HMGB1 in the pro‐inflammatory process we treated hPMVEC with an HMGB1 antibody; and found that the HMGB1 antibody significantly attenuated the increases in HMGB1 and cleaved IL‐1β release, as well as endothelial cell death caused by cytomix treatment. Our results demonstrate that treatment with cytomix results in release of HMGB1, which activates caspase 1 leading to greater HMGB1 production. Furthermore, HMGB1 activation of caspase‐1 leads to cleaved IL‐1β production as well as hPMVEC death. Preventing the interaction of HMGB1 and TLR4 attenuated HMGB1 and cleaved IL‐1β release, and attenuated cytomix‐induced reductions in viable cell numbers. We speculate that the release of damage‐associated molecular patterns (DAMPs), such as HMGB1, may be crucial in the innate immune response of hPMVECs. These findings may have implications for the pathogenesis of ALI in inflammatory lung diseases.
Myeloid Expressed Epidermal Growth Factor Receptor (EGFR) is Critical to Heart Homeostasis and Limits Adverse Outcomes Following Ischemic Attack
Authors: Okyere Ama , Teplitsky David , McEachern Erin , Carter Rhonda , Gao Erhe , Tilley Douglas
Published: May 2022

The healthy heart comprises immune cells, mainly macrophages of differing class and origin. While these myeloid cells remain an area of continuous investigation, we have come to appreciate that a subset of tissue residing macrophages (TRMs) seed the heart during development and are maintained through self‐renewal. Further, TRMs continue to be linked to the maintenance of cardiac homeostasis, for instance, through facilitating crosstalk with other cardiac cells, and participating in immunosurveillance. In the case of injury however, such as a myocardial infarction (MI), TRMs are largely replaced by circulating and recruited myeloid cells. Post‐infarct macrophages (mf) are greater in number and class, and functionally, debris clearance and initiation of cardiac repair mechanisms becomes their utmost priority to limit the severity of heart failure (HF). Given these significant roles, it has become critical to understand how cardiac myeloid cells are regulated, and what mechanisms contribute to their known functions both at steady‐state and following injury. Epidermal growth factor receptor (EGFR) is a cell surface tyrosine kinase receptor known to govern cell function through myriad processes. Although historically studied as an oncogene, EGFR has been shown to regulate cardiac physiology, and notably mf activation, survival and function. However, it is currently unknown whether EGFR influences myeloid homeostasis within the heart. Thus, we have generated myeloid‐specific EGFR knockout mice (EGFRmylKO) by crossing floxed EGFR (EGFRf/f) with LysM Cre mice and performed comparative analyses between the lines at baseline and following injury. In response to MI, EGFRmylKO mice exhibit altered inflammatory responses, resulting in aggravated cardiac dysfunction, exacerbated hypertrophic remodeling, and limited angiogenic repair. These functional and structural outcomes are preceded by a disruption in the post‐injury myeloid cell infiltration continuum that favors adequate repair, resulting in decreased transcripts of reparative factors including IL‐10. Additionally, EGFRmylKO mouse hearts show modest signs of stress at baseline, in the absence of injury, including cardiomyocyte hypertrophy with an associated increase in expression of signature fetal gene transcripts. Whole transcriptome analysis of cardiac resident myeloid (Cd11b+) cells by RNASeq revealed over 700 differentially expressed genes that were significantly altered between EGFRmylKO and EGFRf/f control mice. Among these are IGFBP family members 5 and 7, each known to regulate IGF availability, which itself has been recently shown to influence cardiac hypertrophy. In sum, our results reveal that myeloid cell‐expressed EGFR plays previously unknown roles in the regulation of the cardiac homeostasis baseline as well as the chronic remodeling response to ischemic injury.
Expanding the Utility of 4‐cyano‐L‐phenylalanine as a Vibrational Reporter
Authors: Schick Sarah C. , Papoutsis Brianna M. , Brewer Scott H. , Phillips‐Piro Christine M.
Published: May 2022

Unnatural amino acids (UAAs) have become useful tools for understanding protein structure and local environments. One of the most commonly used vibrational reporter UAAs to study local protein hydration environments is 4‐cyano‐L‐phenylalanine (pCNF) due to the position and sensitivity of the nitrile symmetric stretching frequency as well as the ability to efficiently incorporate this UAA site‐specifically into proteins using the Amber codon suppression methodology. Additionally, pCNF provides promise to function as a distance reporter when coupled with two‐dimensional infrared (2D IR) spectroscopy. Here, a number of superfolder green fluorescent protein (sfGFP) constructs have been generated with pCNF site‐specifically incorporated at two unique sites simultaneously. The pairs of sites were selected to permit the nitrile symmetric stretching frequency of pCNF at each site to be spectrally resolved. The temperature dependent linear IR spectra of these sfGFP constructs with two pCNF UAAs incorporated will be presented, illustrating that the nitrile groups are in different local environments. Preliminary X‐ray crystal structures of these sfGFP constructs will be presented to probe if the UAAs resulted in a structural perturbation and to determine the distance between the nitrile groups of the two pCNF UAAs in each structure. These results will serve as the basis for and calibration of employing pCNF in future 2D IR spectroscopic studies to extract the distances between these two groups based upon coupling between them.
DHT Induces Arterial Stiffening in Female Wild Type Mice
Authors: Horton Alec C. , Wilkinson Mary , Kilanowski‐Doroh Isabella , Ogola Benard , Lindsey Sarah
Published: May 2022

Testosterone is the predominant sex hormone in men and is increased in women with polycystic ovarian syndrome (PCOS). PCOS patients also experience an increased risk for cardiovascular diseases including hypertension and arterial stiffness. Our previous work showed significant downregulation of the G protein‐coupled estrogen receptor (GPER) as well as the nuclear estrogen receptor ERα in cultured vascular smooth muscle cells in response to the testosterone metabolite dihydrotestosterone (DHT), which cannot be aromatized to estrogen. Therefore, we hypothesized that DHT may induce arterial stiffness in female mice via downregulation of vascular estrogen receptors. Two groups of female wildtype mice were treated with DHT via silastic capsule for 4 weeks; one group received a dose designed to mimic DHT levels in men while the other group received twice this dose. While post‐treatment systolic blood pressure was not different between the control, single dose, and double dose groups (111 ± 10, 106 ± 3, 110 ± 7, n=8 per group; P=0.22), intracarotid vascular stiffness measured via pulse wave velocity showed a more than two‐fold increase in single dose (4.3 ± 0.8 m/s) and double dose (4.8 ± 1.0 m/s) DHT treated mice when compared with non‐treated controls (1.9 ± 0.3 m/s; P<0.0001). These findings suggest that high testosterone levels in females may promote arterial stiffening and cardiovascular damage independent of changes in blood pressure. Future studies will include histological analysis of aortic sections using Masson’s trichrome to assess changes in the extracellular matrix as well as droplet digital polymerase chain reaction to assess changes in estrogen receptor expression. In conclusion, these findings of arterial stiffening in response to increased DHT levels in females are important for not only PCOS patients but also women using testosterone for fitness, gender transitioning, or reduced libido.
A Review of MutSα and its Absence in Mismatch Repair Related Ovarian Carcinomas
Authors: Hayes Amy , Jaman Afreen , Johnson Ethan , Rausch Mira
Published: May 2022

This review investigates the roles of microsatellite instability (MSI) and the DNA mismatch repair (MMR) pathway in ovarian carcinomas. MMR promotes genome stability post‐replication by repairing DNA mismatches; defective MMR (dMMR) leaves genomic errors that can cause MSI. To repair these mismatches, the MMR system employs two sets of proteins that form heterodimers: MutL Homolog 1 (MLH1) in complex with post meiotic segregation increased 2 (PMS2) and MutS Homolog 2 (MSH2) in complex with MutS Homolog 6 (MSH6). The MSH2/MSH6 heterodimer (MutSα) recognizes the mismatch, while the MLH1/PMS2 heterodimer (MutLα) repairs the mismatch. The MSH2/MSH6 proteins are each composed of 5 asymmetrical structural domains, the first being responsible for mismatch binding, followed by the connector, lever, clamp, and ATPase domains, or domains I‐V, respectively. The MSH2 mismatch binding domain associates directly with DNA and extends a helix to block the polymerase, while MSH6 associates with DNA via its Phe‐X‐Glu motif. The MutSα complex has two states: an ADP‐bound state in which it searches for mismatches, and an ATP‐bound state in which it signals for repair. Lack of effective repair can contribute to carcinogenesis including ovarian carcinogenesis. Mismatches in microsatellites are phenotypic evidence that the MMR pathway is non‐functional in these tumors, and differences in MMR status are observed across ovarian carcinoma subtypes. For example MSH6 deficiency is associated with three subtypes of ovarian cancer: clear cell, mucinous, and endometrioid carcinomas, and there is a higher survival rate for patients with these MSH6 deficient tumors. Tumor MMR status also influences treatment efficacy and age of disease onset. Interestingly, only in familial carcinomas was MSI demonstrated as a supportive diagnostic tool. Tumors with high MSI can be treated with immunotherapy and immune checkpoint inhibitors; therefore investigating the MMR pathway functionality and MSI status of ovarian carcinomas can shape the course of treatment and patient outcomes.