bluuebunny/crossref_metadata_2025 huggingface dataset:
The GGA Family Proteins Modulate the Cell Surface Transport of α2B‐Adrenergic Receptor through Specific Interactions
Authors: Zhang Maoxiang , Davis Jason , Wu Guangyu
Published: June 2021

Molecular mechanisms governing the cell surface transport of G‐protein‐coupled receptors (GPCRs) remain poorly elucidated. Here, we used α2B‐adrenergic receptor (α2B‐AR) as a model GPCR to determine the role of three Golgi‐localized, γ‐adaptin ear domain homology, ADP ribosylation factor‐binding proteins (GGAs), one family of adaptor proteins for clathrin‐coated vesicles involved in the trans‐Golgi network (TGN)‐to‐endosome transport. It has been well described that the function of GGAs is tightly controlled by interactions via their VHS domain with the acidic LL motifs presented in the C‐terminus of cargo molecules. We found that shRNA‐mediated depletion of individual GGAs strongly arrested α2B‐AR in the Golgi/TGN and significantly reduced the cell surface expression and signaling of the receptor. We further demonstrated that GGAs physically associated with α2B‐AR through specific domains. The GGA‐binding domains were mapped to the C‐terminal regions in the third intracellular loop of α2B‐AR, whereas α2B‐AR‐binding domains were identified to the hinge domain of GGA1, the GAE domain of GGA2 and the VHS domain of GGA3. These studies reveal a novel function of the GGA family proteins in the TGN‐to‐plasma membrane transport of α2B‐AR which is likely mediated through a non‐conventional mechanism. These data also suggest that the GGA adaptor proteins are important mediators of GPCR cell surface targeting.
p‐Cymene Complexed in β‐Cyclodextrin to Cancer Pain Control
Authors: Barreto Andre , Guimarães Adriana , Oliveira Marlange , Serafini Mairim , Araujo Adriano , Barreto Rosana , Quintans Jullyana , Quintans‐Junior Lucindo
Published: June 2021

p‐Cymene (PC) is an alkylbenzene commonly found in cumin oil which has shown antinociceptive activity in animal models. Now, we evaluated the effect of encapsulation of PC in β‐cyclodextrin (PC/β‐CD) on nociception induced by tumor cells (sarcoma 180) in rodents. PC/β‐CD complex was administered (12.5 ‐ 50 mg/kg, p.o.) in mice with tumor (106 cells/25 mL, s.c.) on the hind paw, which were evaluated regarding hyperalgesia (digital von Frey apparatus), spontaneous and palpation‐induced nociception. The experimental protocols were approved by UFS Ethic Committee (CEPA: 19/11). PC/β‐CD (12.5, 25 e 50 mg/kg) was able to reduce significantly hyperalgesia (p < 0.001 or 0.05), and this effect was maintained for 24 hours after treatment. Oral administration of PC/β‐CD complex (12.5‐50 mg/kg) on alternate days also reduced spontaneous and nociception induced by palpation (p<0.001 ‐ 0.05). Free PC (50 mg/kg, p.o.) and PC no complexed with β‐CD, were unable promote significant changes in nociceptive responses. Therefore, results obtained suggest the inclusion complex containing p‐cymene and β‐cyclodextrin may improve the pharmacological characteristics of terpenoids or related compounds with potential applicability in chronic pain, such as cancer.

Financial support: FAPITEC‐SE and CNPq (Brazil).
Deregulation of Bile Acid Homeostasis and Lipid Metabolism in Parenteral Nutrition Mouse Model
Authors: Zhan Le , Yang Ill , Kong Bo , Shen Jianliang , Buckley Brian , Guo Grace
Published: June 2021

Long‐term parenteral nutrition (PN) administration can lead to PN associated liver diseases (PNALD), of which the molecular pathogenesis remains poorly understood. Animal studies in preterm piglets and several mouse models have shed light on the mechanisms underlined. To date, the roles of bile acids (BAs) and their signaling pathways involved in the development and progression of PNALD are still unclear.

In the current study, we have established a mouse PN model with IV infusion of PN solution containing soybean oil based lipid emulsion. Liver mRNA profiling was determined by microarray analysis and serum BA profiling was determined by UPLC‐MS method. PN significantly decreased the mRNA levels of sterol 12‐alpha‐hydroxylase (Cyp8b1) and BA efflux genes, and increased that of cholesterol 7‐alpha‐hydroxylase (Cyp7a1). Consistently, both the levels and the percentages of primary BAs as well as total non‐12α‐OH BAs increased significantly in the serum of PN mice. In addition, PN increased the expression of several key genes involved in lipogenesis, which could be suppressed by farnesoid X receptor (FXR) and/or induced by liver X receptor alpha (LXRα).

This study suggests that PN leads to deregulation of BA and lipid homeostasis, which predispose the subjects to pro‐cholestatic conditions. Determining deficiencies in BA pathway in patients may help to predict probability of PNALD development, and maintaining BA homeostasis by therapeutic agents may represent a novel treatment in the future for PNALD.
Enhanced Epithelial‐to‐Mesenchymal Transition Associated with Lysosome Dysfunction in Podocytes: Role of p62/Sequestosome 1 as a Signaling Hub
Authors: Li Guangbi , Li Cai‐Xia , Xia Min , Abais Justine , Boini Krishna , Ritter Joseph , Li Pin‐Lan
Published: June 2021

Autophagy is of importance in the regulation of cell differentiation and senescence in podocytes. It is possible that derangement of autophagy under different pathological conditions may enhance Epithelial‐to‐Mesenchymal Transition (EMT) in podocytes, resulting in glomerular injury. The present study tested whether deficiency of autophagic flux leads to enhancement of EMT in podocytes. By Western blot and confocal analysis, lysosome function inhibition by a V‐ATPase inhibitor or its siRNA was found to significantly enhance EMT in podocytes, as shown by marked decreases in epithelial markers (P‐cadherin and ZO‐1) and increases in mesenchymal markers (FSP‐1 and α‐SMA). This enhanced EMT was accompanied by deficient autophagic flux, which was demonstrated by increases in LC3B‐II levels and accumulation of p62. We further confirmed that both enhancement of podocyte EMT and deficiency of autophagic flux due to lysosome dysfunction were significantly attenuated by inhibition of autophagosome formation using SP‐1. To explore the mechanisms by which deficient autophagic flux enhances EMT, we tested the role of accumulated p62 as a signal hub in this process. It was found that both Nrf2 and NF‐κB pathways regulated by p62 were not involved in enhanced EMT, while inhibition of CDK1 activity reduced phosphorylation of p62 and enhanced EMT in podocytes, an effect on EMT similar to lysosome dysfunction. Given that lack of phosphorylated p62 leads to a faster exit from cell mitosis, enhanced EMT associated with lysosome dysfunction may be attributed to accumulation of p62 and associated reduction of p62 phosphorylation.
Effect of Leukotriene Modifiers on Transport Activity of Multidrug Resistance Proteins (MRPs)
Authors: Csandl Mark , Conseil Gwenaëlle , Cole Susan
Published: June 2021

Multidrug resistance protein 1 (MRP1), MRP2, MRP3, and MRP4 are membrane transporters that mediate ATP‐dependent efflux of xenobiotics, their metabolites, and many physiological organic anions. MRP1‐4 have distinct substrate profiles; however, they can all transport the glucuronide conjugate of estradiol (E217bG). An established physiological role of MRP1 is to efflux the proinflammatory leukotriene C4 (LTC4). MK‐571 was originally designed as a leukotriene receptor 1 (CysLT1R) antagonist to treat asthma; it is also the most popular MRP1 inhibitor. However, MK‐571 is non‐specific and inhibits most MRP homologs as well as some solute carrier organic anion (SCLO) importers, limiting its usefulness as an experimental tool. Other leukotriene modifiers (LTMs) have been developed to treat inflammatory diseases but little is known of their ability to modulate MRP1 and its homologs. In this study, the effect of a series of LTMs specific for either CysLT1R or CysLT2R on E217bG uptake into MRP1, MRP2, MRP3, or MRP4‐enriched membrane vesicles was measured. The IC50 values for the 5 LTMs tested ranged from 0.91 to 25.27 uM; MRP1 and MRP4 IC50 values were the most alike. In contrast, some LTMs stimulated MRP2 and MRP3. Thus, LY171883 stimulated MRP2 and MRP3‐mediated E217bG uptake by about 2.3 and 4‐fold, respectively, while Montelukast modulated MRP2 in a biphasic manner. CysLT1R specific LTMs were generally a less potent class of MRP modulators. These data suggest that like MK‐571, most LTMs are non‐selective modulators of MRP1‐4 transport, and should therefore be used with caution because of their potential to confound data interpretation.

Supported by Canadian Institutes of Health Research MOP‐133584
Genome‐Wide Mapping of RNA Polymerase II Termination Sites
Authors: Corden Jeff , Merran Jonathan , Schaughency Paul
Published: June 2021

We have used in vivo RNA cross‐linking to map the position of RNA polymerase II (Pol II) in the yeast S. cerevisiae. Pol II terminates transcription of coding transcripts through the polyadenylation (pA) pathway and non‐coding transcripts through the non‐polyadenylation (non‐pA) pathway. Using PAR‐CLIP we have mapped the position of Pol II genome‐wide in living yeast cells after depletion of components of either the pA or non‐pA termination complexes. The results indicate that depletion Nrd1, a non‐poly(A) termination factor leads to widespread read through of non‐pA terminators. Depletion of the RNA helicase Sen1 also leads to read through at non‐pA terminators. In contrast to Nrd1 this read through appears more susceptible to pausing. These results provide a map of in vivo Pol II non‐pA termination regions and reveal sequences that signal termination of different classes of non‐pA transcripts. Ysh1 is a component of the cleavage and polyadenylation machinery and is responsible for cleavage at the pA site and subsequent removal of Pol II from the template. When Ysh1 is depleted from the nucleus Pol II does not read through the termination site but rather accumulates just downstream of the polyadenylation site. We will discuss possible mechanisms that distinguish termination of pA and non‐pA transcripts.
Functional Interplay between Epigenetic DNA Modifications and Transcription Machinery
Authors: Wang Dong
Published: June 2021

Dynamic regulation and faithful maintenance of proper DNA methylation and demethylation are essential for many cellular functions. 5‐Hydroxylmethylcytosine (5hmC), 5‐formylcytosine (5fC) and 5‐carboxylcytosine, newly discovered oxidized forms of 5‐methylcytosine (5mC) are involved in active DNA demethylation process. The roles of these oxidized species of 5mC in epigenetic and transcription regulation have been an area of intensive study recently. Here we report our recent effort in understanding the functional interplay between these different forms of cytosine in DNA on RNA polymerase II transcription elongation. Our results providie new insights into functional relationship between cytosine methylation status and transcription. In addition, we will discuss our recent effort in developing new chemical tools that target the formylcytosine sites and modulate 5fC‐related biological processes.
Plant‐derived protein for potential anti‐clot application
Authors: Wang Kevin , Dickey Alexia
Published: June 2021

Lumbrokinases, a group of fibrinolytic enzymes from the earthworm, has been widely investigated as a thrombolytic natural treatment for cardiovascular problems. However, traditional product extracted and purified from earthworm is complex which contains contaminants that cause side effects. In this study, we cloned both codon‐optimized and original gene fragment of Lumbrokinases for both transient and stable expression in plant. Protein purified from transgenic plant can degrade artificial fibrin. Our study showed that recombinant lumbrokinase caused plant tissue damage. This project was supported by the National Institute of General Medical Sciences of the National Institutes of Health through Grant Number 8P20GM103447.
Novel Negative Gating Modulators of KCa2/3 as Pharmacological Tools for Novel Treatments
Authors: Oliván‐Viguera Aida , Valero Marta , Badorrey Ramón , Wulff Heike , Köhler Ralf
Published: June 2021

Introduction

KCa2/3 channels are present in many organs and tissues such as vascular endothelia, intestinal and bronchial epithelia, neurons or blood cell lineages. Therefore, they play important roles in cardiovascular, immunological and neurological functions and emerge as therapeutic targets.

Existing KCa2/3 pore blockers, due to its mechanism of action, could exert unspecific blocking effects at high dosages. Negative‐gating modulators, which modulates the regulatory mechanism of the active channel, seem to be better pharmacological tools.

Methods

We synthesized new small molecules and performed patch‐clamp experiments to evaluate their potency. We also evaluated their functional activity by isometric tension myography on porcine coronary arteries. We determined cardiovascular safety and efficacy by telemetric cardiovascular monitoring on mice.

Results

Our novel entities met Lipinski's rule of five and we identified RA‐2 as KCa2/3 pan‐negative gating modulator, with potencies in the nM range and with no considerable effects on distantly related human K channels. RA‐2 blocks EDH‐type relaxation and it is apparently safe in‐vivo, with no effects in blood pressure, although it moderately reduces heart rate.

Discussion

We identified new molecules that could serve as pharmacophores for structurally new chemical entities for specific negative‐gating modulation of KCa2/3. RA‐2 has nanomolar potency, and ex‐vivo and in‐vivo activity, and it could be a tool to study physiological and pathophysiological roles of the KCa2/3 channels and may be of therapeutic utility.
Small‐Molecule Inhibition of the Androgen Receptor (AR)‐RUNX2 Interaction in Prostate Cancer (PC)
Authors: Vuong Winston , Tew Ben , Little Gillian , Frenkel Baruch , Jones Jeremy
Published: June 2021

The advent of prostate carcinogenesis is associated with alterations in AR transcriptional activity to promote cancerous phenotypes. In particular, the transcription factor RUNX2 has been shown to modulate AR activity in a locus‐dependent manner. The AR and RUNX2 interact to inhibit a majority of genes they co‐regulate, dubbed Type I, including the tumor‐suppressor NKX3.1. Interestingly, they cooperatively express a small set of genes in a synergistic manner (Type II) including the oncogenes, SNAI2 and SOX9. We devised a robust fluorescent cell‐based assay (z‐score >0.7) to screen for inhibitors of the oncogenic AR‐RUNX2 interaction in the C4‐2B PC cell line with doxycycline‐inducible RUNX2. These cells were transfected with a type II/YFP reporter and a control mCherry plasmid. The cells were treated with doxycycline to induce RUNX2 expression and DHT to activate AR then analyzed in a 96‐well plate format on a Tecan fluorescent plate reader. We identified two compounds from the NCI Developmental Therapeutics Program compound library that inhibited the AR‐RUNX2 interaction; which were validated by a dose curve in the same assay and qPCR for activity against synergistic activation of SNAI2 and SOX9. One compound was reported to inhibit the epidermal growth factor receptor (EGFR) so we sought to investigate the role of EGFR signaling in the AR‐RUNX2 interaction. Knockdown of EGFR changed the regulation of SNAI2 from that of a Type II gene to a canonical Type I gene, suggesting that EGFR signaling is necessary for the synergism. Moving forward, we aim to identify the component of EGFR signaling that facilitates Type II gene regulation as a potential therapeutic target in prostate cancer.
Preclinical Development of Recombinant Human Tissue Inhibitor of Metalloprotease‐2 as a Novel Biologic Therapy for Cancer
Authors: Chowdhury Ananda , Wei Beiyang , Noonan Silvia , Stetler‐Stevenson William
Published: June 2021

Tissue Inhibitor of Metalloprotease‐2 (TIMP‐2), a 21.75 KDa protein, has a unique dual activity and mediates its anti‐cancer properties by metalloprotease (MMP) inhibition and as an anti‐ angiogenic agent with direct anti‐proliferative effects on tumor cells. TIMP‐2 is a promising candidate for further pre‐clinical studies and clinical development. Recombinant human TIMP‐2‐His tagged protein (rh‐TIMP‐2‐6XHis) was expressed in solution culture adapted HEK 293 cells in sequentially expanding culture volumes up to 5 liters in wave bioreactor and purified in two steps by affinity and revered phase chromatography. rh‐TIMP‐2‐6XHis can be obtained to up to 97% purity with a maximum associated loss of about 25% and final yields of >25mg/liter. Purification steps and final TIMP‐2 protein were characterized by SDS‐PAGE, Western Blotting as well as ESI‐TOF mass spectrometry, and quantified by Sandwich ELISA assay. These analyses revealed an efficient purification procedure and high degree of monodispersity of the final product. Circular Dichorism spectroscopy exhibited alpha‐helical content of ~33% for TIMP‐2 at pH‐7 and overall pH dependent change of secondary structure. Purified rh‐TIMP‐2 demonstrates inhibition of MMP‐2 activity and inhibition of EGF stimulated proliferation of A549 human lung cancer cell line. Peptide gel encapsulated rh‐TIMP‐2 dosage forms are being tested for loading capacity and release rates. These results demonstrates a straightforward and robust development route for a novel biologic therapeutic for cancer treatment.
Targeted Delivery of TNF‐Alpha and IFN‐Gamma by Tumor Targeting Peptide Inhibited Orthotopic Colorectal Cancer Growth
Authors: Shen Jing , Lu Lan , Li Long Fei , Wu William Ka Kei , Li Zhi Jie , Cho Chi Hin
Published: June 2021

Objective

To determine the in vivo anti‐tumor activities of TNF‐alpha and IFN‐ gamma given alone or in combination and compare with the effects of their conjugates with tumor targeting peptide in a mouse colorectal tumor model.

Methods

A mouse orthotopic colorectal tumor model was established. Treatment was given by a single i.v. injection. Mice were sacrificed and examined for their tumor sizes and other immuno and biological parameters in tumors 7 days after treatment. Apoptosis was determined by the TUNEL assay. Immunotherapeutic response was determined by immunofluorecence using antibodies against T lymphocytes, macrophages and NK cells.

Results

TNF‐alpha and IFN‐gamma showed marginal inhibitory effects on tumor growth, while their conjugates with tumor targeting peptide further decreased tumor sizes. Compared with the treatment with non‐targeted TNF‐alpha plus IFN‐gamma, targeted delivery by the peptide markedly alleviated the systemic toxicity as shown by a significant reduction in mortality after treatment. Moreover, targeted delivery of TNF‐alpha plus IFN‐gamma synergistically and significantly inhibited tumor growth.

Conclusions

Targeted delivery of TNF‐alpha plus IFN‐gamma exhibited promising tumoricidal activity and at the same time alleviated the systematic toxicity induced by the immuno‐modulators in mice.

Acknowledgements: The study was supported by the Innovation and Technology Support Programme and the General Research Fund from the Innovation and Technology Commission and the Research Grant Council from Hong Kong.
Electrophilic Nitroalkenes Cause Degradation of NFκB RelA Protein in Triple Negative Breast Cancer Cells
Authors: Woodcock Chen‐Shan , Woodcock Steven , Davidson Nancy , Huang Yi , Freeman Bruce
Published: June 2021

Triple negative breast cancer (TNBC), which lacks estrogen receptor (ER), progesterone receptor (PR) and Her2/Neu, is an aggressive and metastatic cancer. Current therapy for TNBC is limited, and it is an unmet need for novel therapeutic agents with improved efficacy. Electrophilic fatty acids are endogenously‐generated signaling mediators that modulate cell differentiation and proliferation via post‐translational modification of nucleophilic amino acids (Cys, His) of transcriptional regulatory proteins such as NFkB, PPARgamma, and Keap1/Nrf2. Preliminary studies have revealed that an exemplary electrophilic fatty acid nitroalkene, nitro‐oleic acid (OA‐NO2), displays potential therapeutic value in TNBC cells, by preferentially reducing the growth and viability of two human TNBC cell lines, but not normal breast cells. Essential to proliferation and survival, NFkB signaling is highly activated in ER‐negative breast cancer cells and primary tumors. Notably, NFkB RelA protein level was diminished in TNBC cells upon OA‐NO2 treatment. Therefore, it is hypothesized that electrophilic fatty acids reduce TNBC cell proliferation and survival through down‐regulation of NFkB expression and signaling. We have demonstrated that OA‐NO2 down‐regulates transcript levels of NFkB –regulated genes, cyclin D1 and the pro‐survival genes, bcl‐xL and survivin, in TNBC cells. Moreover, OA‐NO2 promotes RelA protein polyubiquitination via nitroalkylation of RelA in TNBC cells. Overall, this study will serve as a prelude to the clinical study of electrophilic nitrated lipids as therapeutic agents that may display a high selectivity for killing TNBC tumors.
Calcium Signaling in the Bronchopulmonary Dysplasia Rat Heart
Authors: Haraldsdottir Kristin , Farrell Emily , Sobotik Atzie , Eldridge Marlowe
Published: June 2021

Bronchopulmonary dysplasia (BPD) is a chronic lung disease associated with premature birth. It is a significant contributor to perinatal morbidity and mortality. The disease results in disrupted alveolar and pulmonary vascular growth, and may lead to pulmonary hypertension (PH) and right ventricular hypertrophy (RVH) during infancy. Although recent studies have shed light on the pathophysiology of BPD, much remains unknown about the long‐lasting effects of the disease into adulthood. Previous studies on heart failure, PH and RVH have shown that there are alterations in Ca2+ signaling proteins in the right heart myocardium. The objective of this study is to identify whether there are alterations in Ca2+ signaling protein expression in right ventricular (RV) myocytes in rats with BPD. We hypothesize that there is decreased expression of Ryanodine Receptor (RyR2), SERCA2A and L‐type Calcium Channel (LTCC), and an increased expression of protein kinase A (PKA), Na‐Ca exchanger (NCX) and phospholamban (PLN). A previously established rat model of BPD was used, where BPD rats were exposed to 85% O2 for 14 days and controls were exposed to 21% O2 for 14 days. 78 rat hearts in total were harvested during the study at p0, p7, p14, p21, p28, p56, p90, p180. Relative protein expression differences were determined using Western Blots with antibodies against key Ca2+ signaling proteins in control and BPD RV homogenate.
The Role of MiR‐21 in Right Ventricular Remodeling Secondary to Pulmonary Arterial Pressure Overload
Authors: Chen Michael , Chang Weiting , Mohebali Jahan , Felder Amy , Laurence Rita , Yang Yanfei , Qiu Yiling , Noonan Madyson , Chen Frederick , Liao Ronglih
Published: June 2021

Pulmonary arterial hypertension (PAH) prognosis is related to the degree of right ventricular (RV) dysfunction. While the effects of pulmonary pressure overload on RV remodeling are largely unknown, microRNA‐21 (miR‐21) has been identified as a factor involved in PAH vasculature remodeling and cellular hypertrophy. The goal of this study is to characterize regional structural and functional change in RV after PAH and identify the role of miR‐21. Pulmonary arterial constriction (PAC) induced PAH in 5‐month‐old adult male sheep. Longitudinal echocardiography was used to monitor functional and structural changes. After four weeks, hearts were harvested for histological and molecular examination. Isolated neonatal rat ventricular myocytes (NRVM) were used to define the role of miR‐21 in cardiomyocytes (CM). Elevated PA pressure led to increased RV weight and thickness in PAC sheep. Decreased systolic function was most profound in the RV basal region. Following changes in regional function, miR‐21 was also differentially upregulated in the basal region as determined by RT‐PCR. Interestingly, NRVM treated with a miR‐21 mimic increased mitosis but not cytokinesis compared to controls. Congruently, RV basal CM isolated from PAC animals revealed increased elongated multinuclear cells, supporting the notion of RV pressure overload‐induced hypertrophy. We conclude that PAC led to regional alterations and contributes to adverse global functional changes. The differential upregulation of miR‐21 in basal and altered RV CM remodeling may indicate PAC‐induced regional and global structural and functional RV remodeling.
Immunohistological and Morphological Analysis from a Skeletal Muscle Microbiopsy
Authors: Townsend Jeremy , Fragala Maren , Oliveira Leonardo , Jajtner Adam , Gonzalez Adam , Wells Adam , Stout Jeffrey , Fukuda David , Hoffman Jay
Published: June 2021

The study of skeletal muscle often requires invasive muscle biopsies, traditionally with the Bergstrom technique. Microbiopsies, or fine‐needle biopsies are used with many biochemistry applications but are scarcely used for histology due to inconsistencies with sample weight and integrity. Thus, the aim of the current study was to develop procedures for muscle fiber morphology and immunohistological analysis from a microbiopsy technique.

METHODS

1,366 muscle fibers were analyzed from 8 muscle biopsy samples obtained from four healthy men (26.8 ± 5.4y, 175.9 ± 4.3 cm, 91.9 ± 5.3kg). Percutaneous microbiopsies were taken of the vastus lateralis with a 14‐gauge microbiopsy needle on two separate occasions. Samples were removed from the needle and promptly weighed. The tissue was oriented in a cryomold, embedded in Tissue‐Tek® then frozen in liquid nitrogen cooled isopentane. The muscle sections were stained with haematoxylin and eosin or anti‐laminin and counterstained with AlexaFlour 488® and viewed under 4x magnification. Fiber number and mean fiber area was determined by Image J.

RESULTS

The mean ± SD (range) microbiopsy sample weight was 18.3 ± 2.9 mg (14‐22mg). Fiber number within the microbiopsy specimens was 150.4 ± 120.6 (64–366). All viable fibers were measured in each sample and the mean fiber area was 4385.1 ± 1265.8 µm2 (977.0 – 10,132.93 µm2). The intra‐subject coefficient of variation for mean fiber area was 7.2%.

CONCLUSION

Results show the potential of a minimally invasive muscle biopsy technique for use in immunohistological and morphological analysis. This could allow future advances in the study of skeletal muscle by allowing for smaller muscle groups to be examined.
Acute Whole‐Leg Pneumatic Compression Alters Nerve Growth Factor mRNA and Akt‐mTOR Signaling in Human Skeletal Muscle
Authors: Kephart Wesley , Mobley Christopher , Fox Carlton , Santucci Vincent , Wilson Trent , Goodlett Michael , Sefton Joellen , Pascoe Dave , Kavazis Andreas , Roberts Michael , Martin Jeffrey
Published: June 2021

We sought to investigate recovery markers following a single bout of whole‐leg external pneumatic compression (EPC). Post‐prandial (> 4 hours) and rested participants (N=12) were treated with 1‐h of whole‐leg EPC using a peristaltic pulse dynamic compression device with target inflation pressures of ~70 mmHg. Vastus lateralis muscle biopsies were obtained prior to, 1 h and 4 h following EPC. Samples were analyzed for select mRNA markers of angiogenesis, growth factors, metabolic, and pro‐inflammatory markers as well as various phospho (p)‐proteins related to Akt‐mTOR, Jak‐Stat, and apoptosis signaling. Nerve growth factor (NGF) mRNA exhibited a trend towards up‐regulation from pre to 4 h post compression (p = 0.06), though there was an up‐regulation from 1 h to 4 h post‐EPC (p = 0.04). From pre to 1 h post‐EPC there was a decrease in p‐Bad (Ser112) (p = 0.05) which was still significantly decreased 4 h post‐EPC (p=0.004) phospho‐Stat1 (Tyr701) decreased from 1 h to 4 h post‐EPC (p = 0.03). P‐Akt (Ser473) also significantly decreased from 1 h to 4 h post‐EPC (p = 0.04). P‐ribosomal protein S6 (Ser235/236) was transiently increased 1 h post‐EPC (p = 0.02). P‐p70s6k (Thr389) was decreased 4 h post‐EPC (p = 0.02). P‐proline rich protein‐40 (Thr246) was also significantly decreased 4 h post‐EPC (p = 0.02). These data indicate that an acute bout of EPC up regulates NGF mRNA as well as affecting Akt‐mTOR signaling substrates. Further investigations should employ exercise‐related stimuli in conjunction with EPC to investigate potential recovery‐enhancing effects.
Defective EC coupling is partially responsible for impaired contractibility in hindlimb muscle of Stac3 knockout mice
Authors: Cong Xiaofei , Doering Jonathan , Grange Robert , Jiang Honglin
Published: June 2021

The SH3 and cysteine rich domain 3 (Stac3) gene was recently found to be exclusively expressed in skeletal muscle and essential for skeletal muscle contraction and postnatal life in mice. Data from Stac3 mutant diaphragm suggested that impaired muscle contractility was due to defective EC coupling. In this study, we compared the contractility of hindlimb muscle of embryonic day 18.5 Stac3 knockout and control (heterozygous) mice. Twitch responses were evident in control but undetectable in Stac3 mutant muscle. In response to frequent electrical stimulation, the maximum tension generated in Stac3 mutant muscle was ~20% of control. High [K+]‐induced maximum tension in Stac3 mutant muscle was ~29% of control. The maximum tensions induced by caffeine and 4‐chloro‐m‐cresol in Stac3 mutant muscle were ~55% and ~58% of control, respectively. These data confirm a role for Stac3 in EC coupling, but also suggest that defective EC coupling is only partially responsible for impaired contractility in Stac3 mutant muscle. Myosin‐ATPase and NADH‐tetrazolium reductase staining revealed that hindlimb muscle of Stac3 mutant mice contained 60% more slow fiber‐like fibers than control. qPCR analyses revealed that fast muscle‐prevalent mRNAs were less abundant in Stac3 mutant muscle than in control. These and previously discovered structural abnormalities (centrally located myonuclei and streaming Z lines) in Stac3 mutant muscle further suggest that Stac3 may regulate skeletal muscle contraction through mechanisms in addition to mediating EC coupling.
Prolonged Diaphragm Weakness in Acute Lung Injury UL1TR000117, HL112085, HL113494
Authors: Callahan Leigh Ann , Song Xiao‐Hong , Wang Lin , Supinski Gerald
Published: June 2021

Acquired skeletal muscle weakness, a devastating complication of critical illness, affects both limb and respiratory muscles. Diaphragm weakness is especially important, persisting for days to weeks, prolonging duration of mechanical ventilation and increasing in‐hospital mortality. Currently no animal model of critical illness replicates the sustained diaphragm weakness observed in patients. We sought to establish an animal model that parallels the severity and duration of diaphragm weakness observed in critically ill patients using male C57BL6 mice (n=50) treated with a single intratracheal injection of lipopolysaccharide (IT‐LPS) which induces acute lung injury (ALI). We first measured diaphragm specific force in mice treated with varying doses of IT‐LPS at several time points to determine the dose that would produce sustained diaphragm injury. We then examined diaphragm force‐frequency relationships in mice treated with 30,000 EU/gm of IT‐LPS. ALI invoked significant loss in animal body weight and hypothermia. In addition, ALI markedly reduced diaphragm specific force generation which persisted at Day 10 (150 Hz force averaged 26.4±0.5,17.7±1.7, 15.6±1.9, 15.9±1.8, 15.7±12.1, and 16.7±2.1 N/cm2 for controls, Day 1, Day 2, Day 4, Day 7, and Day 10 groups, respectively, p<0.001). This is the first study to show that a clinically relevant animal model of critical illness, acute lung injury, induces diaphragm weakness in a pattern and time course that parallels data obtained from human studies. We speculate that this model may prove useful for determining the mechanistic basis for prolonged respiratory muscle weakness as well as the pathways required for recovery of diaphragm function in critically ill patients.
Characterization of a Kbtbd13‐Deficient Mouse Model That Recapitulates Nemaline Myopathy With Mutations in The KBTBD13 Gene
Authors: de Winter Josine , Joureau Barbara , Stienen Ger , Granzier Henk , Ottenheijm Coen
Published: June 2021

Background

Nemaline myopathy (NM) is the most common non‐dystrophic congenital myopathy and is characterized by muscle weakness. Recently a novel implicated gene was discovered ‐ KBTBD13 – of which the function of its protein product is unknown. NM patients with KBTBD13 mutations have muscle weakness and show a typical slowness of movement. Here, we generated a mouse model that lacks the kbtbd13 gene to obtain insight into the role of this new gene and its protein product and to elucidate the pathophysiology of this new form of NM.

Methods

Kbtbd13‐KO mice are viable and live into adulthood. Muscle weights of cardiac and skeletal muscle were collected. Intact soleus muscles of 3 month‐old male kbtbd13‐KO and wild‐type (WT) mice were used for in vitro experiments. Muscle tendons were attached to a force transducer and a length motor. Muscles were electrically activated by field stimulation at 30°C.

Results

Maximal tetanic force at 150Hz was reduced in kbtbd13‐KO (n=10). The relative force‐frequency relationship in kbtbd13‐KO mice was shifted to the left compared to WT mice. Both activation and relaxation kinetics were slower in kbtbd13‐KO mice. Muscle weight data reveal atrophy of skeletal muscles, whereas the cardiac muscle shows hypertrophy of the left ventricle.

Conclusion

Kbtbd13‐KO mice phenocopy the normal life span and typical muscle weakness observed in NM patients with mutations in KBTBD13. Hence, we present a novel NM mouse model that is a useful tool to elucidate the role of this new gene and its protein product and to study the mechanism underlying muscle weakness observed in patients.
Atorvastatin‐Attenuates Mechanical Stretch‐Induced Vascular Smooth Muscle Remodeling
Authors: Ghantous Crystal , Soudani Nadia , Farhat Zein , Zeidan Asad
Published: June 2021

Mechanical stretch‐sensitive protein synthesis in vascular smooth muscle (VSMC) is the basis of hypertensive vascular disease. Its mechanisms involve activation of different signaling pathways including Rho/ROCK, MAPK, actin cytoskeleton remolding and production of leptin. However the detailed signaling pathways are still not clear. Recent studies have linked leptin to cardiovascular disease such as hypertension and vascular hypertrophy. In the present study, we used rat portal vein (RPV) organ culture to investigate whether atorvastatin (12 mM) can ameliorate the effect of mechanical stretch on leptin synthesis, the reactive oxygen species (ROS) production, ERK1/2 and SRF activation. RPV were either unstretched (control) or stretched by weight at different time intervals (0, 15, 30, 60 min and 24 hr). Protein expression was assessed using Western blotting and immunohistochemistry methods. Mechanical stretch significantly up‐regulated leptin's expression, ERK1/2, RhoA and cofilin phosphorylation. Atrovastatin significantly attenuated mechanical stretch‐induced leptin expression, ERK1/2 and cofilin phosphorylation and ROS production. Exposing the RPV to mechanical stretch for 1h significantly increased nuclear translocation of the transcription factor SRF which was attenuated by the co‐administration of an anti‐leptin receptor antibody and the atrovastatin. Moreover, mechanical stretch‐induced VSMC hypertrophy and protein synthesis were inhibited by pre‐treatment with atorvastatin.Our results indicate that the therapeutic effect the antihypertensive drug “atrovastatin” in attenuating vascular remodeling might be through the inhibition of leptin and ROS production, ERK1/2, SRF and cofilin phosphorylation.
Selective interaction of CaMKIIδ2 and Fyn mediates vascular smooth muscle (VSM) cell motility
Authors: Ginnan Roman , Zou Xiaojing , Singer Harold
Published: June 2021

Our previous study demonstrated that CaMKIIδ2 and Fyn physically interact via CaMKIIδ2's C ‐terminal “tail”. We also reported that this interaction between CaMKIIδ2 and Fyn positively mediates VSM cell motility. The purpose of this study is to better understand the mechanistic relationship between CaMKIIδ2 and Fyn and to clarify their cooperative role in migrating VSM cells. With immunofluorescence, we show that SiRNA depletion of CaMKIIδ in VSM cells attenuated PDGF‐dependent translocation of Fyn to peripheral membranes. Similarly, overexpression of CaMKIIδ6, which does not interact with Fyn, also reduced Fyn's ability to translocate to the plasma membrane in response to PDGF. Furthermore, overexpression of CaMKIIδ6 reduced VSM cell motility in both a scratch wound assay and a transwell assay. The rate of focal adhesion protein recruitment to focal adhesions is directly linked to cell motility. Using TIRF/FRAP in cells overexpressing GFP‐tagged Paxillin, we show that SiRNA mediated loss of CaMKIIδ2 or Fyn disrupted the kinetics of focal adhesion turnover. Stimulation of VSM cells with PDGF results in tyrosine phosphorylation of P130Cas and Paxillin, both known substrates of Fyn. Along with preventing Fyn translocation to the plasma membrane, overexpression of CaMKIIδ6 inhibited this PDGF‐dependent phosphorylation of P130Cas and Paxillin. Therefore, we conclude that CaMKIIδ2 “delivers” Fyn to peripheral membranes proximal to focal adhesions where Fyn modulates focal adhesion dynamics and subsequent VSM cell motility.
Reduced Common Carotid Artery Blood Flow in Spontaneously Hypertensive Rats is Unaffected by Aging and/or Chronic In Vivo Resveratrol Treatment
Authors: Denniss Steven , Smith Christopher , Mariani Rachelle , Wood Laura , Rush James
Published: June 2021

This study examined common carotid artery (CCA) blood flow (using a perivascular flowprobe) and pressure (using a catheter‐tip transducer) in anesthetized Adult (20‐24 wk old) and Aging (60‐70 wk old) Wistar Kyoto rats (WKY) and Spontaneously Hypertensive rats (SHR) following 28‐days of in vivo treatment (in drinking water, ab libitum) with either no trans resveratrol (RSV; No‐RSV), a moderate RSV dose (Mod‐RSV; ~0.075 mg/kg/day; to mimic daily red wine consumption), or a high RSV dose (High‐RSV; ~7.5 mg/kg/day). CCA mean, maximal systolic, minimum diastolic, and pulse pressure, and heart rate, were all significantly elevated in SHR vs. WKY (p<0.01). Aging was associated with a significant increase in pulse pressure in WKY (p<0.01). CCA mean, maximal systolic, and minimum diastolic blood flow, and vascular conductance, were all significantly reduced in SHR vs. WKY (p<0.05). Aging did not have a significant effect on the flow parameters in either SHR or WKY. Neither Mod‐RSV nor High‐RSV treatment had a significant effect on the CCA blood pressure and blood flow variables measured. These data demonstrate that the reduction in CCA blood flow found in SHR is unaffected by aging or chronic in vivo RSV treatment. Further investigation into what can account for and alter the reduced CCA blood flow found in SHR is warranted.

Funded by NSERC.
The Role of ATP and P2X7 in Upregulation of Vasocontractile ET‐B Receptors in Basilar Arteries
Authors: Haanes Kristian , Edvinsson Lars
Published: June 2021

Cerebrovascular disease is one of the main causes of death in the developed countries. Several G‐protein coupled receptors, such as the Endothelin‐B receptor (ET‐B), are upregulated after a stroke. These receptors cause constriction in the blood vessels and reduce blood flow, causing further damage and reduced chances of recovery. Our group has previously shown that MEK/ERK1/2 inhibition greatly reduced the receptor upregulation and the cerebral damage. Extracellular ATP is a short‐range signal important in the regulation of both the blood flow and also in long term cellular modifications. One of the receptors for ATP, the P2X7 receptor, is of high interest, as it can be involved in apoptosis, proliferation, and ERK1/2 signaling. We hypothesized that ATP could be an important activator of MEK/ERK1/2 and contribute to the upregulation of contractile receptors. Ex vivo ET‐B receptor upregulation in basilar arteries (assessed by the specific agonist sarafotoxin 6c (S6c) in a myograph) can be induced by incubation of artery segments in DMEM media for 48 hours (organ culture). The changes are similar to those observed in vivo in experimental stroke. Surprisingly, adding apyrase (an ATP degrading enzyme) further increased the contraction induced by S6c and was nearly mimicked by the specific P2X7 antagonist Az10606120. The addition of a P2X7 agonist (10µM BzATP) during organ culture had little effect, indicating that the lower ATP concentrations are the important stimuli. In conclusion, ATP and P2X7 appear to be important in the upregulation of ET‐B receptors during organ culture and play a beneficial role because inhibiting them causes an increased upregulation of contractile ET‐B receptors.

Funding: Lundbeck foundation (R167‐2013‐15379) and Lundbeck Grant of excellence
Structural Studies on Glutamate Receptor Gating
Authors: Mayer Mark , Meyerson Joel , Chittori Sagar , Subramaniam Sriram
Published: June 2021

A combination of structural, biophysical and functional assays, pursued over a 30 year period of research, has culminated in a gating mechanism for glutamate receptor ion channels. They key advance which led to mechanistic insight was the use of single particle cryo‐EM techniques to study the structure of individual receptor assemblies trapped in well defined conformations using appropriate combinations of high affinity ligands. At the current time we understand the broad principles, and large scale domain movements, underlying activation, desensitization, and allosteric modulation for AMPA and kainate receptors. Due to the availability of crystal structures at resolutions as high as 1.3 Å, the chemistry of ligand selectivity and in some cases the mechanism of allosteric regulation by drugs and modulatory anions and cations is understood in great detail. By contrast, the details of ion permeation remain poorly understood, due to the low resolution of ion channel domains in the receptor assembly.
Dynamic imaging of compartmentalised intracellular free Zn2+ concentrations in rat ventricular cardiomyocytes
Authors: TUNCAY Erkan , CHABOSSEAU Pauline , LYON Alexander , TURAN Belma , RUTTER Guy
Published: June 2021

Several Ca2+ binding proteins in cardiomyocytes bind Zn2+ suggesting that Zn2+ can modulate the structure and function of many of these. Consequently, Zn2+ may play an important role in excitation‐contraction coupling. The total intracellular free Zn2+ content is comparable to that of Ca2+ and it has been shown that about 70% of the cellular Zn2+ appears to be compartmentalized in organelles. However, very little is known about the mechanisms that control the cellular distribution of Zn2+ and/or its role in cardiac function. Newly‐developed genetically‐encoded FRET‐based sensors (eCALWYs) are a useful tool for monitoring intracellular and compartmentalized free Zn2+. In this study, we measured cytosolic (CYS), mitochondrial (MitC) and endoplasmic reticulum (ER) free Zn2+ by using genetically‐encoded Zn2+ sensitive probes bearing suitable motifs to address sensors with a range of Zn2+ affinities to the ER‐ or mitochondrial matrix. Primary rat cardiomyocytes were infected with adenoviruses expressing CYS‐, MitC‐, or ER‐targetted eCALWY probes for 24 hours, and free Zn2+ levels subsequently monitored by digital imaging. Probe localisation to the appropriate sub‐cellular locale was confirmed by immunocytochemical analysis. We observed that ER free Zn2+ levels were markedly higher than cytosolic and mitochondrial levels. In summary, this approach reveals significant differences in free Zn2+ concentrations among different cellular compartments in cardiomyocytes. (Supported by grants of TUBITAK SBAG‐113S466 & IDRFP‐2214)
Wireless power source for implantable telemetry devices
Authors: Davtyan Armen , Pitsillides Koullis
Published: June 2021

Recent advancements in biotelemetry systems allow measurements of blood flow, blood pressure, ECG and temperature in small animals such as rats (EG3‐Vx‐Sx‐Mx, Transonic EndoGear Inc) with minimal intrusion into their environment. However, these implants cannot be used continuously for extended periods of time, because they use finite capacity batteries. Different size batteries maybe used for different size animals, which also dictates the maximum duration of measurements. To provide telemetry monitoring over extended periods of time, we developed Wireless Power Supply (WPS) that uses inductive power technology to send power to an implanted 3‐dimentional Wireless Power Receiver (WPR) that is used instead of the battery. This WPR can provide continuous power to a telemetry system used in small animals such as rats. The WPS uses a self‐tuning design that adjusts for maximum power delivery even when multiple animals are present in the same cage. This is also possible due to the self‐regulation of the WPR that does not depend on back‐telemetry and regulation of the WPS. These advancements make it possible to have two, or more instrumented animals in the same cage. The WPR receives a maximum of 20mA at 3.5V up to a distance of 15cm, in all 3 dimensions, in a standard size rat cage. This is more than enough to power a telemetry system with the capability to measure advanced cardiovascular parameters such as blood flow, blood pressure, ECG and temperature.
Circulating MicroRNAs Characterize Insufficient Coronary Collateral Artery Development in Patients with Chronic Total Occlusion
Authors: Hakimzadeh Nazanin , Nossent A Yaël , van der Laan Anja , Schirmer Stephan , van Royen Niels , Quax Paul , Hoefer Imo , Piek Jan
Published: June 2021

Coronary collateral artery growth, arteriogenesis, significantly impacts mortality after an acute coronary event. We aimed to identify microRNAs (miRNAs) that differentiate between insufficient or sufficient coronary collateral artery development in patients with chronic total occlusion (CTO).

CTO patients (n=41) were dichotomized based on their pressure‐derived collateral flow index (CFI = (wedge pressure – central venous pressure)/(aortic pressure – central venous pressure)) into low (CFI< 0.39) and high arteriogenic responders (CFI > 0.39). MiRNA profiling on aortic plasma and verification by qPCR, revealed higher levels of miR423‐5p (p < 0.05), miR10b (p < 0.05), miR30d (p < 0.05) and miR126 (p < 0.001) were present in low arteriogenic responders. Receiver operator characteristic curves showed that only miR126 can distinguish patients with low arteriogenic response (area under curve 0.812, p < 0.01, cut‐off 0.037).

In conclusion, we have identified miRNAs linked with poor collateralization and found miR126 as the most suitable circulating biomarker to distinguish patients with poor or adequate coronary artery collateral capacity.

Supported by the Center for Translational Molecular Medicine (EMINENCE).
Release of microparticles from native coronary arteries and saphenous vein grafts in patients with elective stent implantation under protection with an aspiration device
Authors: Kleinbongard Petra , Baars Theodor , Horn Patrick , Kahlert Philipp , Heiss Christian , Westenfeld Ralf , Kelm Malte , Erbel Raimund , Heusch Gerd
Published: June 2021

Objective

Stent implantation into coronary vessels impacts on downstream microvascular function and induces the release of particulate debris and soluble substances, which differs between native right coronary arteries (RCAs) and saphenous vein grafts on right coronary arteries (SVGRCAs). We have now quantified the release of microparticles (MPs) during stent implantation into RCAs and SVG‐RCAs.

Methods

Patients with a stenosis in their RCA or SVG‐RCA, respectively (n = 14/14), were enrolled. Coronary aspirate was retrieved during stent implantation with a distal occlusion/aspiration device. Platelet‐derived MPs (PMPs) were distinguished by flow cytometry as CD41+, endothelium‐derived MPs (EMPs) as CD144+, CD62E+ and CD31+/CD41‐, leukocyte‐derived MPs as CD45+, and erythrocyte‐derived MPs as CD235+.

Results

Intracoronary PMPs and EMPs were increased after stent implantation into their RCAs and SVG‐RCAs (CD41+: 2729.6±645.6 vs. 4208.7±679.4 and 2355.9±503.9 vs. 3285.8±733.2 nr/μL; CD144+: 451.5±87.9 vs. 861.7±147.0 and 444.6±74.8 vs. 726.5±136.4 nr/μL; CD62E+: 1404.1±247.7 vs. 1844.3±378.6 and 1084.6±211.0 vs. 1783.8±384.3 nr/μL, P<0.05), but not different between RCAs and SVG‐RCAs.

Conclusion

Stenting in RCAs and SVG‐RCAs is associated with a substantial release not only of PMPs, but also of EMPs.