bluuebunny/crossref_metadata_2025 huggingface dataset:
Appropriate Cardiac Anatomy
Authors: Mori Shumpei
Published: April 2020

Various approaches, including inspection, palpation, percussion, auscultation, chest radiography, electrocardiography, echocardiography, computed tomography, cardiac magnetic resonance imaging, and other techniques for cardiac imaging, are the methodologies used to assess the inside of the heart from outside the body. It follows that exact knowledge of intrinsic cardiac anatomy is fundamental for accurate diagnosis. In addition, any kind of invasive cardiac procedure, requires an exact knowledge of cardiac anatomy so as to ensure safety of the investigation. In the light of the significant advances made in the resolution of the techniques for imaging now used by modern‐day cardiologists, far more detailed anatomical knowledge is required so as to obtain full value from the investigations. A basic knowledge of cardiac anatomy is initially achieved in the dissection lab. However, once the heart is removed from the donor, it is often oriented and studied in an attitudinally inappropriate manner when compared to its in‐situ orientation. This can lead to confusion and an incorrect understanding of the true location of cardiac components. For the purpose of not only maintaining, but improving the quality of anatomical knowledge, techniques based on modern cardiac imaging can provide a supplement to the dissection experience. In clinical settings, cardiac computed tomography can now provide ideal spatial resolution, at levels equal to or less than 0.5 mm. This permits the evaluation of the arrangement of even thin structures such as the valvar leaflets. Currently, by using datasets obtained from routine clinical scanning, all cardiologists have the ability to provide in‐depth three‐dimensional knowledge of intrinsic cardiac anatomy. This is achieved by interrogation of three‐dimensional reconstructions of multi‐planar two‐dimensional images, thus replicating real cardiac dissection. By means of such “virtual dissection”, the cardiac components can be shown in the blood‐filled living situation. By combining the reconstructed images, the anatomy of intrinsic features can be shown in relation to the pertinent surrounding extracardiac structures. Sections can be repeatedly acquired, using any desired plane viewed from multiple directions. Such “virtual dissection” is obviously performed without extracting the heart from the thorax, presenting an obvious advantage over the traditional cadaveric approach. All the analyses, furthermore, are performed in attitudinally appropriate fashion, without losing any of the coordinates relative to the components of the thoracic cage. Despite the obvious values of “virtual dissection”, it does not replace real dissection. It is the accurate knowledge based on real dissection that provides the basis for proper evaluation of the virtual images. This means that anatomists must themselves keep abreast of the advances made in modern clinical imaging. An appropriate collaborative approach between anatomists and cardiologists will self‐evidently continue to improve the educational values of the understanding of cardiac anatomy.
Hybrid Rat Diversity Program (HRDP): A rat resource for mapping complex traits
Authors: Dwinell Melinda R , Takizawa Akiko , Lazcares Lynn , Schilling Rebecca , Hoffman Matthew , Geurts Aron , Shimoyama Mary
Published: April 2020

Rats are a major model for studying complex disease mechanisms, behavioral phenotypes, environmental factors, and for drug development and discovery. Inbred rat strains control for genetic background and allow for repeated, reproducible molecular, cellular, and whole animal phenotyping. Genetic susceptibility to disease, sensitivity to environmental elements, and pharmacogenomics are critical components of the concepts of precision medicine. A resource that combines the power of genetic stability within strains, power for genomic mapping strategies, and strength in an animal model that mirrors many human disease traits is the goal of this project. Through the development of the Hybrid Rat Diversity Program (HRDP), a powerful panel of inbred rats with genomic, physiologic, and behavioral phenotype data is beginning to serve as a catalyst for research in many fields. When complete, the HRDP will consist of 96 inbred rat strains to maximize power to detect specific genetic loci associated with complex traits while maximizing the genetic diversity among strains. The 96 strain panel consists of 33 genetically diverse inbred strains and two panels of recombinant inbred panels: FEXL/LEXF (33 strains, Japan) and HXB/BXH (30 strains, Czech Republic). Embryo resuscitation and breeding is well underway at the Medical College of Wisconsin (MCW). Whole genome sequencing for 32 of the 96 strains is complete, having been generated on am Illumina NovaSeq S4 to achieve 30X coverage. Liver and brain transcriptome analysis is underway through PhenoGen (
http://phenogen.ucdenver.edu) at the University of Colorado. Genomic, phenotype, and strain information will be made available through the Hybrid Rat Diversity Panel portal at the Rat Genome Database (
http://rgd.mcw.edu). The HRDP will provide a genetically stable population of rat strains with fully sequenced genomes, transcriptomes for brain and liver, and general phenotypic characterization to be used for systems genetic studies and fine mapping of complex traits.

Support or Funding Information

National Institutes of Health, Office of the Director, R24OD024617
Assessing CMPI potentiation of (α4)3(β2)2 nicotinic acetylcholine receptor using the Monod‐Wyman‐Changeux allosteric model.
Authors: Deba Farah , Akk Gustav , Hamouda Ayman K.
Published: April 2020

Positive allosteric modulators (PAMs) of neuronal nicotinic acetylcholine receptors (nAChRs) enhance ACh potency and/or efficacy by binding at site(s) distinct from the ACh, i.e., orthosteric binding sites. Previous work has established CMPI (3‐(2‐chlorophenyl)‐5‐(5‐methyl‐1‐(piperidin‐4‐yl)‐1H‐pyrrazol‐4‐yl)isoxazole as a nAChR PAM that selectively potentiates (α4)3(β2)2 nAChR. In (α4)3(β2)2 nAChR, co‐application of CMPI maximally potentiated responses to ACh EC10 by ~400% with potentiation EC50 of ~0.3 μM. CMPI produced a left‐shift of the ACh concentration‐response curve of (α4)3(β2)2 nAChR enhancing ACh potency at (α4)3(β2)2 nAChRs by ~100 fold with no significant effect on the ACh maximal response. Mutational and computational analyses have identified a CMPI binding site that partially overlaps the ACh binding site (ABS) at the α4:α4 subunit extracellular interface which exists only in the (α4)3(β2)2 nAChR. Here, we are characterizing CMPI potentiation of 4 classes of (α4)3(β2)2 nAChR agonists: 1) a full agonist binding at α4:α4 ABS (ACh); 2) a full agonist not binding at the α4:α4 ABS (5I‐A85380); 3) partial agonists binding at the α4:α4 ABS such as cytisine; and 4) partial agonists not binding at the α4:α4 ABS such as TC‐2559. CMPI potentiation of nicotine, cytisine, and 5‐A855380 was similar to that for ACh (potentiated responses to low but not saturating agonist concentrations enhancing the potency but not the efficacy of these agonists). In contrast, CMPI potentiated responses to low and high concentration (up to 30 μM) of TC‐25559 enhancing its efficacy. However, in (α4)3(β2)2 nAChR containing alanine substitution at α4H116 which allows TC‐2559 to bind at α4:α4 ABS, CMPI enhanced potency but not the efficacy of TC‐2559‐induced currents. Analysis of the findings in the framework of the Monod‐Wyman‐Changeux model indicates that CMPI acts at the α‐α interface with a gating efficacy that is equivalent to that of ACh but higher than the gating efficacy of cytisine at this site. At the α4H116A mutated α‐α interface, TC‐2559 has a higher efficacy than ACh or CMPI. The precise nature of the modulatory effect is thus multifactorial, dependent on the concentrations and nature of the combined agonists.

Support or Funding Information

National Institutes of Health/National Institute of Neurological Disorders and Stroke [Grant NS‐093590] (A.K.H).
4‐HNE Unfavorably Alters Muscle Mitochondrial Bioenergetics and Cell Viability
Authors: Jacobsen Samuel , Walton Chase , Saito Erin , Hansen Jason M. , Bikman Benjamin T.
Published: April 2020

Linoleic acid consumption has increased by orders of magnitude over the past century, eliciting an increasing awareness of and appreciation for its metabolites, particularly 4‐hydroxynonenal (4‐HNE). Elevated 4‐HNE formation is causally implicated in myriad neurodegenerative, cardiovascular, autoimmune, and metabolic disorders. The purpose of this project was to identify the effect of 4‐HNE on muscle cell mitochondrial bioenergetics. Our primary observation was that physiological levels of 4‐HNE compromised muscle cell mitochondrial oxygen consumption, leading to reduced oxygen flux despite a substantial production of reactive oxygen species (ROS). Indeed, when viewed in light of the reduced oxygen consumption, the relative production of ROS with 4‐HNE was even more pronounced. These findings could explain the significant reduction of cell viability demonstrated. In conclusion, this work joins the growing body of evidence suggesting a need to scrutinize dietary linoleic acid consumption.
Defining the Molecular Basis of KIR Channel Mechanosensitivity in Vascular Smooth Muscle
Authors: Fletcher Jacob , Welsh Donald G.
Published: April 2020

In cerebral arteries, the inwardly rectifying potassium channel (KIR) contributes to the setting of membrane potential and development of myogenic tone. KIR channels have been recently identified as a target of mechanical regulation in vascular smooth muscle, by which their activity is suppressed by pressure. However, the mechanism underlying this process remains unknown. The actin cytoskeleton and caveolae are often part of signaling domains implicated in established models of mechanosensitivity. Consequently, this study investigated whether these distinct structural components enable KIR pressure sensing in rat cerebral arterial smooth muscle. Whole‐cell patch clamp electrophysiology was used to monitor KIR activity and initial experiments confirmed that this current was suppressed by mechanical stimulation. Pre‐treatment of cells with actin‐disrupting agents, Latrunculin A and Cytochalasin D, prevented the suppression of KIR, indicative of the cytoskeleton’s participation. As KIR channels reside in caveolae, we next assessed the involvement of caveolin proteins in mechanoregulation. As expected, inhibition of caveolin‐1 signaling with blocking peptides diminished the ability of KIR channels to respond to pressure. The current data highlights that the actin cytoskeleton and caveolae form key parts of a signal transduction structure that confers mechanosensitivity to KIR. Ongoing work seeks to identify other KIR‐protein interactions in cerebral arterial smooth muscle using pull‐down and proximity ligation assay techniques. The functional impact of signaling complex disruption will also be assessed with vessel myography.

Support or Funding Information

Research supported by the Canadian Institutes of Health Research.
Regulation of presynaptic CB1 receptors in persistent inflammation
Authors: Bouchet Courtney A. , Suchland Katherine L. , Ingram Susan L.
Published: April 2020

Opioids are the golden‐standard for pain treatment and therapeutics targeting the cannabinoid system are on the rise; yet the molecular mechanisms by which these systems are regulated is not fully understood. Opioids and cannabinoids can elicit analgesia through activation of mu‐opioid receptors (MOR) or cannabinoid 1 (CB1) receptors, respectively, in the ventrolateral region of the periaqueductal gray (vlPAG). Both receptors suppress GABA release from the presynaptic terminal under basal conditions. Our studies examine how persistent inflammation affects presynaptic MOR and CB1 receptor signaling in the vlPAG. Adult male and female Sprague Dawley rats (postnatal day 30–60) are naïve or injected with Complete Freund’s Adjuvent (CFA) into one hindpaw to induce inflammation for 5–7 days. Using electrophysiology to measure miniature inhibitory postsynaptic currents (mIPSCs), we find that CB1‐mediated inhibition of GABA mIPSC frequency is significantly reduced in inflamed rats compared to naïve rats. MOR‐mediated inhibition of mIPSC frequency is not altered. This effect is consistent in both males and females. There are no effects of inflammation on mIPSC amplitude. Previous work has shown that neuropathic pain, chronic THC exposure, or persistent inflammation reduces CB1 receptor protein levels while mRNA levels are unchanged suggesting that CB1 receptors may be regulated via local protein synthesis. We find that protein synthesis inhibitors, anisomycin (20 μM) or cyclohexamide (80 μM) reduce presynaptic CB1 receptor‐mediated inhibitionof GABA release in the vlPAG but do not affect presynaptic MOR function. These results provide evidence that presynaptic CB1 and MOR are regulated differentially under basal conditions and in persistent inflammation. These data have important implications for the future treatment of pain with pharmaceuticals targeted at either CB1 or MOR receptors.

Support or Funding Information

NIDA T32 Training Grant; NIH NIDA R01DA042565
HSP27 Mediates PAR1‐stimulated Pro‐inflammatory Vascular Responses
Authors: Rada Cara
Published: April 2020

Endothelial cells line the lumen of blood vessels and create a semi‐permeable barrier that is disrupted during vascular inflammation resulting in increased permeability, tissue edema, and subsequent organ failure. Endothelial dysfunction also results in cytokine production and leukocyte recruitment. However, the molecular mechanisms responsible for the pro‐inflammatory state remains to be elucidated. Protease‐activated receptor‐1 (PAR1) is a G protein‐coupled receptor for the coagulant protease thrombin and mediates hemostasis, thrombosis, and inflammatory response to vascular injury. However, the distinct mechanism of how endothelial PAR1 promotes vascular pro‐inflammatory responses remains poorly understood. We recently discovered that thrombin activation of PAR1 stimulates p38 activation through a non‐canonical pathway mediated by TAB1 to promote endothelial permeability. PAR1‐induced p38 activation has been linked to vascular barrier disruption; however, the mechanism by which p38 signaling contributes to pro‐inflammatory responses is not known. We found through a phosphoproteomics screen that thrombin‐stimulated p38 signaling relies on phosphorylation of heat shock protein 27 (HSP27). Moreover, we found that this pathway is physiologically relevant by showing PAR1‐activated HSP27 modulates the closing of the endothelial barrier in vitro and vascular leakage in vivo. We show HSP27 activity is both p38‐ and PAR1‐dependent. Finally, we show PAR1‐induced p38 signals to HSP27 through two p38 downstream kinases, MAPKAPK2 (MK2) and MAPKAPK3 (MK3), to directly phosphorylate HSP27 on specific serines. Together these studies reveal a novel pathway in which thrombin‐induced p38 signals through MK2 and MK3 to regulate HSP27 activity leading to recovery of the endothelial barrier and promoting resolution of pro‐inflammatory responses.

Support or Funding Information

T32 Pharmacological Sciences Training Grant (NIGMS T32 GM007752) Tobacco Related Disease Research Program (TRDRP) Pre‐doctoral fellowship (27DT‐0009)
Yerba mate increases mitochondrial uncoupling in adipose tissue
Authors: Walton Chase M. , Saito Erin , Jacobsen Samuel , Hansen Jason M. , Bikman Benjamin T.
Published: April 2020

Yerba mate is commonly consumed in cultures throughout Central and South America with decades of research revealing numerous effects, including improvements in cardiometabolic outcomes. Among these benefits is an increased resistance to weight gain, as well as altered energy expenditure. However, additional research is needed to elucidate the effects of yerba mate on mitochondrial parameters. Through cell culture and rodent models, we tested mitochondrial respiration and ATP production in adipose to determine the direct effects of yerba mate on mitochondrial coupling status. We found that adipocytes experienced a substantial increase in oxygen flux, without a comparable increase in ATP generation, indicating increased mitochondrial uncoupling. Similar findings were observed in subcutaneous adipose tissue in rodents fed yerba mate chronically. These observations in subcutaneous adipose, which is unique in its capacity to “beige” or undergo mitochondrial biogenesis and uncoupling. Altogether, these results suggest that a portion of the metabolic benefit of regular consumption of yerba mate may be a result of increased adipose mitochondrial uncoupling.

Support or Funding Information

None.
COMBINED FARNESOID X RECEPTOR AGONIST AND SOLUBLE EPOXIDE HYDROLASE INHIBITOR TREATS PROGRESSIVE RENAL FIBROSIS
Authors: Khan Md Abdul Hye , Stavniichuk Anna , Jankiewicz Wojciech K. , Vogel Amy N. , Nolan Benjamin J. , Savchuk Olexiy , Merk Daniel , Imig John D.
Published: April 2020

Renal fibrosis plays a critical role in the progression of chronic kidney disease (CKD) to end‐stage renal disease (ESRD) and is considered an independent predictor for clinical outcomes. Unfortunately, there has been little success in developing anti‐fibrotic therapies that can slow the progression of CKD to ESRD. We investigated the efficacy of a dual‐acting molecule, FXRA/sEHi, in arresting renal fibrosis progression using a unilateral ureteral obstruction (UUO) renal fibrosis model. FXRA/sEHi acts simultaneously as farnesoid X receptor agonist and a soluble epoxide hydrolase inhibitor. C57BL/6J male mice went through either UUO or sham surgery (n=8/group). Mice were treated with either FXRA/sEHi (10mg/kg/d) or vehicle given in drinking water. Interventional FXRA/sEHi treatment was started 3 days after UUO induction and continued for 7 days. Plasma and kidney tissue were collected at the end of the experimental period. Biochemical, histopathological, immunohistopathological, and gene expression studies were carried out to determine the antifibrotic actions for FXRA/sEHi. The UUO group exhibited elevated BUN compared to the sham group (77±10 vs. 44±15 mg/dL) and FXRA/sEHi treatment reduced BUN by 40%. UUO mice demonstrated marked renal fibrosis with higher kidney hydroxyproline content (267±46 vs. 53±14 μg/mg protein) and collagen positive area (4.3±0.1% vs. 0.7±0.3%) compared to sham. UUO mice demonstrated renal inflammation with elevated kidney MCP‐1, renal CD45 positive immune cell infiltration, and renal profibrotic gene expression (TNF‐a, IL‐6, IL‐1b). Interventional FXRA/sEHi treatment reduced renal inflammation in UUO mice. Renal fibrosis in UUO was associated with epithelial‐to‐mesenchymal transition (EMT) as UUO mice exhibited a 2 to 20‐fold higher protein and gene expression of mesenchymal markers a‐SMA, FSP‐1, and FN, as well as a marked decrease in the epithelial marker, E‐cadherin compared to the sham group. UUO mice treated with FXRA/sEHi had markedly reduced EMT. UUO mice also had tubular epithelial barrier injury with increased renal KIM‐1, NGAL expression and lower claudin‐1 and claudin‐3 expression compared to sham. FXRA/sEHi treatment reduced tubular injury markers by 25–50% and maintained tubular epithelial integrity by restoring claudin expressions in UUO mice. Vascular inflammation was evident in UUO mice with 9 to 20‐fold higher ICAM and VCAM gene expression compared to sham that was reduced by 40–50% with FXRA/sEHi treatment. Peritubular vascular density assessed by CD31 expression was reduced by 35% in UUO mice and FXRA/sEHi mitigated vascular loss. Overall, these findings demonstrate that FXRA/sEHi is a promising dual‐acting antifibrotic agent that arrests the progression of epithelial and vascular renal fibrotic pathological events.
Mechanical Stimulation of Piezo1 Induced Protein Expression and Intracellular Trafficking of Aquaporin‐2 in IMCD Cells
Authors: Zhao Xiaoduo , Kong Yonglun , Hu Shan , Li Suchun , Liang Baien , Wang Weidong , Li Chunling
Published: April 2020

Pressure and shear stress‐induced stretch of tubular epithelial cells is relevant to both physiological and diseased kidney. Piezo1 is a mechanically‐activated cation channel stimulated by membrane tension and stretch; it is highly expressed in the inner medulla of the kidney. Aquaporin‐2 (AQP2) is located in the principal cells of the collecting ducts in the kidney, which is a key aquaporin determining final urine output. The current study aims to investigate whether activation of Piezo1 induces AQP2 expression in the collecting duct principal cells. In human kidney biopsy specimen, Piezo1 was found expressed mainly in the collecting ducts where it co‐localized with AQP2. Upon treating primary cultured rat inner medullary collecting duct (IMCD) cells with Piezo1 activator Yoda1 (0.01μM to 1μM), the protein expression of AQP2 was markedly increased. Activation of Piezo1 by Yoda1 was also associated with intracellular F‐actin depolymerization and dramatically increased accumulation of AQP2 on the apical plasma membrane of IMCD cells. Upregulation of AQP2 protein was also observed when Piezo1 was stimulated with mechanical cyclic tensile strain at 5% elongation, 1Hz for 3hour in IMCD cells, which was markedly inhibited by GsMTx4 (0.1μM to 5μM), a Piezo1 antagonist. Both stretch and Yoda1 treatment induced elevation of intracellular calcium levels and upregulation of calpain2 protein expression (calcium‐activated non‐lysosomal thiol‐proteases) as well as phosphorylation of Akt protein in rat IMCD cells. Dehydration for 24h (which is presumably leading to the swelling of principal cells, Chou CL, Am J Physiol Renal Physiol, 2008) in mice was associated with increased levels of piezo1 mRNA and protein expression in the kidney inner medulla. In conclusion, activation of Piezo1 by mechanical stretch or activators increased protein expression and intracellular trafficking of AQP2 in rat IMCD cells, which was associated with F‐actin depolymerizing and elevated intracellular calcium levels. These studies indicate that piezo1 may play a functional role in the regulation of urinary osmolarity in pressure‐induced stretch of principal cells relevant to both physiological and pathophysiological conditions.
Genome‐wide Analysis of Heparan Sulfate Biosynthesis
Authors: Liu Qing , Weiss Ryan , Spahn Philipp , Chiang Austin , Li Jing , Hamill Kristina , Tor Yitzhak , Lewis Nathan , Esko Jeffrey
Published: April 2020

Heparan sulfate proteoglycans (HSPGs) are expressed on virtually all animal cells and are involved in many important biological processes. Each HSPG consists of a core protein with one or more covalently attached linear heparan sulfate (HS) chains composed of alternating glucosamine and uronic acids that are heterogeneously N‐ and O‐sulfated. The arrangement and orientation of the sulfated sugar residues of HS specify the location of distinct ligand binding sites on the cell surface, and these modifications can vary temporally during development and spatially across tissues. While most of the enzymes involved in HS biosynthesis have been studied extensively, much less information exists regarding the specific mechanisms that give rise to the variable composition and binding properties of HS. A genome‐wide CRISPR/Cas9 screen was developed to uncover novel genes other than those encoding known HS biosynthetic enzymes. A lentiviral single guide RNA (sgRNA) library was utilized to knock down gene expression across the entire genome in a human malignant melanoma cell line. Subsequently, high‐throughput screens were adapted to identify sgRNAs that induce resistance to cytotoxins whose action depends on HSPGs and decrease binding of HS‐dependent ligands to the cell surface. Among the resulting targets, we identified well‐known HS biosynthesis enzymes as well as candidate genes with a previously unrecognized link to HS biosynthesis. The top overlapping hits from the screens were then characterized based on gene function and validated to regulate HS assembly. Overall, these studies demonstrate novel approaches to uncovering regulatory factors and provide a better understanding of the genetic regulation of HS biosynthesis. Furthermore, these studies lead us to new methods to manipulate heparan sulfate and its activities in other cellular processes that go awry in human diseases.

Support or Funding Information

R01GM33063 (to J.E.), R21CA199292 (to J.E. and N.L.), R35GM119850 (to N.L.), and K12HL141956 (to R.W.)
Profiling of Vascular Reparative Signaling using Sphingosine‐1‐Phospate Receptor 1 Reporter Mice
Authors: Akhter Md Zahid , Joshi Jagdish C. , BR Vijay Avin , Mehta Dolly
Published: April 2020

Loss of endothelial barrier function is a hall mark of acute lung injury, which induces 40% lethality in affected patients. Sphingosine‐1‐phosphate receptor 1 (S1PR1) upon ligating S1P on endothelial cell (EC) surface enhances endothelial barrier function and reverses lung vascular hyper‐permeability in a variety of inflammatory injury including endotoxin, LPS. Whether S1PR1 is activated dynamically following lung injury in ECs and repairs barrier exclusively through EC‐cell surface signaling remains enigmatic. Using S1PR1‐GFP‐signaling reporter mice (S1PR1 reporter) mice, in which nuclear GFP reports for S1PR1 activity, along with FACS and bone marrow transplantation approaches, we show that LPS primarily activates S1PR1 in resident ECs (CD45−CD31+GFP+) which reached its maximal level at 16 h. Intriguingly, S1PR1‐actived ECs showed enhanced proliferation in vivo and in vitro. RNA seq analysis of sorted GFP‐S1PR1‐ECs identified the transcription factor EGR1, SPHK1 and SPNS2 as top 100 hundred genes. We show that S1PR1 induced EGR1 expression which in turn upregulated SPHK1 expression. We also found that S1PR1 induced SPNS2 through STAT3. Upregulated SPHK1 and SPNS2 lead to amplification of S1P generation and transport. Thus, transplantation of GFP‐S1PR1‐ECs in matrigel induced vascularization and these ECs also repaired defective vascular barrier in lungs of mice conditionally lacking EC‐S1PR1. Findings show that S1PR1 induces self‐driven reparative transcriptional signaling for endothelial barrier repair.
Keratan sulfate disaccharide: specific targeting to langerin and possible applications to COPD
Authors: Taniguchi Naoyuki , Ohkawa Yuki , Fujinawa Reiko , Kanto Noriko , Harada Yoichiro , Kizuka Yasuhiko
Published: April 2020

The C‐type lectin receptor, langerin, is expressed in Langerhans cells (LCs) that are located in the dermis along with other dendritic cells (DCs). Although there have been several reports on the sugar‐binding properties of langerin, the physiological and pathological output upon the recognition of each sugar ligand by langerin are still under investigation. We propose here that the keratan sulfate (KS) disaccharide L4 and oligomeric derivatives thereof, triangle‐L4 and pendant‐L4, are functional ligands of langerin that, on binding, evoke anti‐inflammatory responses. ELISA assays revealed that triangle‐L4 and pendant‐L4 showed stronger binding to recombinant langerin than L4, and we here found that these L4 and derivatives thereof suppressed the expression of inflammatory cytokines in langerin‐expressing primary bone marrow‐derived dendritic cells (BMDCs). Furthermore, in an elastase‐induced mouse model of chronic obstructive pulmonary disease (COPD), the administration of L4 mitigated the extent of emphysema in the lung, suggesting that KS‐related langerin ligands might serve as new drug candidates for the treatment of COPD and other inflammatory diseases. We also discuss several unsolved questions that need to be tackled for our understanding of the functions of langerin and the development of langerin‐targeted therapy by using glycomimetics.

Support or Funding Information

This study was supported for Naoyuki Taniguchi by Japan Agency for Medical Research and Development (AMED, 18ae0101070h0003) and Grants‐in‐Aid for Scientific Research (KAKENHI KIBAN B, 15H04700 and Challenging Exploratory Research, 15K14481).
Abundance of Ssh1p modulates Sec61p stability and topogenesis
Authors: Kim Sungmin , Kim Hyun Ah
Published: April 2020

Protein translocation across the endoplasmic reticulum (ER) membrane is mediated by translocons. Two translocons in the ER of Saccharomyces cerevisiae are the Sec61 and the Ssh1 (
Sec
sixty‐one
homolog
1) complexes. They contain Sec61p and Ssh1p as a pore‐forming core subunit, respectively, and share Sss1p as a complex stabilizing factor. Ssh1p is not essential for cell growth in yeast, and it remains unclear why yeast cells keep this non‐essential translocon component.

Upon overexpression of Ssh1p, we observed a severe growth defect and rapid destabilization of Sec61p at high temperature. We reasoned that overexpressed Ssh1p, which is scarce in steady state might recruit Sss1p from the Sec61 complex, impairing its stability. To test this idea, co‐overexpression of Sss1p with Ssh1p was carried out and data show that it partially rescues a growth defect and Sec61 stability. Furthermore, overexpression of the Sss1p‐interaction‐defective mutant of ssh1p had no effect on growth or Sec61p stability. We also assessed synthesis of single‐spanning membrane proteins in SSH1 deletion and overexpression strains and found that Ssh1p is involved in biased topogenesis of single‐spanning membrane proteins.

Our data characterized cellular roles of Ssh1p that it might contribute for □) adjusting Sec61p stability and □) mediating selective topologenesis of membrane proteins.
A Randomized, Placebo‐Controlled Trial Evaluating Effects of Lebrikizumab on Airway Eosinophilic Inflammation and Remodeling in Uncontrolled Asthma (CLAVIER)
Authors: Austin Cary D. , Edick Melissa Gonzalez , Ferrando Ronald E. , Solon Margaret , Baca Miriam , Mesh Kathryn , Bradding Peter , Gauvreau Gail M. , Sumino Kaharu , Fitzgerald Mark , Israel Eliott , Bjermer Leif , Bourdin Arnaud , Arron Joseph R. , Choy David F. , Olsson Julie K. , Abreu Francis D. , Howard Monet D. , Wong Kit D. , Cai Fang D. , Peng Kun D. , Putman Wendy S. , Holweg Cécile T. J. , Matthews John G. , Kraft Monica , Woodruff Prescott G.
Published: April 2020

Background

The anti–interleukin 13 (IL‐13) monoclonal antibody lebrikizumab improves lung function in patients with moderate‐severe uncontrolled asthma, but its effects on airway inflammation and remodeling are unknown. CLAVIER was designed to assess lebrikizumab’s effect on eosinophilic inflammation and remodeling.

Methods

We performed bronchoscopy on patients with uncontrolled asthma before and after 12 weeks of randomised double‐masked treatment with lebrikizumab (n=31) or placebo (n=33). The primary endpoint was relative change in airway subepithelial eosinophils per mm2 of basement membrane (cells/mm2). Pre‐specified secondary and exploratory outcomes included change in IL‐13–associated biomarkers and measures of airway remodeling.

Findings

There was a baseline imbalance in tissue eosinophils and high variability between treatment groups. There was no discernible change in adjusted mean subepithelial eosinophils/mm2 in response to lebrikizumab (95% CI, −82·5%, 97·5%). As previously observed, FEV1 increased after lebrikizumab treatment. Moreover, subepithelial collagen thickness decreased 21·5% after lebrikizumab treatment (95% CI, −32·9%, −10·2%), while fractional exhaled nitric oxide, CCL26, and SERPINB2 mRNA expression in bronchial tissues also diminished. Lebrikizumab was well tolerated, with a safety profile consistent with other lebrikizumab asthma studies.

Interpretation

We did not observe reduced tissue eosinophil numbers in association with lebrikizumab treatment. However, in pre‐specified secondary analyses, lebrikizumab treatment was associated with diminished degree of subepithelial fibrosis, a feature of airway remodeling, as well as improved lung function and reduced key pharmacodynamic biomarkers in bronchial tissues. These results reinforce the importance of IL‐13 in airway pathobiology and suggest that neutralization of IL‐13 may reduce asthmatic airway remodeling.

Clinical trial registration

NCT02099656

Support or Funding Information

The study was sponsored by F. Hoffmann‐La Roche Ltd.
Histidine Reactive Chemicals Modify the Thermus thermophilus CuA Redox Center
Authors: Acevedo Zachary , Hunsicker-Wang Laura
Published: April 2020

The electron transport chain is a series of redox protein complexes that couple the shuttling of electrons with the pumping of protons, in order to create the electrochemical gradient that drives ATP synthesis. The first redox center of the terminal complex from the Thermus thermophilus bacteria has been isolated as TtCuA and has a binuclear copper center bridged by two cysteines and ligated by two histidines and two weak axial ligands. H157 of TtCuA, a solvent exposed ligating histidine, has demonstrated modification by diethyl pyrocarbonate (DEPC), an exogenous chemical modifier, that is facilitated by the residue’s fast proton exchange with the solvent. The H157‐DEPC adduct is reversible over 48 hours at room temperature, as supported by mass spectrometry, electrochemistry, and visible CD spectroscopy. This is one of two modifications, out of 12, that becomes removed. Due to the lability of the H157‐DEPC bond, it is possible that the H157 Nɛ is primed for proton pumping in the electron transport chain.

In order to model a possible in vivo modification, TtCuA was exposed to 4‐hydroxynonenal (HNE), an endogenous lipid peroxidation product associated with multiple metabolic and neurodegenerative diseases. Exposure of TtCuA to 20 equivalents of HNE appears to reduce the protein over 48 hours and four modifications are observed via mass spectrometry. Exposure of the Pseudomonas aeruginosa azurin protein, a mononuclear blue‐copper denitrification protein with similar ligand groups to TtCuA, is not reduced by HNE, despite modification. Azurin also lacks a ligating histidine that can be modified by DEPC. In conclusion, the binuclear copper center of TtCuA has hyperreactivity in comparison to a similarly ligated mononuclear copper center.

Support or Funding Information

Murchison Undergraduate Research Fellowship, Trinity University Chemistry Department, Mach Research Fellowship, Welch Foundation
Search for New KCa3.1‐Targeting Small Molecules and Monoclonal Antibodies
Authors: Shim Hesung , Nguyen Hai , Cui Yanjun , Colussi Paul , Bednenko Janna , Wulff Heike
Published: April 2020

The intermediate‐conductance Ca2+‐activated K+ channel KCa3.1 (also known as SK4 or the Gárdos channel) is encoded by the KCNN4 gene and plays an important role in the activation of T‐ and B‐cells, mast cells, macrophages and microglia by regulating membrane potential, cellular volume and calcium signaling. KCa3.1 is further involved in the proliferation of dedifferentiated vascular smooth muscle cells and fibroblast. Accordingly, KCa3.1 inhibitors are therapeutically relevant as immunosuppressants and for the treatment of a wide range of fibroproliferative disorders. Using the recently solved cryo‐EM structures of the full length KCa3.1 channel in the closed and two open states we virtually screened a small library of clinically used drugs with two software packages (Glide and AutoDock Vina). The top 50 scoring compounds for the triarylmethane binding site in the inner pore varied significantly between the two programs and the three states. We are currently in the process of functionally confirming the more interesting hits using a thallium flux assay and whole‐cell patch‐clamp.

In parallel we are also searching for KCa3.1 blocking monoclonal antibodies, since antibodies constitute attractive alternatives to small molecules because of their exquisite specificity and long half‐lives. Human KCa3.1 was recombinantly expressed in Tetrahymena thermophila, a unicellular eukaryote that has shown a remarkable ability to produce correctly folded human ion channels at high yield. The recombinant KCa3.1 was purified and adhered to magnetic beads that were subsequently used to pan two human phage display libraries. Library screening identified 34 unique anti‐KCa3.1 antibodies, out of which two were found to functionally block KCa3.1 current in whole‐cell patch‐clamp experiments. The selected clone, TTG‐252, inhibits human KCa3.1 current with an IC50 of 1.8 nM while showing no effect on human KCa1.1, KCa2.2, Kv11.1 (hERG) or Nav1.5. The antibody does not cross‐react to mouse or rat KCa3.1.

Support or Funding Information

Funded by R21NS101876.
The effect of heat stress on motor preparation processing
Authors: Shibasaki Manabu , Ishikura Keiko , Sawai Norie , Hirai Minami , Kobayashi Fumino , Nakata Hiroki
Published: April 2020

An excessively increased internal temperature is known to reduce exercise/sports performance. To clarify the effect of heat stress on motor preparation processing using electroencephalography (EEG), subjects performed two types of voluntary isometric exercise before (normothermia) and during heat stress on different days. Fifteen volunteers performed contingent negative variation (CNV) trials in Experiment 1, and fourteen volunteers performed movement‐related cortical potential (MRCP) trials in Experiment 2, while eleven subjects performed both trials. Whole body heating via a water‐perfused suit increased the esophageal temperature by 1.52 ± 0.14 °C in Experiment 1 and 1.40 ± 0.10 °C in Experiment 2. The mean amplitudes of the middle‐ and late‐CNV at Cz and Pz during heat stress were significantly reduced relative to those in normothermic trials (middle CNV, −3.3 ± 2.8 to −0.5 ± 3.2 μV at Cz, −3.8 ± 2.8 to −1.6 ± 3.0 μV at Pz; late CNV, −5.9 ± 3.5 to −3.1 ± 3.1 μV at Cz, −6.6 ± 2.9 to −3.7 ± 2.8 μV at Pz, P< 0.05 for each). However, in Experiment 2, there were no significant differences between normothermia and heat stress regarding the mean amplitudes of MRCPs including the Bereitschaftspotential (BP) and negative slope (NS′). These results suggest that mild hyperthermia influences cognitive processing being reflected by CNV with stimulus‐triggered movement, rather than motor‐related processing reflected by MRCPs related to self‐initiated movement.

Support or Funding Information

Japan Society for the Promotion of Science 18H03166
Elucidating the Effects of Distal Charges on the Reduction Potential of the Rieske Protein
Authors: Lopez Rachel , Hunsicker-Wang Laura
Published: April 2020

The Rieske protein plays a key role in both cellular respiration and photosynthesis, transferring electrons through the respective cytochrome bc1 and b6f complexes of each process. It is characterized by a [2Fe‐2S] cluster and is unique in that one iron is ligated by nitrogens from two solvent‐exposed histidine residues while the other is ligated by the sulfurs of two cysteines. In the electron transport chain (ETC), the Rieske protein oxidizes ubiquinol and reduces cytochrome c, transferring electrons between complexes II and IV.

Previous work has demonstrated how hydrogen bonds, solvent accessibility of the ligands, and the presence of nearby charged residues can impact the reduction potential of the protein. However, the full extent of this third element, how far from the cluster these charged residues can reside and still impact the reduction potential, has yet to be explored. Two types of mutants have been created to probe the effects of these distal charges. First, mutations removing positively charged residues were predicted to decrease reduction potential. Likewise, mutations removing negatively charged residues were expected to increase reduction potential. Each mutation either resides a unique distance from the cluster or produces a different change in overall charge, demonstrating the impact of each factor both individually and additively. Lastly, the pKa values of the histidine ligands were also expected to be inversely related to reduction potential.

Using pH‐dependent UV‐Visible spectrophotometry, the pKa values of these mutants were determined and correlated to these relationships. While the overall trend of pKa values increased as expected for the negative‐charge mutants, confounding elements also appeared, such as a third pKa sigmoid. Furthermore, E146R, a positive‐charge mutant, also demonstrated increased pKa values, opposite to the hypothesized trend. New mutants that implement a neutral to positive substitutions have been developed to probe the effect of making the protein more positive.

Support or Funding Information

Murchison Summer Undergraduate Research Fellowship, Welch Foundation
Get Out of Your Shell: Protein Extraction from Carcinus maenas
Authors: Kelly Lauren , Myracle Angela D. , Grant Jennifer E.
Published: April 2020

The European green crab, a species that is native to Europe and northern Africa, is an invasive species that has overpopulated the coast of Maine. These crabs end up in fisherman’s nets but presently harbor no economic value. Yet, crab shells in general contain some of the highest protein content among shelled organisms. If the proteins and peptides extracted from Carcinus maenas can be extracted from shells inexpensively, it is possible that the European green crab shell could be transformed from a waste to an economic product. The purpose of this research is to examine the peptide and protein content from the European green crab (Carcinus maenas), using SDS‐PAGE electrophoresis.

Protein extraction from shells is a non‐trivial task. The major component of crab shell is calcium carbonate, deposited within a protein/chitin matrix. Challenges to protein purification abound, particularly because calcium is prone to precipitating out when changes to pH or ionic strength are employed to enrich the proteins. This work expands upon our efforts to optimize extraction conditions. We chose to focus on 24‐hour extractions using hydrochloric acid; conditions ranging from 1 M to 0.25 M HCl were tested for efficacy. Protein precipitation using ammonium sulfate was then performed in order to fractionate proteins. Here we present data on the success of this ammonium sulfate‐based approach which resulted in isolation of a solution that was roughly 0.3 microgram/microliter protein.

This research may identify novel bioactive peptides; little work has been done on uses for the European green crab and this approach may generate value‐added economic ideas.

Support or Funding Information

This work was supported by a University of Wisconsin‐Stout Student Dissemination Grant to LK.
Paracrine Secretion of Cardiac‐Derived Erythropoietin is Required for Cardiogenesis
Authors: Allwood Melissa A. , Platt Mathew J. , Huber Jason S. , Jacyniak Kathy , Edgett Brittany A. , Klein Jordynn M. , Marrow Jade P. , Alshamali Razan , Romanova Nadya , Brunt Keith R. , Vickaryous Matthew K. , Simpson Jeremy A.
Published: April 2020

Erythropoietin (EPO) is widely recognized as the principle regulator of erythropoiesis, however, extra‐erythropoietic functions have been identified including cytoprotection, cardiac inotropy, cellular proliferation, and embryonic development. Whole body deletion of either EPO or the EPO receptor is embryonic lethal with impaired cardiogenesis leading to ventricular hypoplasia. While multiple extra‐renal tissue and cell types produce EPO, whether the heart is a direct source remains unclear. Human recombinant EPO increases myocardial contractility and confers cytoprotection against cardiac injury, which suggests a role for EPO signalling in the heart. Our objectives were to (1) confirm whether the heart produces EPO and (2) determine if there is a role for paracrine EPO signalling during cardiogenesis. We generated constitutive, cardiomyocyte‐specific EPO knockout mice driven by the Mlc2v promoter (EPOfl/fl:Mlc2v‐cre+/−; EPOΔ/Δ‐CM). We confirmed that the heart is a source of EPO expression with a distinct circadian rhythm in adult hearts and increased expression during embryonic development. During cardiogenesis, cardiac EPO expression was reduced, but not eliminated, in EPOΔ/Δ‐CM hearts with decreased cardiac cell proliferation. This suggests EPO signalling is partially compensated by an alternate cardiac cell type during cardiac development. In adult EPOΔ/Δ‐CM mice, global cardiac mass was preserved while cardiomyocyte cross‐sectional area was increased. Taken together, cellular cardiomyocyte hypertrophy in the absence of gross organ hypertrophy, and the observed reduction in cardiac cell proliferation during cardiogenesis, points towards a reduction in the overall number of cardiomyocytes in EPOΔ/Δ‐CM mice. Collectively, these data identify the first physiological roles of extra‐renal EPO by confirming that the heart is a source of EPO and that paracrine expression is required for cardiogenesis. Further, cardiac EPO expression is a complex interplay of multiple cell types where loss of cardiomyocyte production results in compensation from other cardiac cell lineages.

Support or Funding Information

This work was funded in part by the Canadian Institutes of Health Research (JAS), the Natural Sciences and Engineering Research Council of Canada (KRB, MKV, and JAS), the Canadian Glycomics Network (JAS), and the Heart and Stroke Foundation of Canada (KRB and JAS). JAS is also a new investigator with the Heart and Stroke Foundation of Ontario.
Gestational High‐Altitude Hypoxia and Metabolomic Reprogramming in Pulmonary Arteries from Fetal Sheep
Authors: Leslie Eric , Jones Breanna , Alvarez Rafael , Dasgupta Chiranjib , Fiehn Oliver , Bosviel Remy , Newman John W. , La Frano Michael , Zhang Lubo , Wilson Sean M.
Published: April 2020

Gestational high‐altitude hypoxia increases the risk of myriad diseases in human infants including pulmonary hypertension of the newborn. Fetal sheep lungs are susceptible to long‐term intrauterine hypoxia, with the lung structure and function exhibiting characteristics associated with the development of pulmonary hypertension. Previous studies showed thickening of the pulmonary arterial medial wall, dysregulation of arterial reactivity, and reduced right ventricular output in fetal lambs following exposure to high altitude long‐term hypoxia. To further explore the mechanisms associated with hypoxia‐induced aberrations in the fetal sheep lung, we examined the premise that metabolomic and proteomic changes are associated with phenotypic transformation caused by intrauterine hypoxia. To address this hypothesis, we performed metabolomic and proteomic analysis on pulmonary arteries isolated from near term fetal lambs that were exposed to intrauterine high‐altitude hypoxia (3801 m) for the latter 110+ days of pregnancy or those gestated near sea level. The data demonstrated gestational hypoxia caused a glycolytic shift, reduced fatty acid oxidation, and elevated pentose phosphate metabolism along with increased histone expression, illustrative of increases in nucleotide synthesis. Increased nucleotide synthesis in response to gestational hypoxia is suggestive of a shift towards a proliferative and synthetic phenotype, providing insight into the mechanisms related to gestational hypoxia‐mediated pulmonary remodeling and hypertension. These studies begin to shed light on the mechanistic underpinnings associated with the phenotypic transformations to the pulmonary vasculature that occur with intrauterine hypoxia.

Support or Funding Information

Supported in part by NIH grants HD083132 (LZ), 1U24DK097154 (OF ) through a pilot project grant to SMW and HD098477 (SMW). Additional support was provided by USDA Intramural projects #2032‐51530‐022‐00D and #2032‐51530‐025‐00D (J.W.N.). The USDA is an equal opportunity provider, and employer.