bluuebunny/crossref_metadata_2025 huggingface dataset:
Aspirin in Combination with Benznidazole During the Acute Phase of Chagas Disease Prevents Cardiovascular Dysfunction and Decrease Typical Cardiac Lesions in Mice Chronically Infected with Trypanosoma cruzi
Authors: Pinge-Filho Phileno , Pereira Rito Santo , Malvezi Aparecida Donizette , Lovo-Martins Maria Izabel , Lucchetti Bruno Fernando Cruz , Santos Jussevania Pereira , Tavares Eliandro Reis , Verri Waldiceu Aparecido , de Almeida Araújo Eduardo José , Lioni Lucy Megumi Yamauchi , Yamada-Ogatta Sueli Fumie , Martins-Pinge Marli Cardoso
Published: April 2020

Chagas disease, caused by protozoan Trypanosoma cruzi, is one of the main causes of death due to cardiomyopathy and consequent heart failure in Latin American countries. The goal of treatment is to eliminate the parasite and decrease the probability of cardiomyopathy and interrupt the cycle of disease transmission. Benznidazole (BZ) and nifurtimox (NFX) are recognized by the World Health Organization as effective drugs for treatment of disease, but both are very toxic with limited efficacy, especially in the chronic phase. Studies have shown that low doses of aspirin (ASA) were protective in experimental T. cruzi Infection. We evaluated the efficacy of BZ in combination with ASA in low doses using a chronic infection model with T. cruzi Y strain. Our results show that ASA treatment prevented the typical cardiovascular dysfunction (hypertension and tachycardia) and cardiac lesions on chronic phase of disease. Moreover, mice treated with BZ+ASA had a smaller cardiac fibrotic area than those of BZ‐treated mice. These results were associated with an increase of eosinophils and reticulocytes and high levels of nitric oxide in plasma and cardiac tissue of animals treated with ASA compared to the controls. The protective effects of ASA or BZ+ASA on chronically infected mice disappeared when we used Boc‐2 (LXA4 receptor antagonist), indicating that the protector effect of ASA was mediated by aspirin‐triggered lipoxin. These results emphasize the importance of exploring new drug combinations in treatments that can be used in the acute phase of Chagas disease that are beneficial to chronic patients.

Support or Funding Information

The present study was supported by grants from Fundação Araucária ‐ chamada de projeto 09/2016 Programa Institucional de Pesquisa Básica e Aplicada ‐ Conv. 001/2017 ‐ protocolo 47.396 ‐ SIT. 31675, Conselho Nacional Desenvolvimento Científico e Tecnológico (CNPq), CAPES. PPF, MCMP, MILM, WAVJ, LMYL, SFYO are research fellows of CNPq. BFCL and ERT are research fellows of CAPES. RPS was supported by Ministério da Ciência e Tecnologia, Ensino Superior e Técnico Profissional de Mocambique.

Graphical abstract

Figure 1
The Role of κ‐Opioid Receptor‐Induced Autophagy in Synaptic Alterations
Authors: Georgoussi Zafiroula-Iro , Karoussiotis Christos , Sotiriou Aggeliki , Papavranoussi-Daponte Danae , Polissidis Alexia Victoria , Nikoletopoulou Vassiliki
Published: April 2020

Autophagy is a lysosomal degradation pathway that eliminates misfolded proteins and dysfunctional organelles to safeguard cellular homeostasis. In neurons, autophagy controls the quality of cytoplasmic proteins through degradation of important synaptic proteins and modulates synaptic organization and morphogenesis
(1). Emerging studies have shown that G protein coupled receptors (GPCRs) are direct sensors regulating the autophagic machinery
(2) and that opioid receptors regulate neurogenesis and neurotransmission with as yet unclarified mechanisms
(3,
4). In this regard, we have recently demonstrated that κ‐opioid receptor (κ‐OR) agonists induce autophagy in neuronal cells in vitro via a PTX‐sensitive G protein mechanism and identified the downstream components involved in this process
(5,
6). However, it is unknown whether κ‐OR agonists also mediate these effects in vivo and, specifically, whether opiodergic alterations mediated by selective opioid receptor agonists may control synaptic function and spine morphology through autophagy induction.

We are now reporting by in vivo studies that brain extracts obtained from mice that have been treated with the κ‐OR selective agonist U‐50,488H display profound increases of specific autophagic markers. This κ‐OR induced autophagy increase is region‐specific and detected only in the hippocampal extracts. Moreover, isolated synaptosomes from saline‐ or U50,488H‐treated mice exhibited a profound decrease of a number of essential neuronal proteins enriched in dendritic spines, suggesting that these proteins are engulfed in the κ‐OR‐induced autophagic cargo. Our results demonstrate for the first time that κ‐OR‐induced autophagy plays a key role in synaptic function and dendritic spine density and may contribute to the mood disorders targeted by the dynorphin‐κ‐OR system.

Support or Funding Information

This work was supported by the GSRT Excellence II 3722 ‐”NO‐ALGOS” to Z.G; the SANITURA (MIS 5002514) grant, implemented under the Action for the Strategic Development on the Research and Technological Sectors & the OPENSCREEN‐GR “An Open‐Access Research Infrastructure of Chemical Biology and Target‐Based Screening Technologies for Human and Animal Health, Agriculture and the Environment” (MIS 5002691) funded by the Operational Programme “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014–2020).
A cell permeant phosphopeptide mimetic of Niban restores endothelial function and inhibits p38 MAPK activation after vascular injury
Authors: Yim Tsz Wing , Perling Daniel , Polcz Monica , Komalavilas Padmini , Brophy Colleen , Cheung-Flynn Joyce
Published: April 2020

Vascular injury leads to membrane disruption, ATP release, and endothelial dysfunction. Increases in the phosphorylation of p38 MAPK and decreases in the phosphorylation of Niban, a protein implicated in ER stress and apoptosis, are associated with vascular injury. A cell‐permeant phospho‐peptide mimetic of Niban (NiPp) was generated. The effects of NiPp in restoring endothelial function were determined ex vivo using intact rat aortic tissue (RA) after pharmacological activation of p38 MAPK and a clinically relevant stretch injury model. Anisomycin increased p38 MAPK phosphorylation and reduced endothelial‐dependent relaxation in RA. Treatment with NiPp prevented anisomycin‐induced reduction in endothelial function and increase in p38 MAPK phosphorylation (Figure
1). NiPp treatment also restored endothelial function after subfailure overstretch injury (Figure
2). Kinome screening experiments indicated that NiPp inhibits p38 MAPK. These data demonstrate that p38 MAPK and Niban signaling have a role in endothelial function, particularly in response to injury. Niban may represent an endogenous regulator of p38 MAPK activation. NiPp may serve as an experimental tool to further elucidate p38 MAPK regulation and as a potential therapeutic for endothelial dysfunction.

Support or Funding Information

This work was supported by the National Institutes of Health grants R01HL70715‐09 to CB and R01HL105731‐01 to JC.

NiPp prevented anisomycin‐induced phosphorylation of p38 MAPK and endothelial dysfunction in rat aorta (RA).

RA rings were treated with anisomycin (200μM) in the absence or presence of NiPp (500μM) pretreatment and endothelial function was determined in a muscle bath. A) Representative muscle bath tracings. B) Percent relaxation induced by 0.5μM CCH. C, D) Phosphorylation of p38 MAPK in RA after treatment as determined by Western blot analysis. n=10,*p<.05 in one‐way ANOVA with Tukey post‐test.

Figure 1

NiPp restored endothelial function and reduced p38MAPK phosphorylation in RA after subfailure stretch injury.

RA was subjected to subfailure stretch and incubated in the absence or presence of NiPp (500μM) for 1 h. A) Endothelial‐dependent relaxation was determined in the muscle bath. n=5 rats; #p<.05, two‐way ANOVA. B, C) Phosphorylation of p38 MAPK in RA after stretch injury and NiPp treatment as determined by Western blot analysis n= 10. *p<.05, in one‐way ANOVA with Tukey post‐test.

Figure 2
Substrate‐Bound Complex of DesD, a Model Enzyme in the Virulence‐Related NIS Synthetase Family
Authors: Hoffmann Katherine
Published: April 2020

The understudied NIS Synthetase family have been increasingly associated with virulence in bacterial species, due to their key role in the synthesis of hydroxamate and carboxylate “stealth” siderophores. We have identified a model family member, DesD from Streptomyces coelicolor, to characterize structurally using a combination of wild‐type and Arg306Gln variant in apo, cofactor product (AMP)‐bound with analog cadaverine, and cofactor bound with analog dfoB complexes. Kinetics in the family have been limited by solubility and reporter‐based assays, so we have developed a label‐free kinetics assay utilizing an unusual single‐injection isothermal titration calorimetry based method. We report second order rate constants 50‐fold higher than the previous estimations for DesD in both the cofactor‐limiting and dfoG substrate limiting curves. Our Arg306Gln DesD variant was also tested in identical buffer and substrate conditions, and confirmed undetectable activity. This is the first complete complex structure (cofactor, amine and carboxylate) reported for an NIS synthetase, and the first label‐free assay to unambiguously determine kinetics for the NIS synthetase family. Our future directions aim to quantify binding thermodynamics, characterize the small substrates (two molecules of hydroxysuccinylcadaverine, which must be synthesized) and delineate the active site mechanism with site‐directed mutagenesis.

Support or Funding Information

This research was supported in part by the National Science Foundation (NSF‐RUI grant 1716986 to K.M.H.), by the John Stauffer Charitable Trust.

The DesD dimer has two active sites, only one of which is filled with substrate in the structures (left). The small substrate analog cadaverine (yellow atom coloring) is shown bound to wild‐type DesD at the bottom right, and the large substrate dfoB (magenta atom coloring) is bound to Arg306Gln in the top right. Both structures are shown with cofactor ATP (teal atom coloring) bound as well.

Figure 1
Accurate Annotation of Protein‐coding Small Open Reading Frames in the Human Genome
Authors: Martinez Thomas F. , Chu Qian , Donaldson Cynthia , Tan Dan , Shokhirev Maxim N. , Saghatelian Alan
Published: April 2020

Functional protein‐coding small open reading frames (smORFs) are emerging as an important class of genes. Several smORF‐encoded microproteins have been characterized and implicated in a variety of critical processes, including regulation of mRNA decay, DNA repair, and muscle formation. Thus, rigorous and comprehensive annotation of protein‐coding smORFs is of fundamental importance. Here, we integrate de novo transcriptome assembly and Ribo‐Seq into an improved workflow that overcomes obstacles with previous methods to more confidently annotate thousands of novel smORFs across multiple human cell lines. Over 1,500 smORFs are found in two or more cell lines, and ~40% lack a canonical AUG start codon. Evolutionary conservation analyses suggest that hundreds of smORF‐encoded microproteins are likely functional. Additionally, monitoring RNA expression and translational efficiency during cellular stress revealed regulated smORFs, demonstrating an approach for identifying biologically relevant smORFs. We also find that smORF‐derived peptides are detectable on human leukocyte antigen complexes, positioning smORFs as a source of novel antigens. The annotation of protein‐coding smORFs radically alters the current view of the human genome’s coding capacity and will provide a rich pool of unexplored, functional human genes.

Support or Funding Information

This research was supported by NIH/NIGMS (R01 GM102491, A.S.), Leona M. and Harry B. Helmsley Charitable Trust grant (A.S.), Dr. Frederick Paulsen Chair/Ferring Pharmaceuticals (A.S.), NIH/NIGMS postdoctoral fellowship (F32 GM123685, T.F.M.), George E. Hewitt Foundation for medical research (Q.C.), and the Pioneer Fellowship (D.T.). This work was also supported by the Razavi Newman Integrative Genomics and Bioinformatics Core and the Next Generation Sequencing Core Facilities of the Salk Institute with funding from the NIH‐NCICCSG (P30 014195) and the Chapman Foundation.

Figure 1
Glycosylation of Ganoderic Acid A via Recombinant Glycosyltransferase of Bacillus subtilis Under Acidic Operating Condition
Authors: Wu Jiumn-Yih , Chang Te-Sheng , Chiang Chien-Min , Wang Tzi-Yuan
Published: April 2020

Ganoderic acid A (GAA) is a triterpenoid with identified effective activity, isolated from Ganoderma lucidum. Glycosylation of triterpenoid might improve its anti‐toxin bioactivity and also increase both water solubility and physical‐chemical stability. Glycosylation is carried out by glycosyltransferases (GT, EC 2.4.x.y), a type of enzyme that utilize a nucleotide‐activated sugar donor, such as uridine diphosphate (UDP)‐glucose, to transfer the sugar portion to a sugar‐linked molecule. We have utilized Bacillus sp. GA A07, isolated from intestinal bacteria of Zebrafish, to biotransform GAA to GAA‐15‐O‐β‐glucoside
1. Our previous study also showed that the Bacillus subtilis, ATCC 6633 strain, could biotransform GAA to compound, GAA‐15‐O‐b‐glucoside, and one minor compound in Fig.
1. We selected five GT genes‐BsGT110, BsGT292, BsGT296, BsGT398, and BsGT489 from the used strains, and successfully overexpressed and cloned them in Escherichia coli. Two glycosyltransferases (BsGT398 and BsGT489) have shown the capability to catalyze GAA to GAA‐15‐O‐β‐glucoside under some specific conditions. However, the chemical structure of minor compound
2 and its corresponding enzyme remain elusive. In the present study, we identified BsGT110, a GT from the used B. subtilis strain, for the biotransformation of GAA into this compound under acidic glycosylation. BsGT110 showed an optimal glycosylation activity toward GAA at pH 6 but lost most of its activity at pH 8. Through a designed operating production, this product
2 was successfully isolated using preparative high‐performance liquid chromatography and was identified to be a new triterpenoid glucoside (GAA‐26‐O‐β‐glucoside) by mass and nuclear magnetic resonance spectroscopy. We identified that BsGT110 is an unique GT that have a specific activity on the glycosylation of triterpenoid at the C‐26 position under acidic conditions (in Fig.
2) but lost most of its activity at alkaline ones, suggesting that acidic solutions may enhance the catalysis activity of this and similar types of GTs toward triterpenoids.

Support or Funding Information

This research was financially supported by the grants of the Ministry of Science and Technology, Taiwan (Project No. MOST 108‐2221‐E‐507 ‐008 ‐ and MOST 108‐2221‐E‐024 ‐008 ‐MY2).

UPLC analysis of the biotranformation of GAA by BsGT110

Figure 1

Glycosylation of GAA by BsGT110 under the acidic condition

Figure 2
Hair fiber morphology and function: at the intersection of anthropology, physiology, and engineering
Authors: Lasisi Tina , Smallcombe James , Shriver Mark D. , Kenney W. Lawrence , Havenith George , Jablonski Nina G.
Published: April 2020

Humans are unique among mammals in having a nearly naked body but a fur‐covered head. Furthermore, scalp hair is one of the most variable traits among human populations. However, the evolutionary emergence of scalp hair, the factors contributing to its evolution, and its subsequent diversification remain largely unknown. Among the few discussions of human scalp hair evolution is the hypothesis that hairy scalps may have evolved as protective barriers against solar radiation in bipedal savannah dwellers. Managing thermal load would be particularly important in the context of a large thermogenic and thermosensitive brain. And as the presence and morphology of fur, or hair, is generally of great importance to mammalian thermoregulation, we set out to test the thermal effect of scalp hair (morphology). To do this, we developed methods for the quantification of hair fiber morphology and experimentally tested the effect of scalp hair (and variation in its morphology) on heat gain from solar radiation. Thermal manikins are human models designed for scientific testing of thermal environments and to simulate heat transfer between the skin and the environment in a range of temperatures. Using one such thermal manikin, we collected data on heat flux when the manikin was equipped with no wig, and wigs of various hair textures. Our preliminary results show that tightly curled scalp hair, in particular, minimizes the heat gained from solar radiation (Figure
1). The study is being expanded and experiments repeated to enable statistical testing. We plan to use these data in mathematical models of human physiological response to estimate the impact scalp hair might have had in early human evolution. In this research we combined anthropological theory, physiological models and engineering methods to answer a longstanding question of human evolution ‐ why did humans evolve scalp hair and why does it vary? The innovative work stemming from this collaboration provided the opportunity to tackle old questions in new ways and pave the way for more potential interdisciplinary work.

Support or Funding Information

This work is supported by the National Science Foundation (award number 1847845); the Wenner‐Gren Foundation (grant number 9911); and the Africana Research Center at The Pennsylvania State University.

Solar heat gain in different hair textures at three windspeeds.

Figure 1
PKA RIα phase separation drives cAMP compartmentation and suppresses tumorigenic signaling
Authors: Zhang Jason Z. , Lu Tsan-Wen , Tenner Brian , Yang Jessica , Zhang Jin-Fan , Taylor Susan , Mehta Sohum , Zhang Jin
Published: April 2020

The fidelity of intracellular signal transduction hinges on the organization of signaling molecule activities into a dynamic architecture. Spatial compartmentation was first proposed over 30 years ago to explain how diverse G‐protein‐coupled receptors can regulate specific cellular processes despite converging on the ubiquitous second messenger 3’,5’‐cyclic adenosine monophosphate (cAMP). Recent work has challenged the textbook model that cAMP compartmentation is achieved by its local degradation, yet the specific mechanisms responsible for spatially constraining this diffusible messenger remain elusive. We address this longstanding question by identifying the formation of biomolecular condensates of the type I regulatory subunit of cAMP‐dependent kinase (PKA), RIα, as a key driver of cAMP compartmentation. RIα undergoes liquid‐liquid phase‐separation at endogenous levels as a function of dynamic cAMP signaling to form RIα bodies harboring high levels of cAMP and PKA activity. Importantly, we show that this active cAMP sequestration is critical for effective cAMP compartmentation in cells. The pathophysiological relevance of this compartmentation system is illustrated by its key role in the etiology of the atypical liver cancer fibrolamellar carcinoma (FLC). We show that an FLC‐linked oncoprotein fusion between DnaJB1 and the PKA catalytic subunit (PKAcat) potently blocks RIα phase separation and induces aberrant cAMP signaling. Furthermore, loss of RIα phase separation independent of the fusion oncoprotein in normal hepatocytes increased cell proliferation and resulted in cell transformation. Our work reveals liquid‐liquid phase separation as a principle organizer of signaling compartments and highlights the pathological consequences of dysregulating this activity architecture.

Support or Funding Information

This work is supported by NIH R35 CA197622, R01 DK073368 (to J.Z). J.Z.Z. is supported by a predoctoral fellowship from the National Science Foundation (DGE‐1650112).

In healthy cells, endogenous RIα acts as a dynamic cAMP buffer via phase separation, allowing for PDE‐mediated cAMP compartmentation. For fibrolamellar carcinoma (FLC) patients, DnaJB1‐PKAcat fusion oncogene disrupts RIα phase separation, thus disrupting cAMP compartmentation and leading to tumorigenesis.

Figure 1
Development of “Sharkavir”: A Hypothetical Inhibitor for HIV‐1 Protease
Authors: Hunt Matthew , Abbas Feza , Abbas Lyla , Ghali Mina , Ghali Helana , Chabbra Alesa , Schmitt Lavin Emily F. , Sikora Arthur
Published: April 2020

Human immunodeficiency virus (HIV) affects approximately 37 million people worldwide and results in over 1 million deaths annually. A class of drugs first developed in 1995 inhibits the enzyme HIV‐1 protease, thus preventing the maturation of an HIV precursor protein. There are now over 10 protease inhibitors available to treat HIV. Multiple mutations in the protein have made this treatment less effective. The CREST (Connecting Researchers, Educators, and STudents) team at Nova Southeastern University modeled the interaction between HIV‐1 protease and its competitive inhibitors. Darunavir is currently one of the strongest competitive inhibitors, as it binds effectively to the substrate envelope and has yielded a lower resistance for patients. The most effective treatment is a combination of two of these inhibitors: Darunavir and Ritonavir. In order to explain how these drugs work, the active site of the non‐mutated wild‐type HIV‐1 protease was depicted as a binding box model. The protease inhibitors Darunavir, Ritonavir, and our hypothetical drug, “Sharkavir” were 3D printed to show how they fit into the protease active site. Details of the wild‐type HIV‐1 protease, as well as the drugs Darunavir, and Ritonavir, were found in the Protein Data Bank files, 1T3R, 4DQF, and 1N49, respectively. Both structures were imported into Jmol: a protein visualization software. The new protease inhibitor molecule, “Sharkavir”, was designed as a combination of Darunavir and Ritonavir using Marvin Sketch: a software used to manipulate molecular structures. The molecular structure of the hypothetical drug “Sharkavir” is shown below.

Support or Funding Information

This project is funded in part by NSF‐IUSE #1725940 for the CREST Project.

Molecular Structure of "Sharkavir”

Figure 1
Investigating the Kinetics of β1 and β2 Adrenoceptor Effector Recruitment using Luciferase Complementation Assays
Authors: Dijon Nicola , Holliday Nicholas , Charlton Steven
Published: April 2020

Nanoluc‐based luciferase complementation assays (NanoBiT;
Dixon et al, 2016) provide new approaches to measure G protein‐coupled receptor (GPCR) signalling, by assessing the kinetics of receptor subtype recruitment of effector proteins in real‐time. Here, we use NanoBiT systems to compare β adrenoceptor subtype (β1AR/β2AR), recruitment of β‐arrestin2 and an engineered Gαs GTPase domain sensor (mini Gαs; mGαs)(
Wan et al, 2018).

HEK293T cells stably expressed SNAP‐tagged βAR (β1AR or β2AR) fused to C‐terminal 18 kDa LgBiT luciferase fragment, and the 11 amino acid SmBiT fused to either β‐arrestin2 or mGαs. NanoBiT assays were conducted in cells in 96 well plates in HBSS/0.1% BSA at 37°C. Cells were pre‐incubated with furimazine (5min; 37°C), before the addition of agonist. Luminescence was monitored for 31 min (BMG Pherastar Platereader, 37°C). Concentration response curves were fitted in GraphPad Prism v7, with pharmacology quoted as mean±s.e.m (n≥3)

Both βAR subtypes stimulated transient β‐arrestin2 recruitment upon 10μM isoprenaline stimulation (Figure
1a). At 31 min, both receptor subtypes reported partial agonism of salbutamol and salmeterol, with increased potency and efficacy (relative to isoprenaline) at β2AR compared to β1AR (Figure
1c–d). 10μM Isoprenaline also stimulated recruitment of mGas at each subtype, increasing rapidly between 0 and 5 min post agonist addition. Association of β2AR‐mGαs complexes was maintained stably throughout incubation, whilst at β1AR, mGαs recruitment continued to increase over incubation window, reaching 322.0±39.1% by 31 min (%10μM isoprenaline, at 3 min) (Figure
1b).

NanoBiT complementation assays report real‐time recruitment of β‐arrestin2 and mGαs protein, at both β1AR and β2AR. Transient β‐arrestin2 recruitment was reported at both receptor subtypes responses distinguished full and partial agonists. Further studies of the continued βAR‐mGαs association will probe whether mGαs sensors remain stably associated with membrane or endosomal pools of adrenoceptors.

Support or Funding Information

PhD funded by Biotechnology and Biological Sciences Research Council (BBSRC).

NanoBiT luciferase complementation monitors βAR recruitment of signalling effector proteins. Signalling timecourse of (a.) β‐arrestin2 and (b.) mGαs recruitment, at β1AR and β2AR. Concentration response curves of β‐arrestin2 recruitment, at (c.) β1AR and (d.) β2AR. Data points represent mean±s.e.m.; n≥3.

Figure 1
Estimation of Cerebral Vasomotor Reactivity with Near Infrared Spectroscopy in Young Adults
Authors: Shang Jing , Tomoto Tsubasa , Cardim Danilo , Zhang Rong
Published: April 2020

Cerebral vasomotor reactivity (CVMR) reflects the ability of cerebral blood vessels to regulate cerebral blood flow (CBF) in response to changes in carbon dioxide (CO2). Transcranial Doppler (TCD) measurement of CBF velocity (CBFV) in the large cerebral arteries has been widely used to evaluate CVMR. However, assessment of CVMR in the cerebral microcirculation using near infrared spectroscopy (NIRS) has not been fully explored. The aims of this study are to characterize CVMR during hypo‐ and hypercapnia using NIRS, and to investigate the relationship between CVMRs assessed by NIRS and TCD. Thirty‐six healthy adults aged 21 to 45 (women: n=23) underwent CBFV and brain tissue oxygenation measurements using both NIRS and TCD during hyperventilation (hypocapnia) and modified CO2 rebreathing (hypercapnia). NIRS was placed on the forehead to measure brain tissue oxygenation index (TOI), TCD was used to measure CBFV in the middle cerebral artery, and capnography was used to measure end‐tidal CO2 (EtCO2). The percentage changes in TOI and CBFV relative to the baseline level in response to changes in EtCO2 were calculated to assess CVMR. Both TOI (−6±2 %) and CBFV (−41±10 %) were reduced during hypocapnia, and elevated during hypercapnia (TOI: 6±2 %, CBFV: 62±20 %) (Fig.
1). CVMR assessed by NIRS was significantly lower than that measured with TCD in both hypocapnia (0.33±0.16 vs. 2.21±0.43 %/mmHg, p< 0.001) and hypercapnia (0.52±0.19 vs. 3.88±1.22 %/mmHg, p< 0.001, Fig.
2). CVMRs assessed by NIRS and TCD were correlated in hypocapnia (r=0.40, p=0.02), but not in hypercapnia (r=0.10, p=0.55). These findings suggest that NIRS measurement of TOI in responses to changes in CO2 likely reflects CVMR of the cerebral microcirculation, which is in difference from that measured in the large cerebral arteries using TCD

Support or Funding Information

The study was supported by National Institutes of Health (R01AG033106 and R01HL102457). JingShang was supported by the China Scholarship Council.

The typical recordings of hyperventilation (hypocapnia) and the modified CO2 rebreathing (hypercapnia).

Figure 1

Average values of the relative change in CBFV and TOI in 4‐mmHg bins of ΔEtCO2 during hypercapnia induced by CO2 rebreathing. Error bars indicate standard deviation. ΔEtCO2 refers to change in EtCO2 from baseline.

Figure 2
Mapping the relationship between proliferation and morphology in the developing mouse face
Authors: Green Rebecca M. , Lo Vercio Lucas , Guo Sihan , Dauter Andreas , Marchini Marta , Xhao Xiang , Marcucio Ralph S. , Hallgrimsson Benedikt
Published: April 2020

There is a long‐standing prediction that small changes in proliferation and apoptosis during the time frame of facial morphogenesis act to shape the face. Further, many studies show genetic alterations that cause structural birth defects affect local proliferation or apoptosis. Yet, it is unclear how much of local change in regional proliferation would be necessary to cause a defect. Here, we set out to understand the relationship between growth, morphology and proliferation and test that prediction that targeted proliferation shapes the developing face by quantifying proliferation and apoptosis in 3D and relating it to the growth of the face. We use whole mount staining for proliferation and apoptosis markers, whole tissue clearing methods, lightsheet microscopy and atlas and machine learning based quantification methods to identify individual proliferating or apoptotic nuclei within a 3D tissue structure at a set time point. We also employee geometric morphometric analysis of the same tissue structure to quantify overall morphology. By collecting data at various time points across facial development (E9.5–E11.5) and quantifying the age of each embryo, we are able to relate cell biological level growth to tissue level growth and morphological change and relate these two parameters in a way not performed previously.

Support or Funding Information

NIH NIDCR R01‐DE019638 to RM and BH, NSERC Discovery to BH, and CIHR Foundation grant to BH and RM, CIHR postdoctoral fellowship to RMG.

Atlas based quantification of proliferation: A) Maximum Projection of the embryo highlighting the external morphology ‐ lateral view. B) Proliferation staining (phospho‐Histone H3) ‐ lateral view. C–D) Surface morphology of the atlas (n=5) C ‐ anterior view, D ‐ Lateral view. E–F) Heat map of proliferating cells (no density correction) E ‐ anterior view, F ‐ Lateral view.

Figure 1
Luminal Succinate and Acidosis: Effects on Blood Pressure
Authors: Hering-Smith Kathleen , Huang Weitao , Teran Fred , Sato Ryosuke , Hamm L. Lee
Published: April 2020

Succinate (Suc) is well known as a Krebs cycle intermediate but extracellular Suc has been associated with a variety of pathologic processes. A distinct GPCR receptor for Suc (Sucnr1) was identified in the kidney and some (but not all) studies have documented that Suc infusions increase blood pressure (BP) via paracrine signaling. Luminal Suc has been reported to activate Sucnr1, stimulate renin release and alter BP in certain circumstances.

In the nephron, apical NaDC1 (Na+ dicarboxylate cotransporter) is located only in the proximal tubule, reabsorbs filtered citrate and Suc, and is upregulated in acidosis. Thus NaDC1’s regulation is important in preventing calcium stones and promoting acid‐base homeostasis via effects of citrate reabsorption but its role in BP regulation is not known. Our purpose was to explore the role of NaDC1, luminal Suc, and acidosis in BP regulation.

To address these issues, we used NaDC1 KO and WT mice on normal diet or 72 hr acid diet (0.4 M HCl). In initial clearance (CL) studies, KO mice had increased urinary Suc and citrate but no statistical difference in BP even with the infusion of 1 mmol/kg Suc. Suc infused WT did not have increased BP in CL. Acid diet reduced urinary Suc in WT, but had no consistent effect on BP in either KO or WT in CL.

Since recent reports suggested that Suc may affect BP exclusively during more active periods (nocturnal in mice), we additionally examined BP using telemetry (BP/T) for 24/7 monitoring. During 8 PM‐5 AM on a normal diet mean arterial pressure (MAP) was significantly higher in KO than in WT. Acidosis appeared to lessen these BP/T differences. As an example of this, Figure
1 shows MAP at selected time points in a 24 hour cycle, 8 AM, 9 PM, 5AM and 8AM.

To determine whether Sucnr1 gene expression alterations could influence these results, droplet digital PCR (Bio Rad) was performed. Data are expressed as copy numbers of target gene in 1 ng PCR reaction. Thus with ddPCR we determined on normal diet, Sucnr1 expression levels in KO (1343.6 ± 51) are significantly increased from that in WT (1052.8 ± 50, p< 0.007). Furthermore, on 72 hr acid diet the expression of Sucnr1 in KO (1039.6 ± 33) fell while in WT increased to 1148 ± 50, p <0.006).

In sum, on normal diet, Sucnr1 expression is significantly greater in NaDC1 KO vs WT and in chronic acidosis expression decreases in KO but increases in WT. On normal diet MAP in KO is significantly higher than in WT while acidosis diminishes these differences (by slightly lowering KO MAP but raising slightly WT MAP).

These diverse findings illustrate that NaDC1 (and presumably luminal Suc) can regulate BP but that this regulation varies with time of day (or associated factors such as activity) and acidosis.

Support or Funding Information

NIH, DCI PTRF, LA CaTS

MAP differences in NaDC1 KO mice vs WT mice on a normal diet

Figure 1
Clathrin‐mediated phagosome resolution regenerates lysosomes and maintains degradative capacity of macrophages after phagocytosis
Authors: Botelho Roberto , Fountain Aaron , Lancaster Charlene , Terebiznik Mauricio
Published: April 2020

Phagocytosis is the selective internalization of large particles and is critical for immunity, development and tissue homeostasis. The conversion of phagosomes into microbicidal compartments is a highly regulated process, which has been studied in great detail. Conversely, the fate of phagolysosomes, after their cargo is digested, is poorly understood. By utilizing fluorescent bacteria and labelling phagosomal markers, we followed mature phagolysosomes to their resolution. Despite previous assumptions, phagolysosomes are not exocytosed, but instead undergo a continuous dismembering process resulting in the shedding of myriads of vesicles containing degraded cargo. These phagosome‐derived vesicles resemble lysosomes, based on the acquisition of lysosomal markers, acidification and fusogenicity with newly formed phagosomes. Combining pharmacological and genetic strategies, we found that phagosomal fragmentation is dependent on cargo degradation, clathrin and the integrity of the actin and microtubule cytoskeleton. In addition, we provide evidence that phagosome‐derived vesicles are required to recycle lysosomal components allowing for the formation of new phagosomes and consequently maintaining the capacity of macrophages to ingest targets. Thus, we propose that phagolysosome resolution recycles cellular resources for the continuous generation of new phagosomes and the prevention of this phase will critically impair the immune and housekeeping functions of the phagocytes.

Support or Funding Information

Canadian Institutes of Health Research Canada Research Chairs Program Ryerson University Natural Sciences and Engineering Council of Canada

Clathrin mediates phagosome resolution. A. Macrophages engulfed mRFP1‐expressing E. coli. At 1 h post‐phagocytosis, phagosomes are rod‐shaped, like E. coli and mRFP1 is mostly associated with phagosomes. After 4 h of phagosome maturation, phagosomes shrunk and mRFP1 is found in small vesicular compartments. Clathrin inhibitors, pitstop and ikarugamycin, impair phagosome fragmentation. B. Quantification of phagosome‐derived vesicles, where b is significantly different from other conditions.

Figure 1
Development of Potent Inhibitors of the Human Microsomal Epoxide Hydrolase
Authors: Singh Nalin , Barnych Bogdan , Negrel Sophie , Zhang Yue , Magis Damien , Roux Capucine , Hua Xiude , Ding Zhewen , Morisseau Christophe , Tantillo Dean J. , Siegel Justin , Hammock Bruce D.
Published: April 2020

Microsomal epoxide hydrolase (mEH) hydrolyzes a wide range of epoxide containing molecules. Although involved in the metabolism of xenobiotics, recent studies associate mEH with the onset and development of some diseases. This phenomenon is partially attributed to the significant role mEH plays in hydrolyzing endogenous lipid mediators, suggesting more complex and extensive physiological functions. In order to obtain pharmacological tools to further study the biology and therapeutic potential of this enzyme target, we describe the development of highly potent inhibitors of the human mEH with IC50 values in the low nanomolar range, around 2 orders of magnitude more potent than previously obtained mEH inhibitors. Rationalization of binding through docking calculations of the inhibitors to a mEH homology model revealed that an acetamide, with a bulky aliphatic substituent at the α‐position, as well as an aryl ring 3–4 bonds away function as key pharmacophore units. No interaction was observed with the thioether moiety, suggesting it can be replaced by more metabolically stable (e.g. methylene) groups in future studies.

Support or Funding Information

National Institute of Health (NIH) R35 ES030443 and R01 GM076324‐11 grants; National Institute of Environmental Health Sciences (NIEHS) Superfund Research Program P42 ES004699 grant; National Science Foundation Awards 1827246, 1805510 and 1627539; Computation support from Rosetta Commons and the US National Science Foundation’s XSEDE program

Compound 62: The most potent mEH inhibitor

Figure 1

Modeling of key molecular interactions in one binding mode. Left: interactions between amide group of 62 with D226, Y299 and Y374 side chain. Right: π–π stacking between 62 and W227.

Figure 2
Mapping the Multi‐Modal Distribution of Craniofacial Phenotypes in NOSIP Mutants
Authors: Richbourg Heather A. , Saliou Alexa , Devine Jay , Green Rebecca , Hoffmeister Meike , Oess Stefanie , Hallgrimsson Benedikt , Marcucio Ralph
Published: April 2020

Nitric Oxide Synthase Interacting Protein (NOSIP) is a E3 mono‐ubiquitin ligase that regulates the activity of protein phosphatase 2A. Deletion of NOSIP leads to severe and variable malformations of the craniofacial complex. Our goal was to use NOSIP mutant mice to understand of mechanisms of phenotypic variation. The phenotype of an individual embryo is the product of the developmental trajectory that it previously traversed. We propose that this concept of canalization is essential for understanding the etiology of complex phenotypic outcomes. We have previously shown that the brain patterns the face, so sequential perturbations affecting the brain and face may create unique developmental trajectories that each embryo transits. We tested this by creating a 3D morphospace of NOSIP heterozygous and homozygous mutants to determine if embryos occupy unique morphospace regions based on affected craniofacial structures. Embryos between embryonic day 10.5 (E10.5) to E17.5 were collected, fixed, and imaged (mCT). Anatomical features were reconstructed using Avizo. NOISP expression appears to be restricted to the neuroepithelium, lower jaw mesenchyme, and mesenchyme and epithelium in tissue adjacent to the frontonasal ectodermal zone between stage E10.5‐11.5. Embryos with a single telencephalic lobe had more severe facial malformations, including micrognathia and obstructed airway without significant changes in early cell apoptosis (via TUNEL staining). Embryos with normal telencephalic vesicle separation had less severe facial phenotypes, including left‐right mild asymmetry and clefting. Overall, it appears that NOSIP‐KO creates a multi‐modal distribution of craniofacial phenotypes along the developmental timeline by affecting early brain development, suggesting that understanding canalization may provide insight into the complexity of genotype‐phenotype map (Fig.
1).

Support or Funding Information

Funding was provided by NIH F32DE08476 and R01DE019638 grants.

Schematic of the changing phenotypic landscape describing the multi‐modal distribution of craniofacial phenotypes within NOSIP+/− and NOSIP−/− embryos compared to wildtype (light blue).

Figure 1
Novel PAR2 antagonist ameliorates progression of lupus nephritis in NZB/Z F1 mice
Authors: Seo Yohan , Mun Chin Hee , Park Yong-Beom , Lee Sang-Won , Namkung Wan
Published: April 2020

Lupus nephritis (LN) is, the most frequent phenotype in patients with systemic lupus erythematosus (SLE), has a high rate of progression to end‐stage renal disease, even with intensive induction and maintenance therapies, approximately 20% of patients with LN progress to end‐stage renal disease. Recent evidence suggests that protease‐activated receptor‐2 (PAR2) is a therapeutic target for glomerulonephritis. In this study, we identified a novel PAR2 antagonist and evaluated the effect of the PAR2 antagonist on LN in lupus‐prone mouse model. To identify novel PAR2 antagonist, a cell‐based high‐throughput screening was performed. The effect of PAR2 antagonist on podocytes was observed in human podocyte cell line and LN was evaluated in NZB/W F1 mice. We identified punicalagin (PCG, a major polyphenol in pomegranate) as a novel potent PAR2 antagonist. In human podocyte cell line, PCG potently inhibited PAR2 (IC50 = 1.5 ± 0.03 μM) with ~30‐fold selectivity for PAR2 over PAR1 and significantly reduced the PAR2‐mediated activation of ERK1/2 and NF‐κB signaling pathway. In addition, PCG strongly decreased PAR2‐induced increases in ICAM‐1, VCAM‐1, IL‐6, IL‐8, IFN‐γ, and TNF‐α expression. Notably, in NZB/W F1 mice, which spontaneously develop severe autoimmune syndrome resembling human SLE and LN, the intraperitoneal administration of PCG significantly alleviated kidney injury and splenomegaly and reduced proteinuria and renal ICAM‐1 and VCAM‐1 expression. Our results suggest that PCG has beneficial effects on LN via inhibition of PAR2, and PCG is a potential therapeutic agent for LN.

Support or Funding Information

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2017R1D1A1B03029050), a faculty research grant of Yonsei University College of Medicine (6‐2016‐0159) to Sang‐Won Lee, NRF (2015R1D1A1A01057695 and 2018R1A6A1A03023718) to Wan Namkung, and NRF (2019R1I1A1A01061117) to Yohan Seo. This research was supported (in part) by the Yonsei University Research Fund (Post Doc. Researcher Supporting Program) of 2019 (project no.: 2019‐12‐0013).

Schematic overview of inhibitory effect of Punicalagin (PCG) on lupus nephritis in human podocytes and NZB/W F1 mice.

The inhibition of PAR2 by PCG reduced PAR2‐induced ERK1/2 and NF‐κB activation in human podocyte cell line. Notably, PCG ameliorated kidney injury, proteinuria, and splenomegaly in NZB/W F1 mice and reduced ICAM‐1 and VCAM‐1 levels via PAR2 inhibition.

Figure 1
Pharmacological inhibition of soluble epoxide hydrolase alleviates chronic neuropathic pain induced anhedonia symptoms: differential role of AhR and TSPO signaling
Authors: Shan Li , Ailin Luo , Dongyu Hua , Gaofeng Zhan , Hammock Bruce D. , Hashimoto Kenji , Chun Yang
Published: April 2020

Patients who suffer from chronic pain have a higher incidence to develop depression, which needs effective treatments. TPPU, a soluble epoxide hydrolase (sEH) inhibitor, was reported to have analgesia and antidepressant effects in animal models, suggesting its potential improvement in this comorbidity. Here, we sought to investigate: (1) the occurrence of anhedonia symptoms in mice with chronic neuropathic pain and the sEH protein expression in different tissues, including central nervous system and peripheral tissues; (2) the effects of TPPU on nociceptive and anhedonia behaviors in anhedonia susceptible mice, and (3) the involvement of Aryl hydrocarbon receptor (AhR) signaling and translocator protein (TSPO) signaling as possible targets for this comorbidity. Spared nerve injury (SNI) was constructed to mimic chronic neuropathic pain (CNP) model, and that mice in SNI group were classified into chronic pain with or without anhedonia phenotype via hierarchical cluster analysis of sucrose preference test (SPT) results. Protein levels of sEH were increased in medial prefrontal cortex (mPFC), hippocampus, spinal cord, liver, kidney and gut, but not in anterior cingulate cortex (ACC), nucleus accumbens (NAc), striatum, cerebellum, heart, muscle and vessel in anhedonia susceptible mice as compared with sham or anhedonia resilient mice. Chronic treatment with TPPU (3 mg/kg, orally, once per day for 7 days) improved withdraw threshold and SPT scores in anhedonia susceptible mice. Interestingly, the antidepressant, but not analgesia effect, of TPPU seems to be pharmacologically abrogated by pretreatment with AhR agonist (FICZ, orally, 100 μg/kg) via regulating inflammatory cytokines and CYP1A1 levels in the serum as well as neuroinflammation in the microglia and astrocytes. Interestingly, the classical TSPO pathway, a crucial factor in the analgesia and antidepressant effects of TPPU, failed to show any difference in subjects who were treated with drugs targeted at the AhR. Collectively, our findings indicate that TPPU may alleviate depression‐like symptoms in CNP mice through two independent pathways: (1) the direct antidepressant effects via regulating neuroinflammation mediated by AhR, and (2) the improvement of allodynia and depressive symptoms via the TSPO signaling and downstream neurosteroids. Thus, AhR provides a potential therapeutic target for treatment of depression induced by chronic pain, and strategies targeting TSPO signaling could be a vital approach to alleviate the comorbidity of chronic pain and depression.

Support or Funding Information

This work was supported , in part, by grants from the National Natural Science Foundation of China (to A.L., 81771159 and 81571047; to C.Y., 81703482)

The overview of our manuscript.

Figure 1
Structural Characterization of the Apoptosis Signal‐regulating Kinase 1 (ASK1) Domain Responsible for Thioredoxin Binding and its Complex
Authors: Obsil Tomas , Psenakova Katarina , Hexnerova Rozalie , Srb Pavel , Obsilova Veronika , Veverka Vaclav
Published: April 2020

Apoptosis signal‐regulating kinase 1 (ASK1) is a mitogen‐activated protein kinase kinase kinase 5 implicated in p38 mitogen‐activated protein kinase and c‐Jun N‐terminal kinase signaling cascades. ASK1 responds to a wide range of signaling cues and is dysregulated in cancer, neurodegeneration and cardiovascular diseases. The catalytic activity of ASK1 is tightly controlled through oligomerization and binding of several cofactors, among which thioredoxin stands out as the most important ASK1 inhibitor. Interestingly, only the reduced form of thioredoxin inhibits ASK1 by direct binding to its N‐terminal domain and oxidation‐driven thioredoxin dissociation is the key event in oxidative stress‐mediated ASK1 activation. Despite extensive efforts, the molecular basis of this key cellular regulatory mechanism has remained unknown until now, primarily due to the dynamic properties of the ASK1 N‐terminal domain, which has prevented its structural characterization by X‐ray crystallography. Here, we report the structural characterization of the ASK1 domain responsible for thioredoxin binding and of the ASK1: thioredoxin complex using NMR spectroscopy and chemical cross‐linking, which provides the molecular basis for complex dissociation under oxidative stress conditions. Our data reveal that the N‐terminal domain of ASK1 adopts a fold resembling the thioredoxin structure while retaining substantial conformational plasticity at the thioredoxin‐binding interface. Although oxidative stress induces relatively moderate structural changes in thioredoxin, we show that these changes lead to a substantial conformational rearrangement of the thioredoxin‐binding interface on ASK1 driven by the formation of intramolecular disulfide bridges. Lastly, we demonstrate that the cysteine residue at the position 250 of ASK1 is the key element of this molecular switch.

Support or Funding Information

This work was supported by the Czech Science Foundation (Project 19‐00121S), the Grant Agency of the Charles University (Project 368216), the Czech Academy of Sciences (Research Projects RVO: 67985823 of the Institute of Physiology) and partially also by the European Regional Development Fund; OP RDE; Project: ChemBioDrug (No. CZ.02.1.01/0.0/0.0/16_019/0000729).

Structural model of the ASK1‐TBD:TRX complex calculated using the program HADDOCK with the NMR‐based structural model of ASK1‐TBD, and crystal structure of reduced TRX1 (PDB ID: 1ERT).

Figure 1