bluuebunny/crossref_metadata_2025 huggingface dataset:
Pontine Parabrachial Nucleus (PBN) Neuron Subtypes Involved With the Control of Breathing Frequency
Authors: Zuperku Edward J. , Hopp Francis A. , Stuth Eckehard A. , Stucke Astrid G.
Published: April 2020

A subregion within the medial PBN plays a key role in the control of expiratory duration (TE) and breathing frequency (fB)
1. Drug‐induced increases in neuronal activity decrease TE, while depression of these neurons increases TE. Activity from these neurons is relayed with short latencies to neurons in the preBötzinger/Bötzinger region
2. This study analyzed the relationship between charateristics of the discharge frequency (Fn) patterns of I, E, phase spanning and non‐respiratory modulated (NRM) PBN neurons and changes in fB induced by local application of neural transmitter agonist and antagonist. Multichannel neuronal recording with highly localized microinjections into the PBN were performed in decerebrate, vagotomized dogs during isocapnic hyperoxia. The phrenic neurogram was used to assess fB responses and identify neuronal discharge patterns. Plots of fB vs. neuronal Fn during induced fB responses produced by microinjections of AMPA and the GABA‐A receptor antagonist bicuculline (BIC) were linear with high correlation values (r > 0.75). Furthermore, it appears that neurons with little or no respiratory modulation appear to be more highly correlated with fB than neurons with strong phasic patterns, suggesting that these neurons may be responsible for the pontine control of fB.

Support or Funding Information

Supported by Department of Veterans Affairs grant 2 I01BX000721‐08 (Zuperku) & R01GM112960‐01A1 (Stucke)

Example of breathing frequency response (BPM) to microinjected AMPA (green) in PBN and corresponding neuronal responses (red) for 2 neurons (panel A and panel B). Lower: Plots of BPM vs discharge frequency (Fn = Fmax) for two simultaneously recorded neurons, phasic‐I (A) and NRM (B) with corresponding slope and r‐squared values. Top: Cycle‐Triggered Histograms (CTHs)

Figure 1
Impaired Microvascular Endothelial Function in Preeclampsia
Authors: McIntosh Jennifer Jury , Derayunan Alexa , Hader Shelby , Lohr Nicole , Beyer Andreas , Gutterman David
Published: April 2020

Rational

Women with preeclampsia (PE) have compromised endothelial function (assessed by brachial artery flow‐mediated dilation) compared to normal women. Placental and uterine spiral artery endothelial dysfunction is seen in PE pregnancies. Human studies remain incomplete, as most approaches have been unable to eliminate neural, humoral, paracrine influences on vessel response. Direct evaluation of the placental arterioles will allow a more direct analysis of vasodilatory responses as related to the endothelial dysfunction in PE.

Objective

To study placental endothelial function in preeclamptic (PE) and normal placental vessels.

Methods

To examine the impact of PE on the microvascular function, isolated vessels from placentas of healthy and PE pregnancies were used for videomicroscopy. Endothelial‐dependent dilation to flow (FMD), acetylcholine (ACh) and smooth muscle‐dependent dilation to papaverine were evaluated. Data are presented as a percentage of maximum diameter and were analyzed with 2‐way ANOVA RM with statistical significance set at *p<0.05; data are expressed as mean ± SE.

Results

Vessels from PE patients resulted in impairment of FMD (control 71.% ±6.6%, PE 31.6%±10.7%*) and dilation to Ach (control 63.6%±9.4, PE 32.7±8.83%*). Smooth muscle‐dependent dilation to papaverine was unchanged between the two groups (Figure
).

Conclusion

Preliminary studies show impaired endothelial function in placental vessels of subjects with PE, compared to healthy controls, while smooth muscle function was not affected. Understanding the underlying mechanism(s) of vasodilation in placental vasculature will further the understanding the pathophysiology of PE.

Support or Funding Information

The project described was supported by the National Center for Advancing Translational Sciences, National Institutes of Health, Award NumberUL1TR001436. The content is solely the responsibility of the author(s) and does not necessarily represent the official views of the NIH. This project is funded by the Research and Education Program Fund, a component of the Advancing a Healthier Wisconsin Endowment at the Medical College of Wisconsin.

Vessels from PE patients resulted in impairment of FMD and dilation to ACh. Smooth muscle‐dependent dilation to papaverine was unchanged between the two groups. Statistical significance denoted with *

Figure 1
CFTR‐mediated transepithelial bicarbonate conductance is affected differently than chloride conductance by CFTR correctors and potentiators
Authors: Schultz Bruce D , Bridges Robert J
Published: April 2020

The advent of targeted CFTR therapeutics allows us to assess differences in epithelial Cl− and HCO3− conductances that might be attributed to unique combinations of CFTR variants (mutations), CFTR modulators (corrector(s) and potentiators) as well as secretory agonists. To address these questions, Fisher rat thyroid cells expressing wt or mutant CFTR were cultured on permeable supports for the measurement of transepithelial conductance (Gt) in defined ionic conditions. Initial experiments demonstrated stepwise reduction in the forskolin (Forsk)+ivacaftor (VX770)‐stimulated change in Gt as bath NaCl was partially replaced isosmotically with mannitol. Subsequent experiments were conducted in buffers containing 100 mEq/l Cl− with the addition of either 25 mM alternative anions (Na+ salts) or 50 mM mannitol. Outcomes showed forskolin‐induced Gt changes consistent with reported CFTR permselectivity: Cl− > NO3− ≥ Br− > isethionate ≥ gluconate > mannitol. All subsequent experiments were conducted to assess Gt with 100 mEq/l Cl− plus either 25 mEq/l HCO3−, 50 mM mannitol or 25 mEq/l Cl−, in a 5% CO2 atmosphere. Epithelia expressing wtCFTR respond to forskolin with a substantial increase in Gt. The response in HCO3− was ~75% of that observed with Cl−. Exposure to VX770 increased conductance, but a disproportionately lesser change was observed in HCO3− (vs. Cl−), providing an initial suggestion that VX770 promotes differential ion selectivity.

Exposure of cells expressing F508del CFTR to correctors, either C17 or C18, allowed for forskolin‐induced stimulation of Gt with a modest increment of HCO3− conductance. In these conditions, VX770 induced additional Cl− conductance, but no enhancement of HCO3− conductance could be detected. Alternatively, cells exposed to the corrector combination (C17+C18) showed a substantial forskolin‐stimulated increase in anion conductance and a modest increase in CFTR‐associated HCO3− conductance. Thus, the combination of C17 and C18 gives a different outcome regarding F508del‐CFTR HCO3− conductance than either corrector alone.

Cells expressing G551D CFTR exhibited little response to forskolin, but substantial responses to VX770. Responses to VX770 in HCO3− approached the magnitude of the responses observed in Cl−.

Taken together, the results suggest that CFTR correctors and potentiators can enhance CFTR‐mediated anion conductance, but that significant differences in the conductances of HCO3− relative to Cl− are observed between CFTR variants and that responses can be influenced by the correctors employed. Ultimately, these results suggest that CFTR‐associated pH regulation will be impacted by both the patient’s mutation and the CFTR modulator treatment. Furthermore, additional studies are required to evaluate the CFTR modulators for their effects on Cl− and HCO3− conductances on the many CF causing CFTR variants.

Support or Funding Information

CFF BRIDGE18XX0; K‐State CVM SMILE
Variation in Fractional Flow Reserve (FFR) and Its Effects on Stenting Decision Making
Authors: Handly Neal , Heyndrickx Guy R. , Kerkhof Peter L.
Published: April 2020

FFR is an important construct using intra‐arterial pressures to describe blood flow past partial stenoses in coronary arteries to determine the need for percutaneous coronary intervention. Point estimates of the FFR can be compared to two threshold values to determine three decision "zones ": FFR <0.75 such that stenting (S) of this lesion should be performed, 0.750.80 which indicates that no stenting (NS) of the lesion should occur at this time.

Variation of FFR measurements would be expected due to factors at the operator, protocol, and instrument levels. Subject physiology, such as the level of venous pressure, collateral flow, heart rate and size /geometry of the stenosis, can also have an effect. This variation might lead to errors in the decision making process. However, FFR variability is poorly described in the literature and trade documents.

This study was designed to explore the role of FFR variability on probabilities of errors in decision making when measuring a FFR value that was considered to be normally distributed about 0.74, 0.78 and 0.81 (being examples of true values that may occur in each of the decision zones) and with four different standard deviations (SD) in the measure of FFR.

Normal distributions of these FFR were generated with SD of 0.01, 0.02, 0.029 and 0.05 with resampling software (J. Grosberg, Statistics101 Version 4.9). These SD values were based on repeated FFR measures on 6 subjects (Aalst, GRH), from which an average deviation of 0.029 was found. The 0.01, 0.02 and 0.05 SD values were included to further explore their effect on FFR distributions. Probabilities of FFR measures that would be found in zones S, AE and NS were generated to show the frequency of erroneous versus true FFR values.

The results for each chosen FFR and SD combination are given in the accompanying table, showing probabilities of FFR measures in the S, AE and NS zones.

In general, the greater the variation as described by the SD, the greater is the probability of any single measurement being incorrect (i.e., being found in a zone different from its true value). The variability in FFR measurement should be quantified since it has bearing on possible errors in decision making as well as serving guidance in technique improvement. Efforts to assure low variability are important.

FFR 0.74

SD

Zone

S

AE

NS

(true S)

0.01

0.841

0.159

0.000

0.02

0.692

0.307

0.001

0.029

0.635

0.346

0.019

0.05

0.579

0.306

0.115

FFR 0.78

SD

(true AE)

0.01

0.001

0.976

0.023

0.02

0.066

0.775

0.158

0.029

0.151

0.604

0.245

0.05

0.275

0.380

0.345

FFR 0.81

SD

(true NS)

0.01

0.000

0.158

0.842

0.02

0.001

0.308

0.691

0.029

0.019

0.345

0.636

0.05

0.115

0.306

0.579
The Correlation between Common Variable Immunodeficiency and Atrial Fibrillation: Cardiac Implantable Electronic Devices‐associated infection by Stenotrophomonas maltophilia: A case report
Authors: Tsai Jeffrey , Nguyen Nam H. , Dylewski John R.
Published: April 2020

Background

Common variable immunodeficiency (CVID) is associated with systemic infections and autoimmune diseases which may elicit and perpetuate atrial fibrillation via inflammatory and oxidative mechanisms. CVID patients with early‐onset atrial fibrillation (AF) and chronic heart failure (CHF) are refractory to standardized cardiac ablation and frequently need sophisticated cardiac resynchronization therapy. Moreover, CVID‐CHF patients with cardiac implantable electronic devices (CIEDs) are vulnerable to opportunistic infection, especially Stenotrophomonas maltophilia bacteremia, which has high resistance to a variety of empirical antibiotics against gram negative bacteremia.

Case presentation

31 year‐old female with a past medical history of chronic refractory AF, CHF, thyroid cancer, non‐Hodgkin lymphoma, and factor V deficiency presented to the hospital with a fever of 104.0F for 2 days after recent dual‐chamber biventricular defibrillator replacement 2 weeks ago. 2 months ago, the patient was diagnosed with CVID due to recurrent cardiopulmonary infections. Upon this admission, the patient’s blood culture demonstrated S. maltophilia which was likely from the complications of CIEDs placement.

Conclusion

Early‐onset Atrial fibrillation in young population may be a feature of CVID. Early recognition of CVID in these patients, especially with comorbidity of CHF, can mitigate the progression of AF with antioxidants as well as promptly identify and manage CIEDs‐associated infections, such as S. maltophilia.

Figure 1
Understanding the Molecular Underpinnings of Neuronal Kinase CDK5 mediated Metabolic Vulnerabilities in Neuroendocrine Tumors
Authors: Gupta Priyanka , Strange Keehn , Carter Angela , Telenge Rahul , Meijer Laurent , Ghayee Hans , Pacak Karel , Reddy Sushanth , Bibb James
Published: April 2020

Background

Aberrantly activated neuronal kinase CDK5 invoke alternations in mitochondrial dynamics and metabolic dysfunction associated with neurological disorders. Although pro‐tumorigenic role of CDK5 has been recently defined but there is insufficient data to understand the mechanistic role of CDK5 in cancer associated mitochondrial structure and function.

Hypothesis

In this study we intend to investigate putative signalling hotspots downstream of aberrantly activated CDK5 in neuroendocrine tumors of adrenal glands called Pheochromocytoma (PC).

Approach

Our preliminary data for the first time showed deregulation of Ca2+ homeostasis and overexpression of CDK5 and its co‐activators‐p35/25 in malignant PC tumors inflicted with loss‐of‐function mutations in mitochondrial enzyme‐Succinate dehydrogenase B (SDHB). To elucidate role of CDK5 in tumorigenesis, we created a novel bi‐transgenic mouse model where doxycycline inducible overexpression of CDK5/p25 (p25OE) developed organ specific PC lesions.

Results

Phopshoproteomic analysis on p25OE PC tumors unraveled a tumor suppressive mechanism modulated by novel phosphorylation site on metabolic protein‐PRKAG2 (S‐65). PRKAG2 is a non‐catalytic regulatory subunit of AMP‐activated protein kinase (AMPK). S‐65 site on PRKAG2 were found downregulated in human PC tumors compared to normal adrenal medulla. Further biochemical characterization uncovered CDK5‐GSK3β‐AMPK crosstalk underlying human PC cellular proliferation. Picking CDK5 pocket with a novel CDK5 inhibitor ‘MRT3‐007’ induced anti‐proliferative effects with simultaneous increase in phosphorylation states of PRKAG2(S‐65) / AMPK(T‐172) sites followed by excessive mitochondrial fission.

Conclusion

These intriguing initial findings set the ground to discover therapeutic vulnerabilities associated with CDK5/p25/PRKAG2/AMPK signaling cascade and Mitochondrial Dynamics in Neuroendocrine tumors.

Schematic depiction of working model demonstrating a novel crosstalk between a metabolic sensor ‘PRKAG2/AMPK’ and ‘Mitochondrial Fission’ synapsed via neuronal kinase ‘CDK5/p25’ in Pheochromocytoma (PC tumors) inflicted with mutations in SDHB gene.

Figure 1
Time Constant and Length Constant: Graphical Illustrations for Effective Teaching and Learning
Authors: Luedtke Alyssa , Lewis Austin , Kuang Serena Y.
Published: April 2020

Introduction

This is a teacher‐guided students’ presentation. The time constant (τ) and the length constant (λ) are the cell membrane’s passive cable properties that influence the velocity of the spread of graded potentials and thus the velocity of the propagation of action potentials. In teaching and learning the electrophysiology of the cell membrane, we noticed that the information about the two concepts in many current physiology textbooks are incomplete and inconsistent. Some books introduce both τ and λ, whereas some other books do not introduce them. Some books introduce only τ, whereas some other books introduce only λ. Some books use the rising phase of a graded potential to illustrate τ, whereas some other books use the falling phase. Graphical illustrations about these concepts are generally not available in these physiology textbooks. This situation makes it confusing for students to understand the two concepts. In this presentation, we address the above problems and share our figures to facilitate the learning of the two concepts.

Method

Textbook comparisons and graphical illustrations.

Results

1) The incompleteness and the inconsistency about the two concepts will be addressed in a table using a few sampled textbooks. 2) A set of figures (not shown) are used to provide a complete illustration about τ in both the rising phase and the falling phase of a graded potential; and the rising phase is then used to graphically illustrate how τ affects the propagation of an action potential (Figure
1). 3) How λ affects the propagation of an action potential is illustrated graphically using the falling phase of a graded potential (Figure
2).

Conclusion

Graphical illustrations are of great value for the effective teaching and learning of the concepts of time constant and the length constant.

Time constant (τ) determines how fast a local current depolarizes Vm to Vthreshold to trigger an action potential.

Figure 1

Length constant (λ). A: The λ influences the spread of a local current (or the graded potential it generates). The greater the λ, the farther the local current (or the graded potential) spreads. B: The greater the λ, the farther the local current (or the graded potential) spreads, so the more voltage‐gated Na+ channels (VGSCs) in a regional area are likely to open and the faster the AP propagation. VGKC: voltage‐gated K+ channels.

Figure 2
Reductions in Microvascular Function can be Detected by Near‐infrared Spectroscopy (NIRS) following Ischemia‐Reperfusion in Early Postmenopausal Women
Authors: Somani Yasina B. , Soares Rogerio N. , Gosalia Jigar , Delgado Jocelyn , Murias Juan M. , Proctor David N.
Published: April 2020

The onset of menopause and accompanying changes to ovarian hormones often precedes endothelial dysfunction in women. In particular, accelerated impairments in microvascular function coincides with the loss of estrogen, as does impaired endothelial resilience to ischemia‐reperfusion (IR) injury. In early postmenopausal women (4 ± 1 years since menopause), we tested the hypothesis that the NIRS‐derived measurement of vascular responsiveness (reoxygenation slope, %.s−1) in the forearm could detect reductions in microvascular function following whole‐arm IR injury. In conjunction, brachial artery flow‐mediated dilation (FMD) and shear rate (area under curve; AUCpeak) were measured. Thirteen healthy, normotensive (116 ± 11/72 ± 9 mmHg) postmenopausal women (57 ± 3 years) were tested before (PreIR), after (PostIR), and 15 minutes following IR (Post15). The IR injury was achieved by inflating a cuff around the upper‐arm to 250 mmHg for 20 min followed by 15 min of reperfusion. The NIRS‐derived reoxygenation slope was lower PostIR (1.02 ± 0.56%.s−1 vs. 1.23 ± 0.64%.s−1 PreIR; p =0.04), but not at Post15 (1.17 ± 0.51%.s−1; p =0.08). Brachial artery FMD was lower PostIR (3.57 ± 0.52% vs. 8.14 ± 2.44% PreIR; p < 0.001), but not at Post15 (7.61 ± 1.89%; p = 0.54). Shear AUCpeak were similar across tests (p >0.05). Our findings suggest that the NIRS‐derived reoxygenation slope is a non‐invasive method that may detect transient impairments in forearm microvascular function, and may also be used to assess efficacy of therapeutic interventions in improving both vascular function and resilience in this population.

Support or Funding Information

Penn State Human Health and Development Endowment Award

Schematic of study timeline

Figure 1
Anticancer mechanisms determination of L‐aminoacids compounds serie on MDA‐MB‐231 cell line TNBC
Authors: Martinez Doris Cruz , Jiménez Elvia Mera , Percino Teresa Mancilla , Pérez Martha Edith Macías , Rodriguez Maricarmen Hernández
Published: April 2020

Breast cancer represents the second most frequently diagnosed neoplasm worldwide of which approximately 15% is subclassified as triple negative breast cancer (TNBC)
[1], characterized by the absence of estrogen receptors, progesterone and growth factor Her2/neu. TNBC is considered as one of the most aggressive cancers, due to its null response to current endocrine therapies and trastuzumab
[2]. Breast cancer is a pathology associated with aging and unhealthy lifestyles, it has been proven through in vitro studies that MDA‐MB‐231 TNBC cells overexpress HDAC8 as a consequence of epigenetic deregulation mechanisms, linked to the high proliferative capacity and therefore, a high risk of recurrence and low survival
[3]. In this context, our work group have evaluated a serie of compounds on the MDA‐MB‐231 cell line, these compounds have been designed, synthesized and evaluated as iHDAC8 by Docking. Therefore, this work presents the cytotoxicity and evaluation of the antitumor effect on the MDA‐MB‐231 cell line of three compounds derived from L‐amino acids, BisLys, GluEt and I1, which contain the isoindolinyl group and I1 which has a carboxybenzoyl group. To evaluate citotoxicity of BisLys, GlutEt and I1 compounds, MDA‐MB‐231 cells were seeded and incubated at 37°C in a 5% CO2 with DMEM F12 medium supplemented with FBS. Once the cells reached 95% confluence they were treated for 24 hours adding 8 different concentrations of each of the compounds; DMEM F12 medium was used as a negative control and SAHA as a positive control. The proliferation and viability of TNBC cells was quantified by the colorimetric for 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. Type of cell death whether it was necrosis or apoptosis was determined using Annexine V and propidium iodide by flow cytometry. Finally, the blocked cell cycle progression by those compounds was determined using Cayman 10009349 Kit by flow cytometry. The BisLys, GluEt and I1 compounds showed IC50 values of 0.83, 0.66 and I1 3.42 mM, respectively, and inhibited cell proliferation and viability in a dose‐dependent manner on the MDA‐MB‐231 cell line of TNBC. The GluEt compound induced death mainly by necrosis and the BisLys and I1 compounds by apoptosis. The BisLys, GluEt and I1 compounds induced cell cycle arrest in a dose‐dependent manner at the expense of the subG0/G1 phase. The compounds that induced the greatest apoptosis in MDA‐MB‐231 cells were BisLys and I1 which makes them leading compounds in suppressing the growth of MDA‐MB‐231 TNBC cells.
Structural Basis of Protein Kinase C Tumor‐Promoting Response And Membrane Recruitment
Authors: Igumenova Tatyana I. , Katti Sachin , Yu Binhan , Cole Taylor R.
Published: April 2020

Protein Kinase C (PKC) is a family of Ser/Thr kinases that regulate a multitude of cellular processes through participation in the phosphoinositide signaling pathway. The hallmark of their activation is translocation to lipid membranes. Dysregulation of PKC signaling has been implicated in cancer progression, cardiac disease, and Alzheimer’s disease. One of the major challenges in the field is to understand the structural basis of PKC activation and regulation ‐‐ at the level of atomic resolution.

Here, we applied NMR and FRET spectroscopy to gain insight into the structure and protein‐membrane interactions of the regulatory domain of PKC that consist of three peripheral membrane modules: the tandem conserved homology‐1 (C1) and conserved homology‐2 (C2) domains. The C1 domains undergo membrane insertion upon binding their natural agonist, diacylglycerol, as well as tumor‐promoting phorbol esters and other small‐molecule agonists. C2 associates with anionic membranes upon binding Ca2+. Simultaneous association of all domains with membranes ensures the coincidence of four signals: Ca2+ ions, diacylglycerol, phosphatidylserine, and phosphatidylinositol‐4,5‐bisphosphate.

Here, we report the first structure of the C1 domain complexed to a potent tumor‐promoting agent, phorbol 12,13‐dibutyrate (PDBu), in the membrane‐mimicking environment. The structure of the complex shows that C1 has to undergo a conformational change in order to capture the membrane‐embedded ligand. The structure also revealed the role of individual amino acids in the interactions with PDBu and membrane‐mimicking environment. To probe how different agonists modulate the interaction of C1 with membranes, we conducted NMR paramagnetic relaxation enhancement (PRE) experiments with bicelles doped with paramagnetic lipid. The PRE patterns revealed that the C1 domains adopt tilted rather than vertical orientation in the membrane. Our data on four different agonists indicate that C1 domains dictate the general orientation of the agonist complexes in the membrane, but the agonists determine the depth of insertion. This provides a plausible explanation for the differences in the potency of these ligands to achieve the acute and chronic activation response of PKC.

To gain insight into the mechanism of coincidence detection, we developed protein‐to‐membrane FRET assay that we used to characterize the interplay of the C1 and C2 domains in the membrane association step. The FRET experiments revealed a clear picture of how tumor‐promoting agents can elicit constitutive activation of PKC through simultaneous recruitment of C1 domains to membranes and sensitization of the C2 domain to Ca2+.

Support or Funding Information

NIH R01 GM108998; NSF CHE‐1905116; Welch Foundation A‐1784

Agonist‐guided membrane interaction of C1B domain of PKCδ studied by paramagnetic NMR. (A) Schematic representation of the approach. (B) Chemical representations of the different agonists used in the study.

Figure 1
Pancreatic Stellate Cells Serve as a Brake Mechanism on Pancreatic Acinar Cell Calcium Signaling with Modulation by Methionine Sulfoxide Reductase Expression
Authors: Cui Zong Jie , Liu Jin Shuai
Published: April 2020

Although methionine sulfoxide reductase (Msr) is known to modulate activity of multiple functional proteins, roles of Msr in pancreatic stellate cell physiology have not been reported. In the present work we investigated expression and function of Msr in freshly isolated and cultured rat pancreatic stellate cells. Msr expression was determined by RT‐PCR, Western blot and immunocytochemistry. Msr over‐expression was achieved by transfection with adenovirus vectors. Pancreatic stellate cells were co‐cultured with pancreatic acinar cells AR4‐2J in monolayer culture. Pancreatic stellate and acinar cell function was monitored by Fura‐2 calcium imaging. Rat pancreatic stellate cells were found to express MsrA, B1, B2, their expressions diminished in culture. Over‐expressions of MsrA, B1, or B2 were found to enhance ATP‐stimulated calcium increase but decreased reactive oxygen species production and lipopolysaccharide‐elicited IL‐1 production. Pancreatic stellate cell‐co‐culture with AR4‐2J blunted cholecystokinin‐ and acetylcholine‐stimulated calcium increases in AR4‐2J, depending on acinar / stellate cell ratio, this inhibition was reversed by MsrA, B1 over‐expression in stellate cells or by Met supplementation in co‐culture medium. Different from the pancreatic acinar cells, although pancreatic stellate cells also express the cholecystokinin 1 receptor, the receptor is not coupled to calcium signaling. In addition, Msr‐overexpression only partially restored cholecystokinin 1 receptor coupling to the calcium signaling pathway in pancreatic stellate cells, whereas addition of free Met in the culture medium had no effect. These data suggest that Msr play important roles in pancreatic stellate cell function and the stellate cells may serve as a brake mechanism on pancreatic acinar cell calcium signaling modulated by stellate cell Msr expression. Pancreatic stellate cell cholecystokinin 1 receptor is de‐coupled from calcium signaling, Msr overexpression only partially restores cholecystokinin‐stimulation of calcium increases at high CCK concentrations in the stellate cells.

Support or Funding Information

This work was supported by grants from NSFC (31670856, 31971170).

Figure 1
SPARC: Development of Human and Rodent Neuro‐Functionalized Computational Anatomical Models with Detailed Mapping of Peripheral Nervous System
Authors: Lloyd Bryn , Farcito Silvia , Cassarà Antonino , Zhuang Katie , Neufeld Esra , Kuster Niels
Published: April 2020

There is a growing trend within the neuroscience community and the medical and health industries towards bioelectronics medicine: applying electrical signals to the nervous system to control and modulate functions of the body. These so‐called “electroceuticals” include numerous types of neurostimulation devices. To this end, we have developed reference animal and human anatomical models with unprecedented details in the peripheral nervous system, and connectivity to organs and muscles, which were functionalized with compartmental neuronal dynamics models to investigate device interactions with neuronal electrophysiology. The whole body rat model is being segmented from co‐registered high‐resolution (80 × 80 × 80 μm) magnetic resonance imaging (MRI) and computed tomography (CT) images of a female rat. The Visible Korean Human
(1) male and female cryosection data was used as the basis for the new human phantoms, due to the unique resolution (0.1 × 0.1 × 0.2 mm) and quality of these images. To model important peripheral nerves trajectories, the nerves were segmented and anatomically correct trajectories were extracted automatically using a set of anatomical rules specifying, which dorsal or ventral roots are connected to a given nerve. Functionalization is achieved by assigning electrophysiological models of myelinated and unmyelinated axons to axon trajectories within nerve models based on histological investigations documented in the literature
(2).

The new human female and male human models called Yoon‐sun and Jeduk respectively, have been released as part of the Virtual Population
(3) V4.0 model library. The neuro‐functionalized rat will be completed soon. These models will become integration centers for NIH SPARC neuroanatomy and electrophysiology models
(4) and are expected to significantly influence the field of computational neuro‐electrophysiology research enabling studies of multi‐scale models with realistic anatomies and electrophysiology. Simulations will enhance our understanding of mechanisms of neurostimulation, e.g., by MRI gradient fields, provide experimental test‐beds for new therapeutic approaches and devices, and enable study of safety aspects, thus providing a tool to facilitate regulatory submissions and standardization activities.

Support or Funding Information

This work has been done with funding from NIH SPARC (1OT3OD025348‐01S1). The work on human anatomical models also received funding from Innosuisse (25290.1 PFLS‐LS) and KIAT.

Computational rat model is shown with muscle tissue and limbs clipped to show interior tissue structures.

Figure 1

Side‐by‐side visualization of different tissue structures in the female computational human anatomical model ‘Yoon‐sun’. The model contains approximately 1100 separate tissues, with detailed bones, organs, muscles, blood vessels and peripheral nervous system (right).

Figure 2
Exploring the Anti‐Cancer Properties of Efavirenz, in Comparison to Thalidomide
Authors: McNeil Rosie Thecla , Penny Clement Brian
Published: April 2020

Introduction

It has been suggested that “highly active antiretroviral therapy” (HAART) caused resolution of secondary pulmonary cancers. The question then is: ‘Do antiretroviral drugs (ARD) possess anti‐cancer properties, and if so, then how? Efavirenz, a Non‐Nucleoside Reverse Transcriptase Inhibitor (NNRTI) and a first line drug used to manage HIV‐1 infection in both adults and children, is hereby investigated, in comparison with the known anti‐angiogenic and anti‐cancer drug, thalidomide.

Methodology

Embryonic CAMs of 30 fertile eggs of the domestic fowl (Gallus gallus variant Domesticus) were used as in vivo vascular test environment as follows: they were randomly allocated (n=6) to Human recombinant VEGF (R&D Systems), Thalidomide (Sigma‐Aldrich), Control 1 group treated with 70% ethanol (diluent), Control 2 group of embryos that were not manipulated and Efavirenz, (Sequoia Research Products).

Results

Efavirenz suppressed angiogenesis in Chick CAM in 100% of the subjects, showing more anti‐angiogenic properties than thalidomide. Embryonic viability (as a product of CAM angiogenesis) in all the experimental groups (n=6) was as follows: Control 6 (100%); VEGF 5 (83.3%); Thalidomide 1 (17%) and Efavirenz 0 (0.0%). Thalidomide failed to suppress angiogenesis in 17% of the subjects. Moreover, unlike thalidomide, Efavirenz also suppressed erythropoiesis.

Discussion and Conclusion

The role of vascular endothelial growth factor (VEGF) in both developmental and pathological angiogenesis has been studied (e.g. Ferrara, 2000; Hagedorn et al. 2004; Losordo et al., 2004; Maharaj, 2006; Makanya et al., 2007). VEGF is a specific and potent mitogen for endothelial cells that is expressed in a distinct temporo‐spatial pattern during mammalian lung development. Since cancer cell growth and viability depends on neo‐vascularization, this property of Efavirenz, which is antagonistic to VEGF activities, may cause spontaneous resolution of HIV‐associated cancers in patients placed on HAART. In a future work, the mechanism of action of this notable ARD will be further investigated.

Support or Funding Information

Supported by Sefako Makgatho Health Sciences University (SMU), Ga‐Rankuwa, Gauteng province of South Africa CAM Images CAM IMAGES

CAM Images

Figure 1

Results

Group

Mean Angiogenic effect

Standard Deviation

F

P‐value

VEGF

0.83

0.41

Thalidomide

0.17

0.41

9.306

*0.0005

Efavirenz

0

0

Control 1

0.67

0.52

Control 2

1

0

*indicates the p value is significant, p < 0.005. Possessing 17% angiogenicity implies the thalidomide failed as an anti‐angiogenic drug in the one embryo. Moreover, one of the embryos the thalidomide group reached the end of the experiment but died within seconds of exposure. CAM had one short thriving blood vessel on the CAM surface. In Efavirenz‐treated CAMS, no blood vessels were seen and neither was there any sign of erythropoiesis.
Experimental Diabetic Nephropathy is Associated with Increased Expression of Inflammatory Proteins in the Kidney
Authors: Deshmukh Hemalata , Prabhakar Sharma
Published: April 2020

Aims

Diabetic Nephropathy (DN) is the leading cause of end stage renal disease (ESRD) but pathogenesis of DN is far from clear and hence the treatment remains sub‐optimal. Many studies alluded to the role of chronic inflammation in DN. A recent study in human DN identified 17 different inflammatory proteins collectively called Kidney Risk Inflammatory Signature (KRIS) molecules. (Niewczas et al Nat Med 2019) . These proteins, mostly belonging to tumor necrosis factor (TNF) family predicted risk of progression to ESRD. We tested the hypothesis that the expression of some of such KRIS proteins in experimental models including renal cells cultured in high ambient glucose concentration and kidneys from diabetic animal models is increased. 2.

Methods

To test our hypothesis, we used rat mesangial cells (RMC) in culture and ZSF rats —an established animal model for DN. RMC were grown in DMEM media with either low glucose (5.5 mM) or high glucose (25 mM) for 48 hours and expression of these molecules was determined by Western Blot technique and ELISA. Specifically, we examined TNFα R1 and IL15α proteins. In addition RMC grown in low glucose media for a week were incubated in high glucose media for 48 hours and examined for KRIS proteins. ZSF rats (both male and female) were grown on Purina 50008 diet to render them hyperglycemic and euthanized at 40 weeks of age. Male CD rats (non‐diabetic) were used as controls. Blood and urine samples were obtained at the start and end of the study. Kidneys were harvested, tissue was homogenized and homogenates examined for protein expression by WB. 3.

Results

ZSF rats developed obesity, diabetes, hyperlipidemia and proteinuria. Male ZSF rats were heavier (650 gm vs 510 gm) and more hyperglycemic (295 mg/dl vs.210 mg/dl) than females. CD rats were normoglycemic with normal kidney function. Kidney homogenates from ZSF rats demonstrated increased expression of both TNFαR1 and IL15α proteins. These changes were more significant in male than female ZSF rats. RMC exposed to high glucose for 48 hours either directly or after chronic exposure to low glucose demonstrated similar increase in expression of TNFαR1 and IL15α proteins compared to low glucose. 4.

Conclusions

In renal cells exposed to high glucose and in ZSF (diabetic) rats with nephropathy, the expression of TNFαR1 and IL15α proteins, which are inflammatory mediators are increased. Male ZSF rats expressed more significant disease and increase in KRIS proteins indicating the need for further investigations into the role of gender in disease progression in murine models of DN. These results suggest that inflammatory proteins from TNF family are overexpressed in diabetic nephropathy models and that renal injury from chronic hyperglycemia may be mediated by at least in part by chronic inflammation. 5.

Support or Funding Information

This research was funded by Larry and Jane Woirhaye Memorial Research Foundation

Inflammatory Protein Expression in RMC

Figure 1
Reteplase Encapsulated in Minocycline‐Containing Nanoparticles Retains Thrombolytic Activity
Authors: Nong Jia , Marcos-Contreras Oscar A. , Glassman Patrick M. , Muzykantov Vladimir M.
Published: April 2020

Thrombolytic therapy is a critical potion of the treatment regimen for a number of life‐threatening conditions, including cardiovascular disease and stroke. Although thrombolytic drugs have shown profound effect on dissolving clots, short half‐life (t1/2 of minutes) and hemorrhagic complications are significant challenges associated with their use. Nanotechnology has shown to prolong the blood circulation time of drugs and to improve their efficacy and safety. However, leakage of thrombolytic drug‐loaded nanocarriers (NC) from intravascular compartment into brain parenchyma due to compromised blood‐brain barrier integrity following ischemia can still lead to adverse effects. Therefore, it is necessary to combine neuroprotection with thrombolysis therapy in ischemic stroke treatment. Here we have developed NC that contains reteplase, an engineered tissue‐type plasminogen activator, and minocycline, a neuroprotective agent. The formation of the NC is based on metal ion‐assisted self‐assembly of complexes using negatively charged heparin and minocycline. The reteplase is incorporated into the NC through a protein binding site on minocycline. The entrapment efficiency of reteplase and minocycline were 91.8% and 78.3%, respectively. By tracing radiolabeled reteplase, our results showed that the NC significantly increased the blood circulation time of reteplase compared to free reteplase (Panel A). In the in vitro clot lysis assay, the free reteplase or reteplase‐loaded NC (NC(ret)) were incubated with pre‐formed plasma clots and the results indicated that the NC formation did not compromise the thrombolytic function of reteplase (Panel B). In the pulmonary embolism model, free reteplase or NC(ret) was injected 10 min after 125I‐labeled microemboli. Blood and organs were collected 1 hr‐post injection of microemboli to study the retention of clot in the lung after the treatment. Our results showed that the reteplase‐loaded NC significantly reduced the clot retention in the lung (Panel C). All together, we have developed NC containing neuroprotective agent that maintain the function of thrombolytic drug. Future studies will be focused on evaluating the neuroprotective properties of the NC, relative to free drug.

Support or Funding Information

NIH (RO1 HL128398‐02 and RO1 HL 143806‐01), Cardiovascular Institute of Philadelphia (400‐4119‐2)

Characteristics of Reteplase‐Containing Nanocarriers. A: Blood pharmacokinetics of free reteplase (ret) and NC‐encapsulated reteplase (NC(ret)). Comparisons by students t‐test. B: In vitro fibrinolytic activity of ret and NC(ret). C: Lysis of pulmonary emboli following injection of ret and NC(ret). Comparisons by 1‐way ANOVA with Dunnett's test. * denotes p<0.05, ** p<0.01, *** p<0.001.

Figure 1
Mechanism of Activation of the Ubiquitin E3 Ligase, Parkin
Authors: Gehring Kalle , Sauvé Véronique , Sung George , Kozlov Guennadi
Published: April 2020

The Parkinson disease associated proteins, Parkin and PINK1, together comprise a mitochondrial quality control system that promotes neuronal survival through autophagy of damaged mitochondria. In the pathway, PINK1 acts as a sensor of depolarized mitochondria and phosphorylates ubiquitin to recruit and activate Parkin on the mitochondrial outer membrane. Parkin ubiquitinates mitochondrial proteins, which leads to the removal of the damaged mitochondria through mitophagy. We have carried out structural and functional studies of Parkin to understand its regulation and mechanism of activation
1–
3. Parkin exhibits low basal activity as a ubiquitin ligase and requires activation both in vitro and in cells. Multiple features inhibit Parkin activity: the E2‐binding site on RING1 is blocked, the catalytic site is partially occluded, and the incoming E2~Ub linkage is far from the parkin catalytic site (Fig.
1). In response to mitochondrial damage, Parkin is activated by PINK1 via a two‐step mechanism involving binding of Parkin to phosphorylated ubiquitin and a large‐scale conformational change induced by Parkin phosphorylation (Fig.
2). Positive feedback in the Parkin/PINK1 pathway generates a binary, all‐or‐nothing switch for the induction of mitophagy. These structural studies are critical for understanding how Parkin and PINK1 protect neurons and provide a framework for the design of small molecules for treating Parkinson disease.

Support or Funding Information

Supported by the Michael J Fox Foundation, the Canadian Institutes of Health Research, and the Canada Research Chair program.

Autoinhibited conformation of Parkin.

The crystal structure of Parkin revealed that the catalytic and E2‐binding sites are occluded by interdomain contacts mediated by the ubiquitin‐like domain, Repressor Element of Parkin (REP) and RING0 domain
1. Disruption of the inhibitory contacts derepresses ligase activity.

Figure 1

Active complex of Parkin with ubiquitin‐charged E2 enzyme and phospho‐ubiquitin (Ub).

The crystal structure of activated Parkin showed that phosphorylation of the Parkin Ubl domain induces a large conformational change allowing the catalytic RING2 domain to interact with the bound E2~Ub
3. Hydrogen‐deuterium exchange experiments confirm that the RING2 domain is released and accessible upon Ubl binding to RING0.

Figure 2
A Rodent Model of Reperfused Hemorrhagic Myocardial Infarction
Authors: Nair Anand R. , Cokic Ivan , Johnson Eric , Yang Hsin-Jung , Francis Joseph , Dharmakumar Rohan
Published: April 2020

Background

Intramyocardial hemorrhage following reperfusion is strongly associated with major adverse cardiovascular events (MACE) in myocardial infarction (MI) patients. Previous work from by us and others have shown that prolonged ischemic periods prior to reperfusion drives the development of intramyocardial hemorrhage in large animal models, characterized by the presence of iron composites within the infarct zones in the acute and chronic periods following MI. Yet the mechanisms contributing to the adverse outcomes associated with hemorrhagic myocardial infarction (hMI) is not well understood, partly due to the lack of small animal models of hMI. Here, we demonstrate a novel rodent model that mimics reperfused hMI.

Methods & Results

Sprague‐Dawley rats underwent either a 90‐min ischemia and then reperfusion (I/R) (n=22), a 60‐min I/R (n=20) or 30‐min I/R (n=18) of the left anterior descending coronary artery. Sham rats were used as controls. 90‐min I/R always yielded hMI, while 30‐min I/R consistently yielded non‐hMI with no reflow. But, 60‐min I/R resulted in both hMIs (60%) and non‐hMI (40%), with both groups showing evidence for zones of no reflow within MI. Ex‐vivo late‐gadolinium‐enhancement (LGE) and T2* cardiac MRI performed on excised hearts from 90‐min I/R rats revealed colocalization of iron deposits with the scarred tissue. Histological studies verified tissue damage (H&E) detected on LGE and the presence of iron (Perl’s stain) observed on T2*‐CMR. Protein expression of proinflammatory markers (TNF‐α, IL‐1β and MMP‐9) were increased in the 90‐min I/R group when compared to sham‐controls and 30‐min I/R groups. Further, transmission electron microscopy performed on 90‐min I/R myocardium showed accumulation of granular iron particles within the phagosomes.

Conclusion

Short (30‐min) and long (90‐min) ischemic periods before reperfusion consistently resulted in non‐hMI and hMI respectively in rats. This novel rodent model can be a useful tool to elucidate the molecular mechanisms of cardiovascular pathology associated with reperfused hemorrhagic myocardial infarctions.

Late Reperfusion Leads to Development of hMI

Representative LGE and T2*‐w images from ex‐vivo cardiac MRI showing myocardial infarction and hemorrhage respectively from late‐reperfusion (90‐min I/R) and early‐reperfusion (30‐min I/R) groups. Histological images of H&E staining from regions negative for MI (green box) and H&E and Perl’s stained images from regions positive for MI (blue box) are shown.

Figure 1
Leishmania major degrades murine CXCL1
Authors: Yorek Matthew S. , Mazgaeen Lalita , Gurung Prajwal
Published: April 2020

Leishmaniasis is a global health problem with an estimated report of 2 million new cases every year. Innate immune system plays a central role in controlling L. major infection by initiating a signaling cascade that results in the production of pro‐inflammatory cytokines and recruitment of both innate and adaptive immune cells. Upon infection with L. major, CXCL1 is produced locally and plays an important role in the recruitment of neutrophils and macrophages to the site of infection. Herein, we report that L. major specifically targets murine CXCL1 for degradation and this allows L. major to establish infection in the host. The degradation of CXCL1 is not dependent on host factors because L. major can directly degrade recombinant CXCL1 in a cell‐free system. This degradation is specific to murine CXCL1 as murine CXCL2 as well as human CXCL1 and CXCL2 are not degraded. Using mass spectrometry, we have determined that the unknown L. major protease cleaves at the C‐terminal end of murine CXCL1. Using protease inhibitors our data suggest that the L. major unknown protease is a metalloprotease involved in the direct degradation of CXCL1. Finally, we have tested a peptide spanning the cleavage site which can be used to inhibit L. major protease activity and pathogenesis.

Support or Funding Information

This work was supported by NIH K22, NIEHS P30 and University of Iowa Start‐up funds to P.G.

Upon infection with L. major, CXCL1 is produced locally and plays an important role in the recruitment of neutrophils and macrophages to the site of infection. We have identified that a secreted L. major protease cleaves at the C‐terminal end of murine CXCL1. This protease is likely a metalloproteases as Zn2+ chelators inhibit degradation of CXCL1. The degradation of CXCL1 dampens the hosts ability to resolve infection.

Figure 1