bluuebunny/crossref_metadata_2025_split huggingface dataset:
Custom 3D Brain Cancer Model to Test a Novel Anti‐Cancer Electrotherapy
Authors: Deweyert Andrew , Iredale Erin , Xu Hu , Wong Eugene , Schmid Susanne , Hebb Matthew 
Published: April 2020

IntroductionHigh‐Grade Gliomas (HGG) are primary malignant brain tumors with dismal treatment outcomes. Our team has been pioneering a novel implantable biotechnology called Intratumoral modulation therapy (IMT) that distributes non‐ablative electric fields across tumor‐affected brain regions to induce tumor cell death. A systematic means to define the mechanism and optimal treatment parameters for various forms of HGG is needed to advance this promising technology towards clinical application. The aim of this study was to develop a custom, high throughput, patient‐derived 3D HGG model to investigate the therapeutic effects of IMT.MethodsPrimary patient HGG cells (1×104) were implanted into wells of a 96‐well plate containing 200 uL of Matrigel®. The resultant tumor spheroids were characterized over 14 days by observing growth, invasion and viability with MTT assays, confocal microscopy and bioluminescence imaging (BLI). The culture plates were custom adapted to house implantable IMT devices to broadly distribute IMT fields across HGG tumors. Computer‐generated IMT field modeling was performed using COMSOL software to predict field strength and distribution, and to reconstruct IMT fields in the 3D system. Spheroids received 72‐hours of IMT using a spectrum of treatment parameters based upon a continuous, low amplitude sinusoidal waveform.ResultsPatient‐derived HGG spheroids exhibited multi‐layered, progressive growth and invasion of peritumoral matrix over the 14‐day study period. Computer simulation predicted electric field distribution and intensity across patient HGG spheroids for a spectrum of defined IMT parameters. Using the simulation plans to guide treatment settings, IMT produced a significant reduction (>60%) of metabolic viability in HGG spheroids treated with IMT compared to sham conditions. This data was corroborated by BLI which revealed >65% signal loss associated with IMT.ConclusionIMT is a highly promising innovation designed to combat HGGs, the most devastating of primary brain cancers. Our custom in vitro IMT model permits high throughput testing of treatment parameters in 3D patient HGG spheroids. This innovative preclinical strategy will be instrumental in defining the potential and optimizing treatment response across a spectrum of high fatality HGG cancers.Support or Funding InformationThis work was supported by the Cancer Research Society and preformed at Schulich School of Medicine and Dentistry and Lawson Health Research Institute.
A Paradoxical Response of Pulmonary Slowly Adapting Receptor Units During Constant Pressure Lung Inflation
Authors: Yu Jerry , Xu Ling 
Published: April 2020

It is known that activity of pulmonary slowly adapting receptors (SARs) decreases slowly, due to adaptation, during lung inflation with constant pressure. However, it is not uncommon to observe that some SAR units increase their activity instead during the course of lung inflation (we call it creeping). In the current studies, we seek to characterize this phenomenon retrospectively by analyzing previously collected data. From 52 anesthetized, open‐chest and mechanically ventilated rabbits, we examined activities of SAR units from the cervical vagus nerve during different levels of lung inflation with a constant pressure (10, 20, and 30 cmH2O) for 4 seconds. We found that creeping occurred in 46.0% units (64 of 139 units). The increased activity took 3 forms. It could be gradual, abrupt, or a combination of the two (i.e., the unit increased activity gradually first and then abruptly). The creeping occurred more frequently and earlier as the lung inflation pressure increased. For example, creeping occurred in 1.4% of the units and activity started to increase at 3.0 sec at 10 cmH2O (n=2), occurred in 24.5% at 2.2 sec at 20 cmH2O (n=34), and in 41.7% at 1.6 sec at 30 cmH2O (n=58). In many cases SAR activity decreased or stopped completely following the creeping, i.e., the unit deactivated. During a careful literature review, creeping can be identified in different species (opossums, dogs, cats, guinea pigs, and turtles) from the figures reported. However, only one paper discussed this issue as a side finding (1). Farber et al inflated the lung with a constant pressure of 10 cmH2O to examine the adaptation rate of SARs in opossums and found that 9 out of 40 SAR units (22.5%) increased their activity. Undoubtedly, with higher pressure and longer inflation protocols, much more significant portion of the units will exhibit creeping. Clearly, creeping is a common behavior of the mechanosensory unit across species, which has been largely neglected by the scientific community. We speculate that during lung inflation, sudden increases in SAR input will provide a breaking effect (negative feedback) to counter‐react the lung inflation. Certainly, such a mechanism is more beneficial at high pressure levels. Fortunately, such a propencity of activation at higher inflation levels is a built in mechanism of the pulmonary sensory system. Elucidation of underlying mechanisms of creeping will certainly improve our understanding of the mechanosensory function.Support or Funding InformationSupported by a Merit Review Award (PULM‐024‐17S) from Veteran Administration of the United States
Functional Characterization of Fusion Proteins of the Tumor Necrosis Factor Alpha‐Induced Protein 6 (TNFAIP6) Domains
Authors: Hassan Omron , Okeke Theresa , Jauregui Jessica , Klein Elizabeth , Badery Shamla , Glant Tibor , Fulop Csaba 
Published: April 2020

TNFAIP6 is an extracellular protein that is essential for cumulus cell extracellular matrix formation and female fertility in mice. A mammalian expression system was used to generate recombinant proteins of the full length TNFAIP6 and its individual domains, Link and CUB. These proteins contained a short amino terminal sequence essential for catalytic function and a carboxy‐terminal Fc region of mouse IgG2a for easy detection, and were termed TNFAIP6Fc, LinkFc and CUBFc, respectively. Hyaluronan‐binding of the fusion proteins was measured with an enzyme‐linked immunosorbent assay. The catalytic activity of the proteins was assessed by their ability to remove heavy chain 1 (HC1) from inter‐alpha‐trypsin inhibitor (IαI) using western‐blot. The ability of the fusion proteins to restore cumulus cell extracellular matrix formation was tested in a cell culture assay of Tnfaip6−/− cumulus cell‐oocyte complexes (COCs). Both TNFAIP6Fc and LinkFc were able to bind hyaluronan, with LinkFc showing much stronger binding at equimolar concentrations. CUBFc showed no significant binding to hyaluronan even at high concentrations. TNFAIP6Fc catalysed the removal of HC1 from IαI in a concentration dependent manner, indicating that the Fc tag did not interfere with catalytic function. LinkFc, CUBFc, and their combination were unable to remove the HC1 from IαI at high concentrations even though they contained the essential short amino‐terminal region. TNFAIP6Fc successfully restored cumulus cell extracellular matrix formation in Tnfaip6−/− COCs, while LinkFc, CUBFc, and their combination were unable to do so. LinkFc, CUBFc, and their combination did not disrupt normal cumulus cell extracellular matrix formation of Tnfaip6+/− COCs. Our results suggest that the catalytic function of TNFAIP6 requires both of its domains in addition to the previously identified serine residue in the amino‐terminal region, because neither domain alone is able to remove HC1 from IαI or support cumulus cell extracellular matrix formation in Tnfaip6−/− COCs. Furthermore, the absence of catalytic activity of both domains when combined together could imply a pivotal conformational interaction normally achieved by their covalent attachment. The finding that the LinkFc has stronger hyaluronan binding than TNFAIP6Fc could support the presence of such a conformational interaction that weakens the hyaluronan‐binding of the full‐length protein compared to the Link domain alone.Support or Funding InformationSupported by internal funding of Touro University Nevada
Single Cell Transcriptomics Reveals How Hyperlipidaemia Alters Monocyte/macrophage Differentiation At Sites Of Inflammation.
Authors: Purvis Gareth S. D. , McNeill Eileen , Wright Benjamin , Lockstone Helen , Channon Keith M. , Greaves David R. 
Published: April 2020

Monocytes are recruited to sites of inflammation and participate in either a continuation of the inflammatory response or to initiate a programme of pro‐resolution and tissue repair. Using the hCD68‐GFP (CD68GFP) macrophage reporter mouse we characterised the recruitment of monocytes and their differentiation into macrophages in zymosan induced peritonitis (ZIP), a resolving model of inflammation, in mice with normal or elevated lipid levels.We confirmed that the mature macrophages (CD11b+LygC−CD115+) present 48–72hrs after zymosan induced peritonitis (ZIP) differentiate from recruited blood monocytes by adoptively transferring (i.v.) CD68GFP monocytes into ongoing ZIP in wildtype mice.We subsequently performed single cell RNA Seq on monocytes (CD45+CD11b+Ly6G−LygC+) isolated from naïve blood; or blood and peritoneal lavage fluid following ZIP at 2 or 6 hours. Unbiased tSNE plot analysis revealed 8 distinct clusters within the CD45+CD11b+Ly6GLygC+ monocyte population, each with a unique transcriptional profile. These data reveal previously underappreciated heterogeneity with monocytes populations at a single cell level.We next investigated the effects of hyperlipidaemia on the recruitment of monocytes following ZIP. Mice injected with AAV‐8mPCSK9 (1011 particles; i.v.) and fed a high fat diet developed severe hyperlipidemia compared to mice injected with AAV‐8null (1011 particles; i.v.) and fed a chow diet which remain normo‐lipidemic. We reveal that hyperlipidemia increased the recruitment of monocytes into the peritoneum following ZIP. Using single cell RNA Seq, we demonstrate that hyperlipidemia alters the distribution of monocytes within the unique tSNE clusters, and also reveal an altered kinetic in monocyte to macrophage differentiation.In conclusion, we have demonstrated heterogeneity within a previously assumed homogeneous population of newly recruited inflammatory monocytes and that hyperlipidaemia alter both the magnitude and kinetics of the inflammatory process.Support or Funding InformationWe would like to thank the British Heart Foundation Award number: RG/15/10/23915 for funding this work.
Superior Influence of Reindeer compared to Beef Ingestion on Protein Metabolism in Humans
Authors: Coker Melynda Sheri , Ladd Kaylee , Schutzler Scott E. , Park Sanghee , Williams Rick H. , Deutz Nicolaas P. , Wolfe Robert R. , Coker Robert H. 
Published: April 2020

Wild game consumption has been associated with health benefits, but the immediate influence on protein metabolism remains unknown. In order to address the direct effects on protein kinetics, we compared the feeding‐induced response to free‐range reindeer versus commercial beef using stable isotope methodology in humans. Eight male and female participants (age: 38±12 years; body mass index: 24±3 kg/m2) completed two studies using a randomized, crossover design in which they ingested reindeer or beef. L‐[2H5]phenylalanine & L‐[2H2]tyrosine were delivered via primed, continuous intravenous infusion. Blood samples were collected during a 3‐hour basal period and for 4 hrs after consumption of reindeer and beef. A priming dose of L‐[2H4]tyrosine was also delivered. Feeding‐induced changes in whole body protein synthesis (PS), protein breakdown (PB), and net protein balance (NB) were determined via analysis of plasma samples by liquid chromatography‐electrospray ionization‐tandem mass spectrometry (LC‐ESI‐MS). Post‐prandial essential amino acid (EAA) concentrations were higher with reindeer ingestion (P=0.02). PS was not different but PB was reduced with reindeer compared to beef ingestion (P=0.02), contributing to superior level of NB (P=0.001). Reindeer promoted higher plasma EAA levels and NB compared to beef. The amino acid and fatty acid profiles of reindeer may have an immediate and greater benefit on protein metabolism as compared to beef. These data support the potential health benefits of wild game in the preservation of whole‐body protein.Support or Funding InformationResearch reported in this publication was supported by an Institutional Development Award (IDeA) under grant number P20GM103395 and the Biomedical Learning and Student Training Program (UL1GM118991, TL4GM118992, or RL5GM118990) from the National Institute of General Medical Sciences from the National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Kir4.1/Kir5.1 Activity in the DCT is Essential for Angiotensin II Induced Stimulation of Thiazide‐Sensitive NCC Expression/Activity.
Authors: Duan Xin-peng , Meng Xin-Xin , Zhang Dan-Dan , Gu Ruimin , Lin Dao-Hong , Wang Wen-Hui 
Published: April 2020

BackgroundAngiotensin II has been shown to stimulate thiazide‐sensitive Na‐Cl cotransport (NCC) by modulating with‐no‐lysine kinase (WNK) activity. WNK is responsible for the phosphorylation of ste20‐proline‐alanine‐rich protein kinase (SPAK) which regulates NCC activity by the phosphorylation. The basolateral Kir4.1/Kir5.1 channel activity in the DCT plays a role in the regulation of WNK‐SPAK activity by affecting the intracellular Cl concentrations. The aim of the present study is to examine whether Kir4.1/Kir5.1 channel activity in the DCT is required for the effect of angiotensin II on NCC.MethodsWe have used the patch‐clamp methods to examine the effect of angiotensin II on the basolateral K (Kir4.1/Kir5.1) channel activity of the DCT and DCT membrane potential. We also employed immunoblotting to examine the expression of phospho‐NCC (pNCC) and total NCC (tNCC), and used renal clearance method to measure thiazide‐sensitive renal sodium excretion in Kcnj10+/+ (WT) mice and renal tubule‐specific Kcnj10−/− mice (Ks‐Kir4.1 KO).ResultsMale and female mice were continuously perfused with a no‐pressure dose of angiotensin II (200 ng/min/Kg BW) or vehicle through an osmotic pump for 24 hours. Whole‐cell recording showed that angiotensin II infusion significantly increased the Kir4.1/Kir5.1 activity and hyperpolarized DCT membrane in both male and female mice. The effect of angiotensin II on DCT membrane potential was absent in Ks‐Kir4.1 KO mice, suggesting that the stimulation of Kir4.1 was responsible for the hyperpolarization of the DCT membrane. The infusion of angiotensin II also increased the expression of pNCC and tNCC in both male and female mice. Renal clearance study has demonstrated that the thiazide‐induced renal sodium excretion was significantly larger in angiotensin II‐treated mice than those vehicle treated mice, suggesting an upregulation of NCC by angiotensin II. However, the stimulatory effect of angiotensin II on thiazide‐induced natriuresis was significantly diminished in both male and female Ks‐Kir4.1 KO mice, suggesting the role of Kir4.1 in mediating the effect of angiotensin II on NCC.ConclusionAngiotensin II stimulates the basolateral Kir4.1/Kir5.1 channel activity in the DCT. Kir4.1/Kir5.1 activity in the DCT is required for the stimulatory effect of angiotensin II on NCC activity/expression.Support or Funding InformationNational Institutes of Health grants DK54983 (to Dr. Wang)
Increased phosphorylation of ACTN4 leads to podocyte vulnerability and proteinuric kidney disease and is stimulated by high glucose and TGF‐b
Authors: Feng Di , Kumar Mukesh , Muntel Jan , Gurley Susan B. , Birrane Gabriel , Stillman Isaac E. , Ding Lai , Wang Minxian B. , Ahmed Saima , Schlondorff Johannes , Alper Seth L. , Novak Richard , Ingber Donald E. , Steen Hanno , Pollak Martin R. 
Published: April 2020

Defects in the podocyte cytoskeleton contribute to podocyte vulnerability within the glomerulus and proteinuric kidney disease. Genetic mutations in ACTN4—an essential crosslinker of the actin cytoskeleton—have been linked to proteinuric kidney disease in humans. However, how post‐translational modifications of ACTN4 affect podocyte integrity and kidney function remain unknown. We detected phosphorylation of ACTN4 at serine 159 (S159) by mass spectrometry. Our ACTN4 X‐ray crystal structure showed that phosphomimetic S159D ACTN4 changes the charge at S159 without changing the protein’s conformation. Phosphomimetic Actn4 mouse models were generated using CRISPR/Cas9 technology, and these mice developed proteinuria and glomerulosclerosis after subtotal nephrectomy. The link between ACTN4 phosphorylation at S159 and renal pathology prompted us to use targeted mass spectrometry to identify upstream signaling pathways that might increase ACTN4 phosphorylation at S159. Since high extracellular glucose and TGF‐b can both lead to kidney disease and podocyte dysfunction, we examined whether these stimuli lead to increased ACTN4 phosphorylation at S159. We found that mean S159 phosphorylation was nearly 2‐fold higher in podocytes treated with high glucose vs mannitol control (p<0.05). Similarly, mean S159 phosphorylation was nearly 2‐fold higher in the podocytes treated with TGF‐b vs in untreated podocytes (p<0.05). Moreover, we quantified the F‐actin alignment in phalloidin‐stained podocytes. WT podocytes treated with high glucose demonstrated more spatially correlated F‐actin alignment, similar to the alignment seen in phosphomimetic Actn4 podocytes but different from nonphosphorylatable Actn4 podocytes. Our findings suggest that ACTN4 may mediate podocyte injury as a consequence of both genetic mutations and signaling events that modulate phosphorylation. Future studies using whole animal models and human samples should determine the extent to which the detrimental effects of high glucose on the kidney are mediated by phosphorylation of ACTN4.Support or Funding InformationK01DK114329, R37DK059588 and Wyss Institute for Biologically Inspired Engineering at Harvard University
Angiotensin II activates mTORC1 pathway in the kidneys through a pressor‐independent mechanism.
Authors: Shimada Satoshi , Yang Chun , Kumar Vikash , Stumph Megan , Kurth Theresa , Mattson David L. , Cowley Allen W. 
Published: April 2020

BackgroundAngiotensin II (AngII) has been found in vitro to stimulate the mTORC1 pathway and we have observed that AngII treatment enhanced mTORC1 phosphorylation of the ribosomal protein S6 kinase (pS6/S6) in a dose related manner in the NRK cell line. However, the in vivo effects of chronic elevations of AngII upon kidney activity of mTORC1 or upon mTORC2 (pAKT/AKT) have not been determined.GoalTo determine the effects of chronic hypertensive elevations of AngII upon the kidney expression of mTORC1 and mTORC2 in Sprague‐Dawley (SD) rats. Since it would normally not be possible to separate the direct effects of AngII upon mTORC1 activation from the effects of arterial pressure, studies were carried out using a unique servo‐control system that enabled the renal perfusion pressure (RPP) to the left kidney to be maintained at normal levels while the contralateral right kidney was subjected to the elevated systemic pressure.MethodsMale SD rats (n = 15) were maintained on a 0.4% NaCl diet throughout the study. At 9 weeks of age, rats were instrumented with carotid and femoral arterial catheters for measuring blood pressure, a femoral venous catheter for AngII infusion and a balloon occluder cuff was placed around the abdominal aorta between the renal arteries. The carotid and the femoral mean arterial pressures were used as surrogates to the RPP of the right and left kidneys respectively. After 7 days of recovery, baseline blood pressure was measured for 3 days, then the n = 9 of rats were infused with AngII (50 ng/kg/min) for 7 days to induce hypertension. N = 6 of rats were infused with only saline (vehicle control) throughout the study. A computer‐driven servo‐control system was used to inflate the aortic cuff to maintain left RPP to baseline levels while right RPP was left uncontrolled. Following 7 days of Ang II infusion, kidneys were removed for molecular analysis.ResultsPrior to infusion of AngII, baseline RPP values to the right and left kidneys were similar (117.3±3.0 vs 112.2±3.4 mmHg). Following 7 days of AngII infusion, the right RPP was significantly higher than left RPP (162.4±9.0 vs 113.2±2.7 mmHg; p<0.05). Average 24 hr mean arterial pressures (MAP) remained unchanged in saline infused rats throughout the study. Western blot analysis of renal cortical tissue at day 7 found pS6/S6 was higher in both pressure‐uncontrolled and pressure‐controlled kidneys of AngII infused rats (2.25±0.21 vs 2.00±0.23) compared to saline infused rat kidneys (1.42±0.21; p<0.05). In contrast, pAKT/AKT was unchanged by either AngII or differences in RPP.ConclusionChronic elevations of circulating AngII activates mTORC1 pathway in the kidneys through a pressor‐independent mechanism but has little effect on activation of mTORC2.Support or Funding InformationP01 HL116264, RO1 HL137748
Broken heart: disruption of the RNA binding protein rbm24a alters heart morphogenesis and leads to cardiomyopathy
Authors: Brastrom Lindy , Slusarski Diane 
Published: April 2020

Congenital heart disease is the most common birth defect in the United States. While transcriptional control of heart development has been well studied, only recently has post‐transcriptional control started to be appreciated. One mechanism of post‐transcriptional control is through RNA regulation. RNA binding proteins are a class of proteins which regulate RNA through various mechanisms including polyadenylation, stability, and splicing. Recently, the RNA binding protein, rbm24a, has been linked as a key regulator of heart development.Depletion of rbm24a in our model, the zebrafish, leads to cardiomyopathy and cardiac edema. We hypothesize rbm24a plays a key role in atrial development.Utilizing both morpholino knockdown and CRISPR/Cas9 mutagenesis, we find cardiomyopathy and cardiac edema in rbm24a depleted animals. To understand the underlying mechanism of the heart defect, we started by examining the formation of the heart tube. While the heart tube successfully forms and jogs to the left side of the body, the size of the heart tube is comparable to control animals at 12 hours post fertilization (hpf) but is significantly smaller by 24hpf. Previous research has shown the mouse ortholog of rbm24a, Rbm24, binds directly to the atrial‐specific gene Myh6. This suggests the first steps of heart development occur normally with the cardiomyopathy potentially arising from a defect in atrial development. We are currently exploring the possibilities that the atrium has (1) fewer cells and (2) that the decrease in atrium cell number is due to a migration, proliferation, apoptosis, and/or differentiation defect.Here, we explore the mechanism(s) by which the RNA binding protein, rbm24a, is leading to cardiomyopathy and cardiac edema in zebrafish.