bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of the Sural Nerve Complex in North American Adult Cadavers: Distribution, Population Specific Morphology, and Anatomic Variants.
Authors: Steele Robert , Coker Charles , Freed Blair , Brauer Phillip
Published: April 2020

BACKGROUND

The Sural nerve complex (SNC) consists of four components: the medial sural cutaneous nerve, lateral sural cutaneous nerve, sural communicating branch, and sural nerve proper. The sural nerve is a cutaneous nerve that innervates the distal posterolateral leg and dorsolateral foot. The SNC is highly variable and has six defined morphological types each representing different patterns in which the SNC can form the sural nerve proper. Because of variability amongst different populations the SNC may frequently become a site of iatrogenic injury.

PURPOSE

The purpose of this study was to determine if the morphological type and spatial characteristics of the SNC we found in North Americans aligned with that described in the current literature.

METHODS

Six defined morphologic types of SNC have been described, each representing different patterns in which the SNC forms the sural nerve proper (Pirarvin, KR, Annals of Anatomy, 202, 36–44, 2015). Cadaveric data was obtained bilaterally in from 49 formalin‐ and three Thiel‐embalmed body donors (n=104 limbs) from KCUMB via superficial dissection methods revealing the SNC in the posterior leg. Measurements were obtained using forensic rules and calipers and documented using a digital camera. This study first approved by the KCUMB Institutional Biosafety Committee.

RESULTS

Our results showed a predominance of two major types of SNCs. In the first type (53%), the SNC originated from the lateral sural cutaneous nerve or common fibular cutaneous nerve. In the second type (34%), the SNC originated from the medial sural cutaneous nerve independent of the sural communicating branch, with or without the presence of lateral sural cutaneous nerve contribution. We also found a prevalence of bilateral asymmetry (67%) in the formation of the SNC. Lastly, were found higher frequencies of previously described SNC variants including connections of the SNC with the posterior femoral cutaneous nerve (30%) and terminal branches of the medial sural cutaneous nerve connecting with other cutaneous nerves adjacent the lateral malleolus (22%).

CONCLUSION

Our data showed there are two prevalent forms of SNCs within the North American population and that bilateral asymmetry is more common than previously reported. A more complete understanding of the variation in the SNC is important for clinical electrophysiology evaluations, neurological disease testing, lower extremity tissue repair, and sural nerve use in autogenous nerve grafts.
ROLE OF TYROSYLPROTEIN SULFOTRANSFERASE 2 (TPST2) IN PANCREATIC CANCER
Authors: Cai Xinran , Xie Wen
Published: April 2020

Pancreatic cancer is an aggressive disease which has become the third leading cause for cancer deaths in the United States. Tyrosylprotein sulfotransferase 2 (TPST2) is a post‐translational modification enzyme which locates in the trans‐Golgi apparatus and catalyzes the sulfation of proteins’ tyrosine residues. Moreover, tyrosine sulfation has been shown to play a critical role in protein‐protein interaction. By analyzing TCGA data, we found that the expression of several known substrates of TPST2 were affected in pancreatic cancer. However, whether TPST2 affects cancer cell behavior is unclear. To determine the role of TPST2 in pancreatic cancer, MTT cell growth assay and soft agar colony formation assay were performed to assess cancer cell proliferation in MiaPACA2 and PANC‐1 human pancreatic cancer cell lines. Migration and invasion of cancer cells in vitro were evaluated by transwell assay. CRISPR‐Cas9 was utilized to establish TPST2 KO stable cell lines. Total RNA‐seq analysis was performed to analyze the transcriptome in the TPST2 KO and control cells. We showed that knockdown of TPST2 inhibited the proliferation of pancreatic cancer cells both in vitro and in vivo. Knockdown of TPST2 also decreased migration and invasion of pancreatic cancer cells in vitro. RNA‐seq analysis revealed that the extracellular matrix (ECM) receptor interaction pathway was significantly downregulated in TPST2 KO cells. We further demonstrated that expression of intergrin beta 4 (ITGB4), a protein involved in ECM receptor interaction pathway, was decreased when TPST2 was knocked‐down or inhibited. Our studies have uncovered the role of protein sulfation in pancreatic cancer and may establish TPST2 as a therapeutic target for pancreatic cancer.

Support or Funding Information

This work was supported in part by National Institutes of Health grants DK083952, ES023438 to Wen Xie.
Effects of Fat Tissue on Chronic Muscle Injuries
Authors: Quigley Mikayla , Kopinke Daniel
Published: April 2020

During aging and certain muscle diseases such as Duchenne Muscular Dystrophy (DMD), muscle mass declines and is being replaced with fat tissue in a process called fatty degeneration. However, it is unclear if intramuscular fat plays an important role in the initiation and/or progression of fatty degeneration. Recent data suggests that when the fat fraction is greater than 0.5, over half of DMD patients are no longer able to walk. Based on these observations we, therefore, hypothesize that an increase in fat could impair muscle regeneration and function in chronic injuries. In young and healthy muscles, satellite stem cells are required for regeneration. Fibro/adipogenic progenitors (FAPs), which can differentiate into fat tissue and produce fibrotic scar tissue during chronic injuries and aging, aid in the healing process of muscle injuries. While beneficial, the exact relationship between FAPs and satellite stem cells during muscle regeneration is unclear. The purpose of this project was to establish a novel mouse model enabling us to conditionally prevent the differentiation of FAPs into adipocytes to determine the functional role of fat. To prevent FAPs from turning into fat, we conditionally deleted Pparγ, the master regulator of adipogenesis, using the Cre‐LoxP system in the mouse (Pparγ cKO). To induce fat formation, I injected 50% Glycerol into the tibialis anterior muscle, which causes muscle fiber loss and replacement with adipocytes. I stained adipocytes with PERILIPIN via immunofluorescence and quantified the amount of fat that was present in Pparγ cKO and control mice to determine if fat formation was indeed repressed after loss of Pparγ. I found a strong reduction in the amount of adipocytes, which have formed 21 days after a Glycerol induced injury in Pparγ cKO compared to control mice, arguing for efficient repression of adipogenesis. I also looked at fibrosis through Sirius Red staining to determine if the decrease in fat cells resulted in an increase of FAPs developing into fibrotic scar tissue. However, I saw no significant difference in fibrotic tissue between control and Pparγ cKO mice, suggesting that the prevention of fat formation does not cause an increase in the presence of fibrotic tissue.

Thus, our new mouse model successfully blocks fat formation without increasing fibrotic scar tissue. In the future, this new mouse model will allow us to determine if the prevention of fat in a chronic muscle disease model, such as the mouse model for Duchenne Muscular Dystrophy, will have a positive effect on muscle repair and function.

Support or Funding Information

Start‐up grant given to Daniel Kopinke; ASPET SURF Program at the University of Florida funding to Mikayla Quigley
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

Introduction

High‐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.

Methods

Primary 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.

Results

Patient‐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.

Conclusion

IMT 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 Information

This 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 Information

Supported 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 Information

Supported 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 Information

We 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 Information

Research 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

Background

Angiotensin 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.

Methods

We 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).

Results

Male 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.

Conclusion

Angiotensin 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 Information

National 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 Information

K01DK114329, 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

Background

Angiotensin 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.

Goal

To 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.

Methods

Male 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.

Results

Prior 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.

Conclusion

Chronic 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 Information

P01 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.
Angiotensin II Represses Guanylyl Cyclase/Natriuretic Peptide Receptor‐A Gene Expression and Receptor Signaling and Function
Authors: Arise Kiran K. , Kumar Prerna , Samivel Ramachandran , Zhao Hanqing , Lindsey Sarah , Pandey Kailash N.
Published: April 2020

Atrial and brain natriuretic peptides (ANP and BNP) bind to guanylyl‐cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA) and regulate sodium excretion, water balance, vasorelaxation, and steroidogenesis; all directed to combat hypertension. ANP acting through NPRA lowers blood pressure and blood volume and protects the heart and vascular disorders and end‐organ damage. The two vasoactive hormones, angiotensin II (ANG II; vasoconstrictive) and atrial natriuretic peptide (ANP; vasodilatory) antagonize the biological actions of each other. The objective of the present study was to examine whether ANG II plays critical roles in the transcriptional repression of Npr1 (encoding for NPRA) and receptor function. To delineate the mechanisms of ANG II‐mediated repression of Npr1 transcription and function, we performed the promoter deletional analyses, which showed that region −1182/−914 base pairs upstream from the transcription start site of the Npr1 promoter contains cis‐acting elements for ANG II‐regulated repression of Npr1. ANG II significantly decreased the levels of NPRA mRNA (60%), NPRA protein (52%), and intracellular accumulation cGMP (65%) in cultured mesangial cells and attenuated ANP‐mediated relaxation of aortic rings ex vivo. The results showed that transcription factors, CREB and HSF‐4a facilitated the ANG II‐mediated repressive effects on Npr1 transcription and receptor function. The current findings suggest that ANG II‐mediated repressive mechanisms of Npr1 transcription and receptor function may provide new molecular targets for the treatment and prevention of hypertension and renal and vascular disorders.

Support or Funding Information

This work was supported by NIH grant HL062147
ABO Blood Type and Von Willebrand Expression in Pulmonary Endothelial Cells
Authors: Post Ginell R. , Post Steven R. , Murray Glenn
Published: April 2020

ABO blood group antigens are expressed on von Willebrand factor (VWF) and glycosylation patterns influence circulating VWF levels. Retrospective studies performed on patients with deep vein thromboses indicate that individuals with type O blood group have a decreased thrombotic risk secondary to lower von Willebrand factor (vWF) plasma levels compared to type A, B and AB (non‐O) individuals. We examined the effect of ABO blood type on tissue‐associated VWF protein and mRNA levels in formalin fixed paraffin‐embedded pulmonary tissue obtained at autopsy from decedents who died from pulmonary embolism with known ABO blood group phenotype (O, A, B or AB). Tissue microarrays were created and stained with antibodies to VWF and platelet/endothelial cell adhesion marker‐1 (PECAM‐1) as a marker of endothelial cells. A pixel count scoring algorithm was used to quantify VWF and PECAM‐1 staining intensity in pulmonary arterioles in digitized images. Compared with type O, non‐O individuals have a significantly higher amount of endothelial cell‐associated VWF protein expression. However, quantitate RT‐PCR showed that, when normalized to PECAM‐1, VWF transcripts were lower in type A individuals, compared to type O. In conclusion, VWF protein levels associated with pulmonary vascular endothelial cells are influenced by ABO antigenic determinants; however, protein levels are not regulated at the transcriptional level.

Support or Funding Information

Research was supported by funds provided by UAMS Department of Pathology.
Developmental loss of oligodendrocytes hinders adult CNS remyelination and increases astroglial and microglial activation
Authors: Pajoohesh-Ganji Ahdeah , Karl Molly , Garrison Eric , Clarkson-Paredes Cheryl , Miller Robert H.
Published: April 2020

Multiple sclerosis, a neurodegenerative autoimmune disease characterized by loss of oligodendrocytes and myelin in the brain and spinal cord, results in localized functional deficits. Several risk factors have been associated with MS, however none can fully explain enhanced susceptibility to MS. Epidemiological data, based on geographical prevalence studies suggest susceptibility is established early in life and frequently long before disease diagnosis indicating developmental events influence adult disease progression. Here we test the hypothesis that loss of mature oligodendrocytes during postnatal development results in enhanced susceptibility to a second demyelinating insult. A transgenic mouse model was utilized to specifically induce apoptosis in a subset of mature oligodendrocytes (MBP‐iCP9) during the first 2 postnatal weeks followed by either a local LPC spinal cord injection or the induction of EAE in the adult. Immunostaining, immunoblotting, behavioral testing, and scanning electron microscopy were utilized to examine the differences between groups. We show that during development, oligodendrocyte apoptosis results in transient reductions in myelination and functional deficits that recover after 10–14 days. Compared to animals in which oligodendrocyte development was unperturbed, animals subjected to postnatal oligodendrocyte ablation showed delayed recovery from an LPC lesion. Unexpectedly, the induction and severity of EAE was not significantly altered in animals following oligodendrocyte ablation even though there was a substantial increase in spinal cord tissue damage and CNS inflammation. It is currently unclear why these changes are not reflected in enhanced functional deficits. These observations suggest that developmental loss of oligodendrocytes results in long lasting tissue changes that alter its capacity for repair in the adult.

Support or Funding Information

NIH834857
Loss of AC10 impairs neutrophil recruitment into the lung following Pseudomonas aeruginosa infection
Authors: Sayner Sarah L. , Choi Chung-Sik
Published: April 2020

Pseudomonas aeruginosa is a gram‐negative, opportunistic bacteria and a common cause of nosocomial pneumonia in critically ill patients that can progress to acute respiratory distress syndrome (ARDS). These patients have impaired gas exchange leading to hypoxia, altered blood pH and elevated bicarbonate. Bicarbonate, delivered into the cell via sodium bicarbonate cotransporters (NBCs), activates adenylyl cyclase 10 (AC10) to increase intracellular cAMP. Indeed, bicarbonate activation of AC10 is required for LPS‐induced lung endothelial permeability. Neutrophil recruitment into the airspaces plays a central role in the progression of ARDS. Recently, AC10 has been implicated in transendothelial emigration of neutrophils from the blood into the underlying tissue via CD99, a transmembrane protein critical for neutrophil recruitment. We sought to determine whether AC10 is necessary for neutrophil recruitment into the airspaces following P. aeruginosa intratracheal inoculation. Increasing doses of bacteria were introduced directly into the lung of wild type and AC10 and NBCn2 knockout mice. Bronchoalveolar lavage fluid (BALF) was recovered 24 hours later and examined for recruitment of polymorphonuclear cells (PMNs) into the airspaces. The number of PMNs recruited into the airspace following Pseudomonas inoculation increased with bacterial number and reached a maximum at a dose of 1E5 bacteria after which 100% of cells in the BALF were PMNs. Thus, doses upto 1E5 bacteria were used to examine the role of AC10 and NBCn2 in PMN recruitment into the airspace between wild type and knockout animals. Our data reveal a decreased number of PMNs in the BALF of both AC10 and NBCn2 knockout female mice; however, no difference in the number of PMNs recruited into the airspace was detected between male wild type and knockout animals. In addition, we confirmed the expression of CD99 in pulmonary microvascular endothelial cells and whole lung from both rat and mouse. Thus, CD99 is expressed in pulmonary endothelial cells and female mice utilize AC10 and NBCn2 to facilitate neutrophil recruitment into the airspace following a model of P. aeruginosa pneumonia.

Support or Funding Information

NIH R01HL121513
Day‐to‐Day Reproducibility of the Near‐infrared Spectroscopy Venous Occlusion Technique in Young Healthy Adults
Authors: Dennis Joshua J. , Wiggins Chad C. , Smith Joshua R. , Isautier Jennifer M. J. , Johnson Bruce D. , Joyner Micheal J. , Cross Troy J.
Published: April 2020

Background

Previous studies have demonstrated that the near‐infrared spectroscopy venous occlusion technique (NIRS‐VOT) provides a valid assessment of regional forearm muscle blood flow; however, day‐to‐day reproducibility of hemodynamic variables derived from the NIRS‐VOT remains to be determined, particularly across the range of physiological blood flows observed during handgrip exercise.

Purpose

The purpose of this study was to evaluate the day‐to‐day reproducibility of forearm hemodynamics assessed via the NIRS‐VOT.

Methods

Near‐infrared spectroscopy (NIRS) data were obtained from the flexor digitorum superficialis muscle on the right arm of 14 young, healthy adults (age: 27 ± 6 yrs; BMI: 24.6 ± 2.8 kg·m−2). The NIRS‐VOT was performed via rapid inflation of a pneumatic cuff placed on the upper arm for approximately 10 s. The NIRS‐VOT was performed at rest, and immediately after a 15 s isometric handgrip contraction at 20%, 40% and 60% of the subject’s maximum voluntary contraction. Measurements were repeated 3–5 times. Participants returned to the laboratory for a second visit on a separate day, and repeated the same number of measurements. Forearm blood flow (FBF), and blood pressure were measured on a beat‐by‐beat basis, and forearm vascular conductance (FVC) was calculated. FBF and FVC were subsequently compared for day‐to‐day reproducibility.

Results

FBF and FVC significantly increased with the intensity of handgrip exercise (P< 0.05); however, these measures of forearm hemodynamics were not significantly different (P >0.05) between Day 1 and Day 2 at each exercise‐intensity (Table
).

Conclusion

The findings of the present study indicate that the NIRS‐VOT provides a reliable assessment of regional forearm muscle hemodynamics across a physiological range of blood flows incurred by isometric handgrip exercise.

Support or Funding Information

MJJ is funded by the NIH (R‐35‐HL139854) and the Mayo Foundation. CCW is supported by the NIH training grant (5T32DK007352‐39), JRS is supported by AHA (18POST3990251), and TJC is funded by AFAR Postdoctoral Transition Award (DIAMOND18161).

FBF and FVC on experimental days

Day 1

Day 2

P‐value

FBF, mL·min−1·100mL−1 (tissue)

Rest

2.0 ± 0.5

0.8 ± 0.2

0.001

20% MVC

14.4 ± 2.65

15.4 ± 2.4

0.679

40% MVC

26.0 ± 5.19

28.0 ± 4.3

0.606

60% MVC

39.4 ± 6.39

47.4 ± 7.7

0.858

FVC, mL·min−1·100 mmHg−1

Rest

170 ± 47.3

72 ± 16.4

0.001

20% MVC

1297 ± 227.8

1454 ± 209.6

0.380

40% MVC

2496 ± 492.8

2656 ± 434.0

0.912

60% MVC

3820 ± 659.1

4542 ± 744.7

0.535

MVC, kg

49.4 ± 3.0

47.9 ± 2.6

0.721

Abbreviations: FBF, forearm blood flow; FVC, forearm vascular conductance; MVC, maximal voluntary contraction. Values are presented as means ± SE. P values indicate comparison between days 1 and 2.
Tritiated JNJ‐63514230 is a novel GluN2B‐selective radioligand with no affinity to sigma receptors
Authors: Schoellerman Jeffrey , Lord Brian , Bhattacharya Anindya , Bonaventure Pascal , Letavic Michael , Lovenberg Tim , Balana Bartosz
Published: April 2020

Introduction

Commonly used GluN2B radioligands exhibit limited selectivity towards GluN2B receptors as they interact with sigma and other off‐target receptors. Here we describe development and characterization of [3H] JNJ‐63514230, a novel radioligand selective for GluN2B‐containing NMDA receptors.

Methods

The selectivity JNJ 63514230 was determined using functional assays and CEREP panel. Saturation binding and competitive radioligand binding experiments were used to characterize [3H]‐JNJ 63514230 properties. We have also tested utility of [3H]‐JNJ 63514230 as a tracer for ex‐vivo autoradiography in rat brain.

Results

We confirmed that widely used tracers [3H] Ifenprodil and [3H] Ro 25‐6981 have substantial affinity to sigma receptors as measured in radioligand competition experiments (CEREP). In contrast, new tracer [3H] JNJ‐63514230 exhibits no affinity to sigma 1 and sigma 2 receptors in CEREP assays. Saturation binding in rat brain membranes yielded a Kd value of 2.53 nM and Bmax of 2531 fmol/mg. Association constant Kon, dissociation constant Koff, and calculated equilibrium binding constant (Kon/Koff) were 0.0067±0.001 min−1 nM‐1, 0.021±0.0002 min−1, and 3.28±0.61 nM, respectively (average of 3 individual experiments each ± SD). Enhanced selectivity for GluN2B‐containing receptors translates to reduced non‐specific binding and better signal to noise ratio in both in vitro radioligand binding assays and ex vivo autoradiography.

Conclusions

We showed that new radioligand [3H] JNJ 63514230 is superior to previous previously described GluN2B tracers as it permits accurate measurement of affinity to GluN2B‐containing receptors (Ki) without using additional blockers to mask native sigma receptors.

Support or Funding Information

Authors are full time employees of Janssen Research & Development, LLC. The research was funded by the company.
SOARing Achievements: Do Medical Students Think Our New Teaching Strategy Achieved Its Goals?
Authors: Gritton Cory Buenting , Carmichael Heather , Kondo Kimi , Lee Lisa , Royer Danielle
Published: April 2020

In 2018, the University of Colorado implemented Structured Obligatory Application & Review (SOAR) sessions designed around 11 specific goals associated with evidence‐based trends in medical education in order to improve the anatomy block. The sessions, spaced across the block, each included 4 rotations with small group work and hands‐on activities integrating clinical and anatomy content. Rotations included embryonic relationships with gross, anatomy in imaging & physical exam, surgical cases with cadaveric correlation, and team‐based learning of board‐style questions. The objective of this study was to evaluate whether SOAR successfully achieved its goals: peer‐to‐peer learning, self‐directed learning, identifying knowledge gaps, engaging review, long‐term retention, confidence in anatomy, connecting anatomy concepts, exam preparation, clinical relevance, and clinical application.

In an IRB exempt study, 1st year medical students (n=182) were invited to complete a paper survey during the last SOAR in 2019. The survey included 11 Likert items pertaining to stated SOAR goals. Previously, 1st‐year students in the 2018 pilot SOAR (n=184) were invited to complete an online survey after block completion. The 2018 survey included the same 11 items, plus two free response questions that were qualitatively analyzed for themes.

The 2019 survey response rate was 81% (n=149), much greater than 2018 online response rate (31%, n=59). Analysis of 2019 responses revealed student agreement greater than 50% for all 11 SOAR goals. Strong achievement of a goal was determined from upper quartile (75–100%) agreement ratings, which was seen for 4 goals: providing clinical relevance (91%), applying anatomical knowledge to clinical situations (89%), identifying knowledge gaps (82%), and connecting anatomical concepts (78%). Goals were considered moderately achieved with agreement ratings in the next quartile (50–74%), with the remaining 7 SOAR goals within this designation: an engaging way to review anatomy (70%), self‐directed learning (69%), peer‐to‐peer teaching (68%), preparation for written examinations (67%), long‐term retention (57%), improving confidence in anatomy (52%), and preparation for practical exams (51%). Cohort comparison revealed no significant difference in responses between the pilot and subsequent year of SOAR implementation for each of the goals (Mann Whitney U tests, p>0.05). Overall, SOAR goals were strongly to moderately achieved based on student perceptions, with benefits perceived strongest in providing clinical relevance, clinical application of anatomy, knowledge gap identification, and providing an engaging review. This suggests that developing targeted, interactive, small group activities such as those used in SOAR may be a valuable evidence‐based approach to better integrate clinical and anatomical content in the modern pre‐clinical medical curriculum.