bluuebunny/crossref_metadata_2025 huggingface dataset:
Renal blood flow and oxygen metabolism in renovascular hypertension
Authors: Patinha Daniela , Koeners Maarten P.
Published: April 2020

Renovascular hypertension affects ~1–4% of the hypertensive population and represents the second‐leading cause of secondary hypertension. There is an urgent clinical need to gain insight into the aetiology of renovascular hypertension to form the basis of new treatment targets. We hypothesize that high blood pressure in these conditions aims to sustain perfusion thus optimising organ blood flow and tissue oxygen homeostasis.

Male Wistar Rats with (2K1C, n=5) or without (control, n=3) renal artery clip to generate renovascular hypertension were used. Six weeks after renal artery clipping, renal hemodynamics and oxygen metabolism were evaluated under isoflurane anaesthesia. Renal blood flow was measured in the clipped kidney using an ultrasound probe, glomerular filtration rate was measured using FITC‐inulin and the arterio‐venous difference in oxygen content was used to determine the clipped kidney oxygen consumption. Despite mean arterial pressure being higher in the 2K1C group when compared with controls (134±9 vs 97±10 mmHg, p=0.02), blood flow (7±1 vs 7±1 ml/min, p=0.823), glomerular filtration rate (0.42±0.1 vs 0.53±0.1 ml/min, p=0.45) and oxygen consumption (1.0±0.12 vs 0.6±0.1 mlO2/min, p=0.06) were not significantly different between 2K1C and control animals

This preliminary data suggests that high blood pressure in 2K1C animals is sustaining normal renal perfusion, oxygen homeostasis and renal hemodynamics despite the reduction of arterial diameter.

Support or Funding Information

British Heart Foundation funded research.
Discovery and Characterization of a Potent and Selective Inhibitor for Human Phosphoinositide‐3‐kinase γ
Authors: Meleza Cesar Alejandro , Chen Ada , Fournier Jeremy , Mailyan Artur , Mata Guillaume , Jeffrey Jenna , Banuelos Jesus , Zhao Xiaoning , Leleti Manmohan R. , Jin Lixia , Powers Jay P. , Walters Matthew J. , Young Stephen W.
Published: April 2020

Phosphoinositide‐3‐kinases (PI3K) belong to the class 1 family of signal transducing enzymes comprised of PI3Kα, PI3Kβ, PI3Kδ and PI3Kγ. Together they are part of the PI3K‐Akt pathway acting as secondary messengers by catalyzing the phosphorylation of the inositol ring of phosphoinositides to regulate key cell functions such as proliferation, differentiation, and migration.

Of the four isoforms, PI3Kγ has garnered attention for its expression on immune cells and its role in tumor progression, inflammation, and immune response. PI3Kγ activity has been correlated to cell proliferation and tumor growth in human pancreatic ductal adenocarcinoma (PDAC) and in hepato cellular carcinoma (HCC). Additionally, PI3Kγ inhibition is suggested to be a key switch within the tumor microenvironment between immune stimulation and suppression. This suggest that selective inhibition of PI3Kγ may be an effective anti‐cancer strategy.

Previous attempts to selectively target PI3Kγ have proven largely unfruitful due to the poor selectivity profiles of many of the available inhibitors. We have developed a potent, reversible and selective small molecule PI3Kγ inhibitor. This inhibitor (compound 1) demonstrates activity against recombinant human PI3Kγ with sub nanomolar potency as determined by detection of ADP generation measured by luminescence. Additionally, compound 1 is highly selective over the rest of the class 1 PI3K family. Finally, compound 1 shows double digit nanomolar potency in MCP‐1 induced THP cells as measured by phosphorylation of AKT Ser473 using AlphaLisa and low triple digit nanomolar potency in whole blood as measured by flow cytometry.
The microtubule regulator ringer functions downstream from the RNA repair/splicing pathway to promote axon regeneration
Authors: Vargas Ernest John Monahan , Matamoros Andrew , Qiu Jingyun , Song Yuanquan
Published: April 2020

Promoting axon regeneration in the central and peripheral nervous system is of clinical importance in neural injury and neurodegenerative diseases. Both pro‐ and antiregeneration factors are being identified. We previously reported that the Rtca mediated RNA repair/splicing pathway restricts axon regeneration by inhibiting the nonconventional splicing of Xbp1 mRNA under cellular stress. However, the downstream effectors remain unknown. Here, through transcriptome profiling, we show that the tubulin polymerization‐promoting protein (TPPP) ringmaker/ringer is dramatically increased in Rtca‐deficient Drosophila sensory neurons, which is dependent on Xbp1. Ringer is expressed in sensory neurons before and after injury, and is cell‐autonomously required for axon regeneration. While loss of ringer abolishes the regeneration enhancement in Rtca mutants, its overexpression is sufficient to promote regeneration both in the peripheral and central nervous system. Ringer maintains microtubule stability/dynamics with the microtubule‐associated protein futsch/MAP1B, which is also required for axon regeneration. Furthermore, Ringer lies downstream from and is negatively regulated by the microtubule‐associated deacetylase HDAC6, which functions as a regeneration inhibitor. Taken together, our findings suggest that ringer acts as a hub for microtubule regulators that relays cellular status information, such as cellular stress, to the integrity of microtubules in order to instruct neuroregeneration. Future studies include developing small‐molecule inhibitors for Rtca and further elucidating TPPP’s conserved function in regeneration.

Support or Funding Information

Y.S. is a recipient of the National Institute of Neurological Disorders and Stroke (NINDS) Pathway to Independence Award. This work was supported by an Intellectual and Developmental Disabilities Research Center New Program Development Award (CHOP/Penn), an NINDS K99/R00 award (R00NS088211), and a National Institutes of Health grant (1R01NS107392) to Y.S., and a Predoctoral Training Program in Developmental Biology (T32 HD083185) to E.J.M.V.
The Effect of Acute Hypocapnia and Respiratory Alkalosis on Neurovascular Coupling Magnitude
Authors: Ciorogariu-Ivan Anna-Maria , Bader Taylor , Keough Joanna , Donald Joshua , Leacy Jack K. , O’Halloran Ken D. , Wilson Richard J. , Jendzjowsky Nicholas , Day Trevor A.
Published: April 2020

The brain requires well‐regulated cerebrovascular perfusion to match local metabolic demand. Regional coupling of perfusion to metabolic rate is termed neurovascular coupling (NVC). NVC remains stable during blood gas perturbations upon ascent and acclimatization to altitude, where individuals are exposed to antagonistic effects of hypoxia (vasodilation) and hypocapnia (vasoconstriction), where acid‐base variables are likely well‐compensated. Few studies have addressed the effects of acute hypocapnia and respiratory alkalosis independently. The aim of this study was to assess the specific effects of acute steady‐state hypocapnia on NVC magnitude in a laboratory setting. We recruited 10 healthy participants and instrumented the posterior cerebral artery (PCA) with a transcranial Doppler ultrasound. NVC was elicited using a standardized strobe light stimulus (6 Hz; 5×30sec on/off), and both peak and mean absolute responses from baseline (BL) were quantified. Participants were coached to hyperventilate to reach steady‐state hypocapnic steps of Δ‐5 and Δ‐10 Torr end‐tidal (PET)CO2 from baseline levels. PETCO2 levels were significantly decreased from 36.7±4.2 (BL) to 31.0±5.1 and 25.9±5.4 Torr (P<0.05), with estimated arterial pH(a) values of 7.43±0.05 (BL), 7.47±0.08 (Δ‐5) and 7.59±0.11 (Δ‐10), respectively (P<0.05). There was a significant reduction in NVC magnitude from BL (10.2±2.5 cm/s) during controlled hypocapnia at both Δ‐5 (8.3±2.6 cm/s) and Δ‐10 (8.1±2.7 cm/s) in peak PCA velocity (PCAv) (P<0.05), but no significant decrease in mean PCAv (P>0.05). Our study demonstrates that acute respiratory alkalosis attenuates peak NVC magnitude with a floor effect at Δ‐5 Torr PETCO2 (~7.47 pHa), without further attenuation at Δ‐10 (~7.59 pHa). This is one of few studies demonstrating a change in NVC with acute blood gas challenges. However, the effect is small and only observed in peak PCAv responses, with no differences between mild and more severe respiratory alkalosis.

Support or Funding Information

MRU Faculty of Science and Technology, University College Cork, University of Calgary
Determining the Role of Extracellular Matrix Compliance on Facial Morphogenesis
Authors: Hanne Nicholas Jean , Hu Diane , Linde-Medina Marta , Hallgrimsson Benedikt , Marcucio Ralph S.
Published: April 2020

We have previously found that collagen 4 and laminin are thinner in regions of the frontonasal process undergoing growth, suggesting tissue compliance may contribute to regulation of facial morphogenesis. Lysyl oxidase (LOX) initiates enzymatic crosslinking of collagen and elastin. Crosslinking in extracellular matrix fibrils increases tissue stiffness and may constrain growth in certain directions while allowing cells to exert forces required for directional growth. Our goal was to examine the distribution of lysyl oxidase in the frontonasal process (FNP) of chick embryos during growth and fusion of the palate. At Hamburger and Hamilton stage 22, LOX is present in the ectodermal basement membrane along the FNP, but at stage 26 it is present primarily in the basement membrane lining the nasal pits. LOX is also present in the lateral edges of the mesenchyme of the FNP at stage 22, but is more present in the medial and caudal mesenchyme at stage 26. These results mirror the distribution of collagen 4 and laminin, where more crosslinking was observed in regions resisting growth (like the nasal pits) and in regions where cellular forces may be generated to direct growth (ectodermal basement membrane). We next prevented initiation of crosslinking with a small‐molecule from Lathyrus odoratus, beta‐aminoproprionitrile (BAPN), which permanently binds to LOX. Ingestion of L. odoratus causes osteolathyrism, a musculoskeletal degenerative disease, and researchers have found that BAPN administered in high concentrations to embryos causes skeletal and craniofacial deformities. We found that microinjections of BAPN in the caudo‐lateral edge of the FNP at low concentrations (50, 75, and 100 ng/uL) caused mild truncation of the FNP without affecting LOX localization or increasing cell apoptosis (via TUNEL staining). Together, these results provide preliminary evidence that tissue compliance may play an important role in facial morphogenesis. Future work will utilize a LOX RNAi vector to further examine the role of extracellular matrix crosslinking on palate growth and fusion.

Support or Funding Information

Funding was provided by: CIHR, and NIH, NIDCR R21‐DE028198
Perception of the Chaldean Population Towards Whole Body Donation
Authors: Gappy Mavis , Barremkala Malli , Farr A. Celeste
Published: April 2020

INTRODUCTION

The use of human donors for anatomic dissection is a long‐held tradition in medical education. The majority of donors are Caucasian which leads to a lack of racial diversity among the body donors. The aim of this project is to determine the knowledge, attitude, and perception of the Chaldean population towards whole‐body donation. By understanding the perspectives of the Chaldeans relative to whole body donation programs, we may be able to explain their lack of participation in the donation programs.

METHODS

Focus groups were held to ascertain the participants understanding of the body donation process, willingness to donate, and influencing factors. Chaldeans over the age of 18 were invited to participate in the study. During the focus groups, audio recordings were collected for transcription and analysis of responses. They were recruited from the Southeast area of Michigan, which has one of the largest Chaldean communities in the country. Five focus groups have been conducted with a total of 30 participants between the ages of 18–50.

RESULTS

A majority of participants reported that they did not know about the whole‐body donation process and were more familiar with organ donation. They generally agreed that whole‐body donation is valuable for education yet they would prefer organ donation. For either donation type, they generally believed in the conspiracy theory that the healthcare worker would let them die to get their body or organs. The factors influencing their decision to donate are family, providing a benefit to society, religion, having a funeral, and being cremated. The participants were unsure whether Catholicism permits whole‐body donation or cremation (negative factor). The consensus among the groups was that they would never do whole‐body donation but might donate their organs.

CONCLUSION

The results reveal that participants are unwilling to participate in whole body donation programs mostly because of the lack of knowledge on the whole‐body donation process and a conflation with organ donation. Participants commented that they would be interested in more education regarding whole‐body donation. Anatomy dissection programs that are planning to improve diversity in their cadaver population should talk to Chaldean religious leaders to get a further understanding of the religious implication of whole‐body donation and engage in educational programs in the community that reflect and respect those views. Furthermore, education should explain the difference between whole‐body donation and organ donation and the benefits of these donations while also addressing the related conspiracy theory.
The Impact of Temperature on Critical Power Determined by a Three Minute All‐Out Test
Authors: Kaiser Brendan W. , Kruse Ka’eo K. , Gibson Brandon M. , Santisteban Kelsey J. , Larson Emily A. , Wilkins Brad W. , Jones Andrew M. , Halliwill John R. , Minson Christopher T.
Published: April 2020

Critical Power (CP) separates the heavy and severe exercise domains, and work above CP (defined as W′) results in an inexorable progression to maximal oxygen uptake and rise in blood lactate concentration to fatigue. The thermoregulatory demands accompanying a rise in body core temperature (Tc) compounded with the cardiovascular challenges presented by maximal exercise in a hot environment may impact CP and W′. To date, no study has explored the effects of elevated Tc and environmental temperature on CP and W′. The purpose of this study is to determine the effect of high environmental and Tc on CP and W′. CP was estimated as end test power (average of the last 30 sec) from a series of three‐min “all out” tests (3MT). Volunteers (n=7, 3F) performed a 3MT during a familiarization visit and two experimental study days (thermoneutral and hot) in a randomized crossover design. Prior to the 3MT in both experimental conditions, subjects were immersed in either thermoneutral (36°C for 30 min) or hot (40.5°C) water until Tc was ≥ 38.5°C. All 3MT were performed in an environmental chamber controlling for both temperature and humidity (18°C and 45% RH for thermoneutral; 38°C and 40% RH for hot). A paired t‐test was used for statistical analysis and data are presented as mean ± SE. Although variable, CP was similar between thermoneutral (245 ± 22W) and hot (243 ± 25; p=0.83), while W’ was only mildly reduced from thermoneutral (11 ± 1 kJ) to hot (9 ± 2) p=0.17. Total work performed was similar between thermoneutral (55 ± 5 kJ) and hot (53 ± 5) conditions (p=0.27) and peak power was similar between thermoneutral (827 ± 89W) and hot (809 ± 85) p=0.49. These preliminary data suggest that high Tc and environmental temperature, per se, appear to have no significant impact on CP as estimated by a 3‐minute all‐out test.
L‐Plastin, a Novel Regulator of Microglial Activation in Parkinson’s disease
Authors: Budge Kevin , Richardson Jason , Safadi Fayez
Published: April 2020

Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease worldwide behind Alzheimer’s disease. One prominent feature of PD is the marked loss of dopaminergic and motor dysfunction. Currently, there are no therapies to effectively slow disease progression. Neuroinflammation is a major contributing factor in neurodegeneration diseases including PD. Microglia, a type of glial cell, are primary regulators of neuroinflammation and these cells are capable of assuming activation states ranging from inflammatory to anti‐inflammatory and reparative. Thus methods to combat microglial inflammation or promote microglial anti‐inflammatory and reparative effects show promise as treatments to slow neurodegeneration. L‐plastin (LPL) is an acting bundling protein and other research has shown that LPL plays a role in osteoclast function, T‐cell activation, and it may be involved in the NLRP3 inflammasome activation in macrophages. It has been shown in osteoclasts, that LPL phosphorylation at serine 5 plays a role in sealing zone formation. Using a peptide inhibitor to block phosphorylation of serine 5 on LPL prevented sealing zone formation and inhibited osteoclast‐mediated bone resorption. Given the evidence that LPL may play a role in inflammasome activation in macrophages, we investigated if LPL may play a role in microglial activation. To assess the function of LPL in the microglial inflammatory response, we utilized qPCR, Western blot, and immunofluorescence techniques. We show here that lipopolysaccharide (LPS) treatment induces LPL phosphorylation at serine 5, as well as increased Lcp1 mRNA expression. Furthermore, using a TAT‐fused peptidomimetic inhibitor of LPL phosphorylation significantly reduced LPS‐induced inflammatory gene (Il‐1b, Il‐6, Tnfa) expression. For this study, an acute 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) mouse model was also used. Male (10‐week‐old) C57BL/6N mice were treated with four injections (one every two hours) of either saline or 15mg/kg of MPTP. Mice were then sacrificed two days after the last injection and striatum and hindbrain were isolated. Two days after MPTP treatment is when peak inflammation occurs in the brain. We found that Lcp1 mRNA expression was significantly increased in the striatum of MPTP‐treated mice compared to saline‐treated mice. These results suggest that LPL may play a role in the microglial inflammatory response, and that LPL inhibition may be a novel way to combat neuroinflammation and neurodegeneration. We are currently staining for phosphorylated LPL in the microglia of MPTP treated mice. Furthermore, we have obtained LPL KO mice to assess the in vivo function of LPL in mouse models of PD.

Support or Funding Information

Kent State University Biomedical Sciences
Striatal specific orphan GPCRs crosstalk with Dopamine D2 receptors to regulate cAMP signaling
Authors: Felsing Daniel E. , Zamora Joshua , Raval Sweta , Allen John A.
Published: April 2020

The striatum is a subcortical brain region that performs vital functions including movement control, regulation of attention and is a critical component of the reward system. Current drug targets have displayed limited efficacy in treating striatal‐related diseases including for substance‐use disorders, schizophrenia and Parkinson’s disease. G protein‐coupled receptors (GPCRs) are valuable drug targets with ~35% of FDA approved medicines targeting these receptors. However, nearly 120 GPCRs remain orphan receptors, whose endogenous ligands, G protein signaling, and therapeutic potential are unknown. We previously conducted a comprehensive RNAseq genome‐wide survey of human tissue gene expression and identified several orphan GPCRs selectively expressed in the human striatum, namely, GPR6, 52, 88, 101, 139 and 149. Here we elucidate striatal orphan receptors that modulate G protein‐mediated cAMP signaling and examine potential signaling crosstalk between orphan receptors and dopamine D2 receptors (D2R). Biochemical and CRISPR/Cas9 knockout studies in human embryonic kidney 293 (HEK293) cells confirmed GPR6, 52 and 101 increased cyclic adenosine monophosphate (cAMP) via Gs/olf G proteins, while GPR88 decreased cAMP levels via Gi/o G proteins. Notably, when expressed alone, GPR6, 52 and 101 profoundly increased basal levels of cAMP, indicating these receptors are constitutively active. We hypothesized that GPR6, 52, and 101 may set the basal tone for cAMP that would allow robust function of the Gi/o‐coupled dopamine D2R to inhibit adenylyl cyclase and decrease cAMP. When expressed alone in HEK293 cells and without any stimulation to the adenylyl cyclase system, the cAMP inhibition window for the D2R and agonist quinpirole was minimal and difficult to reliably detect (~1000 light counts/sec, Glosensor assay). However, when the D2R was co‐expressed with either GPR6, 52, or 101, the assay window to inhibit cAMP for quinpirole was magnified by over 200X (200,000–500,000 light counts/sec), with agonist potency unchanged. These studies suggest that these striatal specific human orphan receptors may establish dynamic basal cAMP levels and this may facilitate inhibition of cAMP via Gi/o‐coupled receptors, such as the D2R. Our findings also suggest GPR6, GPR52 and GPR101 are potential drug targets to modulate cAMP signaling in striatal specific neurons, which may be therapeutic for treating several neurological and neuropsychiatric diseases.

Support or Funding Information

NIDA T32 DA07287 (DEF) and RSGPT18‐ PhRMA Foundation (JAA).
Longitudinal Analysis of Gut Microbiome Composition After Experimental Cervical Spinal Cord Injury
Authors: Newton Jessica S. , Huffman Emily E. , Britsch Daimen R.S. , Strattan Lydia E. , Maggard Rachel S.J. , Calulot Chris M. , Alilain Warren J.
Published: April 2020

Following an experimental C2 spinal cord hemisection in rats, there is a gradual spontaneous recovery of breathing function that can take place over time. Additionally, interventions at later time points are more effective after injury. What is not known is the mechanism mediating this observation. To begin to answer this question, we investigated the role of the gut microbiome after injury. Recent studies have emerged suggesting that the gut microbiome has critical implications on the proper functioning of the central nervous system (CNS). Indeed, gut dysbiosis, or a microbiome imbalance, can occur which can negatively impact the CNS. Neurotrauma, including spinal cord injury (SCI), can lead to acute gut dysbiosis and impaired recovery. It is our hypothesis that the composition of the gut microbiome is dynamic after injury with dysbiosis improving over time from an acute post‐SCI state. In this study, we build upon these initial studies and investigate the impact of cervical SCI on the gut microbiome over time and up to chronic timepoints. In order to test our hypothesis, we collected fecal samples before and up to 12 weeks after a C2 hemisection in adult female rats, in order to assess microbiome composition at various timepoints post injury. Preliminary results suggest that following cervical SCI, gut dysbiosis occurs immediately after injury but recovers by two months post injury. Future studies will classify bacterial identities and assess the impact of the post‐injury gut microbiome on recovery of respiratory motor function and plasticity.
Ultra‐Small Biomimetic Nanoparticles for Alzheimer’s Disease Targeted Drug Delivery
Authors: Tatiparti Katyayani , Rauf Mohd Ahmar , Sau Samaresh , Alzhrani R. , Iyer Arun
Published: April 2020

Introduction

Alzheimer’s disease (AD) the most common form of dementia worldwide, that affects older population and the 6th leading cause of death in USA. The cost of care involved is about $234 billion. AD is marked by the presence of beta‐amyloid protein plaques outside neurons that leads to cell death and the accumulation of an abnormal intracellular neurofibrillar tangles of hyper phosphorylated τ (tau) protein. This undesired accumulation causes obstruction of nutrition and other essential molecules to the brain. There is no potential treatment in the clinic because of the lack of proper understanding of the pathogenesis, delivery challenges across the Blood‐Brain‐Barrier (BBB), and unfavorable immune responses.

Challenge

In the present research we are developing ultra‐small nanoparticles (USNPs), that are expected to (i) Enhance the circulation half‐life and minimize the side effect of therapeutics; (ii) enable transport of therapeutics across the BBB and promote their accumulation in the desired brain region; (iii) assist in reduction and clearance of beta amyloid plaques; and (iv) promote favorable immune response. In addition when USNPs are combined with an NIR‐dye it may function as a diagnostic agent. In order to test our hypothesis, we designed a new Lactoferrin (Lf) based USNP to target low‐density lipoprotein receptor‐related protein‐1 (LRP‐1) overexpressed in AD.

Results

The TEM analysis showed well‐defined spherical Lf NPs with an ultra‐small size of ~ 20–30 nm that will allow efficient penetration/delivery across the BBB. Further, the data obtained from our preliminary studies in SH‐SY5Y and BV2 brain cells suggest a dose‐dependent cell uptake of DiD‐fluorescence labeled Lf NPs. The cytotoxicity assays on normal brain cells showed them to be safe/non‐cytotoxic with a cell viability around ~ 85–90%. Subsequently, Tauopathy was induced employing okadoic acid in SH‐SY5Y cells to mimic AD under in vitro conditions. This was followed by testing of various combinations of the protein kinase inhibitors to assess the efficacy of the combination therapy on the hyper phosphorylation of Tau protein (pTau) in the brain cells. We also developed conditioned BBB cell culture model to demonstrate the penetration of Lf USNP across the BBB. Our IHC data reveal that it can be used to deliver combination of protein kinase inhibitors into the brain to reduce the tau tangle formation. Thus far, our hypothesis is being supported by the observed results.

Conclusion

The preliminary results looks very promising in the direction of AD combination therapy. The outcome for our study protend to provide a strong rationale to employ Lf USNPs to overcome the BBB for improved AD targeting of the drugs, leading to better therapy outcome.
How Comparable are 2D and 3D Methods for Measuring Bone Laminarity in Developing Pigeons?
Authors: McGuire Rylee S. , Brown Alexandra , Jonglertjanya Kittaporn , Simons Erin L. R. , Lee Andrew H.
Published: April 2020

The cortical bone of birds contains microscopic tunnels called vascular canals, which can be classified based on orientation as: longitudinal, radial, oblique, or circumferential. Circumferential canals are hypothesized to reflect adaptation to flapping flight. This hypothesis is supported by histological surveys of birds that show the proportion of circumferential canals (laminarity) is elevated in bones thought to experience habitual torsional loads. However, the most detailed data on torsional loading during avian flight is from the pigeon, a species in which the adults show low wing bone laminarity. Nevertheless, there may still be support for biomechanical adaptation if the laminarity is greater in adults than in juveniles incapable of flight. Alternatively, if the pattern is reversed, laminar bone may simply be a juvenile trait.

Laminarity has largely been analyzed using 2D histological sections, which may not accurately represent complex 3D structure. High‐resolution micro‐CT is an imaging technique that allows 3D reconstruction, providing greater accuracy and objectivity in the measurement of canal orientation. Here, we compare 2D and 3D methods to clarify how wing bone laminarity develops in pigeons.

We sampled a growth series of four pigeons that were collected from a previous study. A cortical strip (1 × 3 mm) was cut from the right humerus, ulna, and radius of each bird. The strips were scanned with high resolution microCT. 3D analysis involved segmentation, autoskeletonization, and measurement of 3D canal orientation using Avizo. For 2D analysis, five evenly spaced orthoslices were digitally extracted from each strip. Canals were fitted with ellipses and trigonometric equations were used to approximate orientation. In both 2D and 3D methods, canals were categorized and laminarity was calculated. Principal components analysis was performed to convert highly correlated variables (i.e., mass, bone length, and section modulus) into principal components (PCs). The relationship between principal components and laminarity was assessed using beta regression modeling.

2D analysis overestimates laminarity among juvenile specimens. Therefore caution is warranted when applying 2D analysis to complex canal networks. Nevertheless, both methods show that the laminarity of each element significantly decreases with maturity (PC 1). Therefore, torsionally loaded bone is not necessarily more laminar. It seems laminarity is influenced by additional factors that warrant further investigation.

Support or Funding Information

College of Graduate Studies Intramural Funding (Erin L. R. Simons & Andrew H. Lee); Kenneth A. Suarez AZCOM Summer Research Fellowship (Alexandra Brown, Kittaporn Jonglertjanya, Rylee S. McGuire)
Regulation of TAZ Expression by Cyclic Stretch
Authors: Miranda Maria Zena , Turki Razan , Kuebler Wolfgang M. , Simmons Craig A. , Kapus Andras
Published: April 2020

It is increasingly recognized that fibrosis‐related phenotype transitions (e.g. EMT and fibrobrolast‐myofibroblast transition) are brought about by the interplay between chemical (e.g. TGFβ) and mechanical (e.g. stretch) stimuli, which, in part, act through cytoskeleton‐regulated transcription factors. Among these, the Hippo effector TAZ and the Rho/actin‐regulated myocardin‐related transcription factor (MRTF) are TGFβ‐ and mechano‐sensitive transcriptional co‐activators, which play key roles in the pathogenesis of organ fibrosis. Previously we have shown a multilevel crosstalk between TAZ and MRTF. Moreover, we found that TGFβ is a strong inducer of TAZ protein and mRNA expression, an effect mediated by p38‐dependent MRTF activation, independent of a net nuclear translocation of MRTF or TAZ. These results revealed that, in addition to TAZ nuclear translocation, the regulation of TAZ expression (a hitherto poorly characterized process) is also a critical feature of fibrogenesis. Since mechanical stimuli are prominent inducers of fibrosis, we sought to determine if cyclic stretch impacts TAZ expression and to investigate the potential role of the RhoGTPase/MRTF signaling pathway in this process. Cyclic stretch (10%, 1 Hz) increased MRTF‐transcriptional activity, measured by a CArG box‐ (cis‐element for MRTF) containing luciferase reporter construct, between 3 and 6 hr in C3H10T1/2 cells. Overexpression of Rho family small GTPases (RhoA, Rac1 and Cdc42) known to be activated by stretch and to induce MRTF translocation, were sufficient to enhance the activity of a TAZ promoter luciferase reporter (TAZ‐Luc). Consistently, cyclic stretch increased TAZ protein and mRNA expression, and activated TAZ‐Luc. TAZ‐Luc activation was abolished by MRTF inhibition or the mutation of the CArG box within the reporter (mtTAZ‐Luc). The stretch‐induced activation of TAZ expression was a universal phenomenon, since similar effects were observed in Interstitial Rat Mesenchyme Cells (IRMC) and Human Pulmonary Microvascular Endothelial Cells (HPMEC) as well. Finally, since the redox‐regulator pirin was recently suggested to be an upstream regulator of MRTF, we checked its potential involvement in the regulation of TAZ expression. While CCT‐251236, a pirin‐binding compound abolished the TGFβ‐induced TAZ expression, pirin silencing failed to recapitulate this effect. This suggests that the drug has alternative targets and MRTF can be activated in a pirin‐independent manner. Taken together, our studies show that critical chemical and mechanical fibrogenic stimuli increase TAZ expression and MRTF is a key mediator of this effect, both via classic (Rho‐mediated, translocation‐dependent) and by “non‐canonical” (phosphorylation‐induced activation) mechanisms. We propose that the ensuing rise in TAZ expression potently accelerates and exacerbates fibrogenic phenotype transitions.

Support or Funding Information

Kidney Foundation of Canada and Natural Sciences and Engineering Research Council of Canada (AK)
Morphological Grouping and Body Mass Prediction Models
Authors: Lancaster Terry (Bird) Elisabeth
Published: April 2020

Every facet of an organism’s function is affected by its body size, such as heart rate, metabolism, organ size and function, feeding ecology, and locomotion. Using body size to assess the function and size of organs in extant mammals is relatively easy. However, estimating the body size and organ function of transitional fossil species, such as the Eocene whales, is difficult. The Eocene whales are diverse in body size, structure, and habitat, therefore a single allometric model will not provide the most accurate body size estimation for each fossil whale family and therefore less accurate organ function assessment. The goal of my research is to predict the body size of the Eocene whales using morphological models of extant mammals as guides. The first stage is the morphological grouping and body mass prediction models using modern mammal skeletons.

I measured the skull, vertebrae, and appendicular bones of multiple species of modern aquatic, semi‐aquatic, and terrestrial mammals. I calculated an average body mass representative of each species using recorded body weights. Using Principal Component Analysis (PCA), I determined morphological groupings for these mammals from the skeletal measurements. For each PCA analysis completed, I created a linear regression equation to predict body size from the skeletal variables used in each analysis and the calculated average body sizes. I tested these prediction models using the measurements from 12 representative skeletons that were not included in the data set used to create the model, specifically Odocoileus virginianus, Tragulus napu, Hippopotamus amphibus, Tapirus indicus, Sus scrofa, Canis lupus lycaon, Nyctereutes procyonid, Ehydra lutris, Gulo gulo, Neophoca cinerea, Lobodon carinophagus, and Ursus maritimus.

From the PCA, I identified five morphological groups for the mammals selected: Aquatic, Semi‐aquatic, Semi‐terrestrial, Large Terrestrial and Small Terrestrial. For the prediction model using all skeletal variables, the standard error of the estimate (SEE) for all 12 test individuals ranged from 0.3359 – 0.7496. For the skull variables, the SEE ranged from 0.1260 – 0.3197. For the limb variables, the SEE ranged from 0.1716 – 0.3964. For the vertebral variables, the SEE ranged from 0.1511 – 0.2851. These small SEE values and the large sample size substantiate the accuracy of these predictive models and the reliability of their respective confidence intervals (CI) and prediction intervals (PI) for the models. The results confirm that these linear regression models will be appropriate to predict the body mass of the Eocene whales in the second stage of this project.

Support or Funding Information

Kent State University; East Tennessee State University; Sigma Xi
Role of b‐lapachone treatment mediated base excision repair protein PARylation in colorectal cancer cells.
Authors: Singh Naveen
Published: April 2020

Poly (ADP‐ribose) PAR is a protein post‐translational modification catalyzed by a family of enzymes known as PARPs. Most solid tumors overexpress NAD(P)H;Quinone Oxidoreductase 1 (NQO1), which catalyzes a futile redox cycle of b‐lapachone during exposure, resulting in the formation of high levels of reactive oxygen species (ROS). Futile cycle b‐lapachone recycling produces elevated superoxide levels, resulting in massive levels of hydrogen peroxide that lead to substantial DNA damage and endoplasmic reticulum Ca2+ release. DNA damage and Ca+2 entry into the nucleus leads to the hyperactivation of poly(ADP‐ribose) polymerase 1 (PARP1). A majority of parylation is noted when hyperactivated PARP1 self‐parylates, forming extensive Poly(ADP) ribosylated PAR‐PARP1 levels are noted within 5 mins of exposure to 4 μM β‐lap in A549 cells, as noted by Western blotting using an antibody specific for PARylated moieties. PAR‐PARP1 is a protein post‐translational modification catalyzed by a family of enzymes known as PARPs. DNA single strand breaks along with base‐damage and abasic sites together with nuclear Ca2+ levels lead to hyperactivation of PARP1, a DNA repair enzyme that utilizes NAD+ to generate PAR moieties. This is followed by dramatic NAD+/ATP losses and tumor cells die of a special programmed necrosis process, referred to as NAD+‐keresis. PAR‐PARP1 is rapidly degraded by poly(ADP‐ribose) glycohydrolase (PARG) a exo‐ and endoglycosidase.

PARG‐knockdown HCT116 cells were used to gain insight into the function of PARG and whether PARG depletion would enhance b‐lapachone induced cell death by blocking PAR recycling. Proteomic screening after b‐lapachone treatment in colorectal cancer cell line revealed the PARylation landscape of base excision, mismatch and nucleotide excision repair proteins. Twenty (20) of the proteins were found to overlap with H2O2 treatments (200 μM or 2 mM), while one protein (i.e., SPSF) was unique to b‐lapachone treatment. b‐Lapachone treatment affects BER pathways and qualitative analysis of the human Asp‐ and Glu‐ADP‐ribosylated proteome after b‐lapachone treatment indicate major alterations in BER pathways. We confirmed that b‐lapachone mediated cytotoxicity was independent of PARG knockdown or TOPO I or II protein losses. In contrast, RFC1 and PCNA siRNA‐mediated protein knock downs significantly enhanced b‐lapachone lethality, whereas knockdown of Topo I or Topo IIalpha did not affect b‐lapachone lethality. Taken together, these results demonstrated that knockdown of RFC1 enhances lethality of b‐lapachone and could serve as an effective treatment strategy in cancer conditions.

Support or Funding Information

This work was funded by NIH/NCI 5 R01 CA102792‐16 to DAB.
Assessing understanding of structural relationships: early lessons in a large scale virtual reality deployment
Authors: Meyer Carolyn A. , Ivie Kenneth R. , Heise Natascha , Brown Katie , Clapp Tod R.
Published: April 2020

The study and practice of medicine requires a comprehensive understanding of interactions and an accurate perception of spatial relationships within the body. Existing diagnostic, classroom, and patient education materials are limited by 2‐dimensional representations of these 3‐ dimensional associations. Virtual reality is an emerging technology that creates a more lifelike and intuitive learning experience by allowing users to interact with 3D data. This technology can be used to advance the efficiency of understanding the 3‐dimensional associations that constitute the human body.

BananaVision is a networked, multiuser virtual reality tool that renders human anatomical structures for the creation of instructional content. It allows users to quickly explore structural relationships as well as identify and dissect in a virtual environment. Here we describe a large‐scale virtual reality deployment and examine its efficacy as a tool to engage students and aid in preparation for a human dissection course.

Human gross anatomy dissection students were randomly assigned to three groups. Each group was provided different study tools; an anatomy atlas, autonomous use of BananaVision virtual reality, or autonomous use of BananaVision virtual reality along with a pre‐recorded instructional module. After the study period, students were required to complete an assessment of their knowledge of structural relationships. Students found the BananaVision to be an engaging and useful tool. Additionally, the study group given access to BananaVision along with the module were more efficient in their preparation. However, performance on assessments was similar between groups.

As virtual reality technology is implemented in education, the field is in the early stages of evaluating meaningful processes and procedures that demonstrate measurable outcomes. This study begins to address some of the early challenges when integrating a virtual reality deployment in anatomy curriculum. As demands on gross anatomy classrooms continue to increase, it is important to identify technologies and strategies that improve efficiency.
Cranial Base Structures Geometric Morphometry and Description of a Clinical Relevant Anatomical Variation
Authors: Fazan Frederico Sassoli , Assis Cirino Mucio L. , Thomazini José Antônio , da Cunha Tirapelli Daniela Pretti , Tirapelli Luis Feranado , Fazan Valéria Paula Sassoli
Published: April 2020

The foramen magnum is a large median oval opening in the occipital bone in the base of the skull, which leads into the posterior fossa. The foramen magnum has a forwarded shift of its anterior border in humans causing a shortening of the cranial base. The foramen is described as oval, wider posteriorly than anteriorly, and its antero‐posterior diameter exceeds the transversal one. Anteriorly, the margin of the foramen magnum is slightly covered, on each side, by an occipital condyle. These condyles, oval in shape, are located obliquely, so that their anterior end is closer to the median plane. Thirty dry skulls, from adult individuals, present in the anatomy laboratory of our institution were evaluated and a study of the dimensional anatomy of the foramen magnum and the occipital condyles was performed. With the aid of an analog caliper (precision of 0.05mm), the antero‐posterior and transverse diameters of the foramen magnum (posterior to the occipital condyles) and occipital condyles were obtained. Our results showed that the mean antero‐posterior diameter of the foramen magnum was 36.1 ± 0.5 mm and the transverse diameter was 30.0 ± 0.4 mm. The mean antero‐posterior diameter of the right occipital condyle was significantly smaller than that of the left, being 22.2 ± 0.4 mm and 23.1 ± 0.4 mm, respectively. The mean transverse diameter of the right occipital condyle was also statistically smaller than that of the left, being 12.6 ± 0.2 mm and 13.2 ± 0.2 mm, respectively. One of the skulls studied (excluded from the previous statistic) showed anatomical variation in the shape and size of the foramen magnum. This foramen was narrow, with an antero‐posterior diameter of 26.75 mm and a transverse diameter of 22.50 mm. In addition, the occipital condyles were asymmetrical, with the right condyle being practically transverse in relation to the foramen magnum, making a protrusion into its interior, contributing to the transverse narrowing of this foramen. The left condyle presented antero‐posterior (22.95 mm) and transversal (15.95 mm) diameters larger than the right (19.35 mm and 13.10 mm respectively). There were no signs of fractures or previous craniotomies in this particular skull. The present study reports the geometric morphometry of some striking structures at the base of the skull, in addition to describing an important anatomical variation of these structures. We did not find reports of significant difference between the right and left condylar diameters in the literature. The anatomy of the skull base is useful for estimating the race and sex of individuals in forensic medicine. Furthermore, geometric morphometry is an important tool in primatology and anthropology.

Support or Funding Information

FAPESP, CNPq, CAPES and FAEPA
3β‐Diol Attenuates COX‐2 Levels in a Neonatal In‐Vivo and In‐Vitro Ischemic Injury Rodent and Human Model
Authors: Beer Yasmin , Shi Samuel , Rodriguez Janelle , Gonzales Rayna J.
Published: April 2020

Studies using experimental stroke models demonstrate that neuroprotective agents which provide protection from secondary injury, including inflammation, result in improved outcome. Estrogen receptor β (ESR2) has the potential to be a novel therapeutic target in the treatment of ischemic stroke. Previous studies have shown that ESR2 activation elicits beneficial effects in the cerebrovasculature by promoting vasorelaxation and by attenuating pro‐inflammatory mediator production. We previously observed that activation of the androgen, 5α‐androstane‐3β,17β‐diol (3β‐diol), reduced pro‐inflammatory mediator production in adult human vascular smooth muscle cells and rodent cerebral arteries, and this response was ESR2 dependent. The current study was designed to determine if ERS2 is expressed in both a human pediatric brain endothelial cell model and juvenile rat stroke model and whether 3β‐diol will attenuate cyclooxygenase‐2 (COX‐2) expression following ischemic injury. Male rat pups (P10–15) underwent 15 minutes of bilateral internal carotid artery occlusion (BCAO) or sham surgery and 24‐hour reperfusion. At the onset of reperfusion, pups were injected with 3β‐diol i.p. (2.5mg/kg) or vehicle (1.2% EtOH and 98.8% PEG). Next, animals were heavily sedated and whole brain removed at 24 hours. In an in‐vitro model of ischemic stroke, male primary pediatric human microvascular endothelial cells (HBMEC; Cell Systems) were exposed to hypoxia plus glucose deprivation (HGD; 1% O2) or normoxia (21% O2) plus or minus 3β‐diol (10nM) or vehicle (0.0001% ethanol) for 3h at passage 7, using a humidified chamber (BioSpherix®) housed within a 5% CO2 incubator. Total protein was extracted from rat whole brain and human endothelial cells, assayed for protein content, and COX‐2 levels assessed using western analysis. RT‐PCR analysis was used to explore whether 3β‐diol alters expression of its target receptor, ERS2. We observed that ESR2 was basally expressed in the prepubescent brain and HBMECs, and expression was not altered following ischemic injury. Proinflammatory COX‐2 protein levels were increased in both whole brain and HBMEC homogenates from BCAO and HGD groups compared to sham and vehicle controls, and the selective ERS2 agonist, 3β‐diol, attenuated this response. In conclusion, these studies suggest that 3β‐diol may have a role in modulating ischemia‐induced COX‐2 during stroke. Since ERS2 is expressed in the whole brain and brain endothelial cells, and ERS2 levels are not decreased during ischemic injury, ERS2 may be a viable target for future stroke therapeutics in the pediatric population by attenuating inflammatory modulators during ischemic injury in the developing brain.

Support or Funding Information

University of Arizona Valley Research Partnership Grant VRP04 P1 (RJG), American Heart Association 19AIREA34480018 (RJG).
Transcriptome of SHR Carotid Bodies Reveals Novel Peptide Receptors Associated with Hypertension
Authors: Pauza Audrys G. , Paton Julian , Murphy David
Published: April 2020

Background

The epidemic of hypertension demands novel therapeutic strategies and innovative multi‐system approach to clinical management. The carotid body (CB) is the primary site of peripheral chemoreception in the human body. CB dysfunction has been implicated in numerous cardio‐respiratory and metabolic conditions, including essential hypertension, diabetes mellitus, sleep apnoea and heart failure. Recent attempts to modulate CB activity has proven successful in alleviating symptoms despite lacking specificity at the target organ. The ribbon‐cable hypothesis suggests that the CB is composed of specialised chemosensory subunits responsive to a specific stimulus in the bloodstream
1.

Aim

We hypothesised that the hypertensive phenotype is associated with alterations in a subset of chemosensory subunits, identification of which would offer highly specific therapeutic opportunities for clinical intervention.

Methods

We performed a high‐throughput transcriptomic screen of the CBs in the spontaneously hypertensive (SHR) and the normotensive Wistar‐Kyoto (WKY) rats.

Results

Transcriptomic screen of the SHR CBs revealed numerous differentially expressed genes associated with the hypertensive state. Our experimental design uncovered underlying lateral differences between the CBs at the intra‐individual level. Network analysis revealed genes associated with serine metabolism, oxidoreductase activity, and G protein‐coupled receptors to be significantly enriched in the WKY‐SHR comparison. A number of previously unidentified peptide receptors were found to be differentially expressed in the SHR.

Conclusion

Newly identified receptors serve as candidate genes for pre‐clinical proof of concept and feasibility functional studies. This provides a unique opportunity to design novel therapies to abate carotid body tonicity and hyperreflexia in neurogenic and metabolic hypertension.

Support or Funding Information

This work has been generously supported by the British Heart Foundation (BHF)