bluuebunny/crossref_metadata_2025 huggingface dataset:
Vascular and Blood Flow Responses to Acute Upper Limb Exercise: Impact of Sex and Menstrual Cycle Phase
Authors: Garten Ryan , Weggen Jennifer , Hogwood Austin , Decker Kevin , Darling Ashley
Published: April 2020

Purpose

This study sought to determine whether fluctuations in estrogen levels throughout the menstrual cycle (early (EF) vs late follicular (EF) phase) altered the vascular and blood flow responses to acute upper limb exercise and if these responses were different when compared to males.

Methods

Ten young females (YF: 21±3 yrs) were recruited and completed two identical visits (EF and LF) consisting of an acute upper limb exercise bout (progressive, rhythmic handgrip). Peripheral (brachial artery (BA) diameter, shear rate, and blood flow) and central (mean arterial pressure, heart rate) hemodynamic measurements were obtained during the last minute of each 3‐minute handgrip workload (3kg, 6kg, and 9kg) via Doppler ultrasonography and finger plethysmography, respectively. Male controls (YM: n = 21; 21±3 yrs) matched for age completed one visit to provide a biological sex comparison. Data is presented as mean ± standard deviation.

Results

In young females, brachial artery flow mediated dilation (controlled for shear rate) [EF (3kg: 0.006±0.01; 6kg: 0.015±0.01; 9kg: 0.022±0.01); LF (3kg: 0.008±0.01; 6kg: 0.017±0.01; 9kg: 0.024±0.01)], blood flow [EF (3kg: 162±61; 6kg: 236±74; 9kg: 332±104 ml.min−1); LF (3kg: 151±23; 6kg: 237±47; 9kg: 330±104 ml.min−1)], and vascular conductance [EF (3kg: 1.8±0.7; 6kg: 2.5±0.7; 9kg: 3.1±0.7 ml.min−1.mmHg−1); LF (3kg: 1.8±0.4; 6kg: 2.6±0.6; 9kg: 3.3±1.1 ml.min−1.mmHg−1)] were significantly increased with each progressive handgrip workload but were unaltered when comparing the EF and LF conditions. When compared to young males (YM), young females reported no differences in BA FMD [YF vs YM (3kg: 0.006±0.01 vs 0.007±0.01; 6kg: 0.015±0.01 vs 0.013±0.01; 9kg: 0.022±0.01 vs 0.018±0.01)], but did report significantly lower blood flow [YF vs YM (3kg: 162±61 vs 228±64; 6kg: 236±74 vs 347±94; 9kg: 332±104 vs 474±119 ml.min−1)] and vascular conductance [YF vs YM (3kg: 1.8±0.7 vs 2.5±0.7; 6kg: 2.5±0.7 vs 3.8±0.9; 9kg: 4.9±1.1 vs ml.min−1.mmHg−1)].

Conclusion

The findings of this study revealed that fluctuations in estrogen throughout the menstrual cycle do not alter vascular and blood flow regulation during exercise, but, when compared to young males, young female’s exhibited lower blood flow/vascular conductance. Additional research is necessary to determine if these sex differences in blood flow/conductance represent adequate or inadequate blood flow regulation during exercise.
Properties of Lipid Rafts in Two Epigenetically Distinct Subtypes of the Oncogenic Cell‐line SW13
Authors: Pascual Agnes S. , Espejo Luis S. , Moore Jacob L. , Leyva Kathryn J. , Hull Elizabeth E.
Published: April 2020

Introduction

Mutant GOF p53 is an epigenetic regulator which promotes oncogenesis. Many cancers with a GOF mutation accumulate cholesterol and inhibition of cholesterol synthesis with statin treatment reverses some of the pro‐oncogenic properties of GOF p53. Our central hypothesis is that lipid raft signaling is altered by cholesterol accumulation in cells expressing a GOF p53. To address the role of cholesterol in promoting oncogenesis, we use the SW13 cell line bearing a GOF p53 (H193Y). This cell line has two epigenetically distinct cell subtypes that differentially express genes coding for GPI anchors, cholesterol biosynthesis, and sphingolipids, all of which are components of lipid rafts. In addition, the SW13 subtypes have different oncogenic profiles. The SW13(Vim−) subtype has an epithelial morphology and is highly proliferative. The SW13(Vim+) subtype has a mesenchymal like phenotype and a higher metastatic potential. When treated with histone deacetylase inhibitors (HDACi), SW13(Vim−) appear to adopt a SW13(Vim+) phenotype. Therefore, we treat with HDACi to control the subtype conversion and shRNA to knockdown GOF p53 expression. This experimental approach allows the composition of raft fraction to be correlated to GOF p53 expression and oncogenic properties of each SW13 subtype, with and without p53 expression.

Methods

Proliferation rate, MMP expression, and migration of SW13(Vim−), SW13(Vim+), shRNA SW13(Vim−), and shRNA SW13(Vim+) were quantitated by EdU assays and in situ zymography respectively. We have isolated lipid raft fractions by the detergent resistant enrichment technique from SW13 cells of each subtype, with and without GOF p53 expression knockdown by shRNA. Proteins were identified by mass spectrometry analysis. Fluorescent cholesterol assays and p53 western blots were used to quantify cholesterol levels and p53 protein expression, respectively.

Results

Data suggest that there is a shift from planar rafts in the SW13(Vim−) to caveolar rafts in the SW13(Vim+) line. Although the contribution of GOF p53 to this shift is under study, evidence suggests that raft composition is altered in the shRNA lines, in which GOF p53 has been knocked down by ~90%. In addition, p53 knockdown increases the proliferation rate in SW13(Vim−) (p = 0.01) but does not affect SW13(Vim+). In contrast, p53 knockdown decreases MMP activity in SW13(Vim+) (p = 0.05) but not SW13(Vim−).

Conclusions

Our data support the hypothesis that lipid raft signaling is significantly altered between the epigenetically distinct SW13 subtypes and that these differences may correlate to oncogenic potential. In addition, statin treatment, which reverses some of the pro oncogenic properties of GOF p53, may do so, in part, by altering raft composition and signaling.
Regulation of the PAH1‐encoded phosphatidate phosphatase during lipogenesis in the oleaginous yeast Yarrowia lipolytica
Authors: Carmon Taylor , Ukey Rahul , Fakas Stylianos
Published: April 2020

The PAH1 gene encodes for phosphatidate phosphatase (PAP) that plays a vital role in lipid biosynthesis by catalyzing the conversion of phosphatidate (PA) to diacylglycerol (DAG) which is used to synthesize triacylglycerol (TAG). Previous research has shown that PAP activity is elevated during growth in media that induce lipogenesis. In this work, the PAH1‐encoded PAP activity and protein levels were analyzed in wild type and pah1Δ cells during lipogenesis. The results showed that the PAH1‐encoded PAP activity increased in a time‐dependent manner, but the Pah1 levels declined with time. Examination of the subcellular fractions revealed that Pah1 was almost entirely responsible for the membrane‐associated PAP activity. The lipid content of the wild type cells was higher than that of the pah1Δ cells, while the pah1Δ mutation did not have a major effect on the levels of DAG and TAG.

Support or Funding Information

This work was supported, in whole or in part, by National Institutes of Health Grant R15GM124602‐01 from the United States Public Health Service. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Formation of Products similar to Advanced Glycation End products (SAGEs) and Binding Affinities between Keto Acids with Human Serum Albumin
Authors: Cai Ang , Tang Xiaodan , Liu Weixi , Ma Hang , Dain Joel A.
Published: April 2020

Children who have the diabetic ketoacidosis may suffer from cognitive decline. It believes that the cognitive decline is related to carbohydrates metabolism. Keto acids (KAs) play significant roles in biological pathways related to carbohydrates metabolism. Although we have reported that KAs (pyruvic acid, α‐ketoglutaric acid, levulinic acid, and oxaloacetic acid) can react with human serum albumin (HSA) and form similar to Advanced Glycation End products (SAGEs), to be more important, the formation of the SAGEs and binding affinities of KAs with HSA remains unclear. Hence, we evaluated four keto acids including pyruvic acid, α‐ketoglutaric acid, levulinic acid, and oxaloacetic acid for their SAGEs formation and binding affinities of them with HSA. KAs were incubated with HSA at 37 °C for seven days and the formation of SAGEs were measured by visible, fluorescence, and circular dichroism (CD) stability assays, respectively. Pyruvic acid and α‐ketoglutaric acid did not induce the formation of SAGEs while levulinic acid and oxaloacetic increased the formation of SAGEs compared to the control group. MALDI‐TOF was used to provide critical insights of KAs induced formation of SAGEs. Kinetics and binding affinity determination between KAs and HSA were performed by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC).
Optical clearing and 3D‐Imaging Reveals a Sexual Dimorphism in the Structure and Remodeling Response of the Distal Convoluted Tubule
Authors: Tahaei Seyedmohammad Ebrahim , Coleman Richard , Saritas Turgay , Ellsion David H. , Welling Paul A.
Published: April 2020

Introduction

The abundance of the distal convoluted tubule (DCT) thiazide‐sensitive sodium chloride cotransporter (NCC) is well appreciated to be greater in females than males. Because structural remodeling of the DCT is dependent on NCC activity, it has been generally assumed that there is a corresponding sexual dimorphism in DCT morphology. Until now, this has never been directly examined. Here, combining new optical clearing techniques with staining for markers of DCT segments, state‐of‐the‐art high‐speed volumetric imaging and analysis tools we quantitatively assess DCT morphology in male and female mice, and study DCT remodeling response to furosemide.

Methods

Male and Female (3‐month‐old) mice were treated with vehicle or Furosemide administered in the food (100 mg/kg per day) for seven days. Total NCC and phospho‐active NCC abundance (pNCC) was evaluated by Western Blot in one kidney, the other kidney was perfused and fixed for imaging. Kidneys were cleared using the Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis (CUBIC) pipeline, co‐stained with antibodies that label the early DCT (DCT1, parvalbumin) and the entire DCT (DCT1 & 2; NCC), imaged using a high‐speed spinning disc confocal microscope, and processed with 3D rendering and analysis software (IMARIS).

Results

We confirmed previous studies that females have greater NCC expression in the basal state. Surprisingly, the length of the DCT was longer in male (~620 μm) than female mice (~560 μm). Furosemide treatment significantly increased the abundance of NCC and pNCC in both sexes, and this was paralleled by an expansion of the DCT. The remodeling response to furosemide was more profound in females (~20% increase in DCT length) than males (~8%). The DCT elongation effect of furosemide treatment in females stemmed largely from an increase in DCT1 length rather than a change in DCT2. By contrast, furosemide expanded the DCT1 and DCT2 to the same extent in males.

Our data indicate that sexual dimorphism in NCC does not originate from differences in the DCT length. In fact, male have a longer DCT than female mice, which suggests that the density of NCC per unit of length is higher in females. Altogether, the differences in the remodeling response suggests that females have more “plastic” DCTs compared to males.

Support or Funding Information

This study was supported by NIH and Ben J. Lipps postdoctoral Fellowship award.
Spotlight on Tendon: Its Novel Role in Long Bone Growth and Rickets‐Caused Osteosclerosis
Authors: Li Hui , Jing Yan , Ma Chi , Wang Zheng , Feng Jian Q.
Published: April 2020

INTRODUCTION

It has been known for many years that tendon‐attached bone is rough and bump, and that patients with hypophosphatemic rickets (such as DMP1 mutations) develop osteosclerosis in the tendon‐attached area. The goal of this study was to test whether tendon directly forms bone and how hypophosphatemia initiates the onset of osteosclerosis in a Dmp1 KO mouse (a hypophosphatemic rickets model) using cell lineage tracing and multiple imaging techniques.

METHODS

We generated a chondrocyte tracing mouse line (Col 10a1‐Cre;R26R tomato) and a tendon tracing mouse line (Scx‐CreERT2;R26R tomato) with and without a Dmp1 KO background, plus a high Pi rescue diet treatment for seven weeks.

RESULTS

Our key findings were: 1) the non‐tendon attached bone cells were negative in both tracing lines; 2) cells in the tendon‐attached bone were Scx+ but Col X−; 3). In the Dmp1 KO mice, tendon‐attached bone volume sharply increased, in which there were more EdU+ bone cells (i.e., more cell proliferation) with high expression levels of bone markers such as Runx2 and β‐catenin; and 4) a high Pi diet greatly improved the above phenotypes.

CONCLUSION

Tendon (an avascular tissue) directly forms bone cells in the tendon‐bone joint area, which is rough and porous compared to non‐tendon attached bones. Osteosclerosis in Dmp1 KO (a hypophosphatemia rickets model) is caused by accelerated cell transdifferentiation from tendon to bone due to hypophosphatemia.

Support or Funding Information

U.S. National Institutes of Health grants to J.Q.F. (R01DE025014 and DE025659)
The Effect of Diabetic Conditions on the Delivery of a Pancreatic Hormone
Authors: Jacobs Monica , Pinger Cody , Gilmore Abigail , Spence Dana
Published: April 2020

C‐peptide, a hormone that is found in the pancreatic β‐cell, is co‐secreted in equimolar amounts to insulin, and has been shown to alleviate microvascular complications found in diabetes. Prior research has indicated that C‐peptide requires a complex of human serum albumin (HSA) and zinc (Zn2+) to be transported in the bloodstream and elicit its effects. HSA is a prominent plasma protein and is involved in transporting many different molecules throughout the body. Due to the physiological conditions attributed to type 1 diabetes (T1D) there is both an increase in glycated HSA and circulation of free fatty acids; which are commonly bound to HSA. These alterations in homeostasis can inhibit HSA from binding and delivering C‐peptide to elicit its therapeutic effects. HSA was isolated from control (n=3) and diabetic (n=4) samples using antibody‐coated magnetic beads, and characterized through electrospray ionization time‐of‐flight mass spectrometry. A PolyJet 3D‐printer was used to fabricate ultrafiltration devices to measure C‐peptide and Zn2+ binding to HSA in diabetic conditions. Free C‐peptide was quantified using enzyme‐linked immunosorbent assay (ELISA), and free radioisotopic 65Zn2+ was measured on a 2480 WIZARD2 automatic gamma counter. Results indicate disruptions in C‐peptide’s binding affinity for HSA when free fatty acids are in solution (2.92 nM, n=12) as opposed to control conditions (3.68 nM, n=12). In addition, there is an inverse linear correlation with Zn2+ binding affinity and glycation of HSA (R2 = 0.7227, p<0.05). In discovering these correlations, we are better able to understand how diabetic conditions alter HSA’s binding abilities and thus homeostasis of Zn2+ and C‐peptide. Additionally, this has opened an unprecedented area of study beginning to look at the modifications in the diabetic bloodstream that can possibly disrupt the binding and delivery of C‐peptide.

Support or Funding Information

National Institutes of Health
Alanine scan of WDV Rep reveals its ssDNA binding mechanism
Authors: Nelson Andrew Thomas , Tompkins Kassidy , Lopez María Paz Ramirez , Gordon Wendy
Published: April 2020

HUH endonucleases (dubbed “HUH‐tags”) are small protein domains capable of forming covalent adducts with ssDNA in a sequence‐specific manner. The active site of HUH endonucleases utilizes a conserved histidine‐hydrophobic‐histidine motif to coordinate a metal ion that stabilizes a nucleophilic tyrosine necessary for phosphodiesterase activity. Because viral‐derived HUH‐tags are relatively small, react quickly, and require no chemical modifications to their ssDNA substrate, they have great value as protein fusion tags in biotechnologies ranging from genetic engineering to single‐molecule studies. One of the greatest assets of these tags is sequence‐specificity to their unique, native Ori sequence in vivo, introducing the possibility of using multiple HUH‐tags in multiplexed “one‐pot” reactions. However, their mechanism of ssDNA sequence binding and specificity is poorly understood, and there is noted cross‐reactivity between tags of closely‐related species. In order to understand the mechanism of ssDNA binding, we performed an alanine scan along a positively‐charged patch of one such HUH‐tag, wheat dwarf virus replication initiator protein (WDV), and characterized the enzymatic activity in both the rate and extent of the reaction. In molecular beacon Stopped‐Flow experiments, single point mutants of WDV showed a more than 60% decrease in reaction rate constant, and gel shift assays showed an almost complete lack of activity for some variants for single nucleotide ssDNA substitutions. In all, these findings help us to highlight key interactions in WDV‐ssDNA binding, and we gain further insight into potential rational engineering of HUH‐endonucleases to bind desired sequences of DNA.
Zamicastat is a noncompetitive Dopamine‐β‐hydroxylase inhibitor that modulates sympathetic nervous system activity
Authors: Bonifacio Maria-Joao , Sousa Filipa , Igreja Bruno , Loureiro Ana I. , Pires Nuno M. , Soares-da-Silva Patricio
Published: April 2020

Dopamine‐β‐hydroxylase (DβH; EC 1.14.17.1) is an enzyme responsible for the conversion of dopamine (DA) into norepinephrine (NE). It is present in the chromaffin cells of adrenal medulla and in noradrenergic nerve terminals of the central and peripheral sympathetic nervous system. DβH inhibition has been proposed as an alternative mechanism for the treatment of hypertension and chronic heart failure. Zamicastat ((R)‐5‐(2‐(benzylamino)ethyl)‐1‐(6,8‐difluorochroman‐3‐yl)‐1H‐imidazole‐2(3H)‐thione) is a novel DβH inhibitor. The aim of this study was to characterize the pharmacodynamics of Zamicastat in vivo, both in Wistar Han and in spontaneously hypertensive rats (SHR), and on DβH in human cells. DβH activity was measured by a modification of the Nagatsu and Udenfriend
(1) spectrophotometric method, based on the conversion of tyramine into octopamine either in adrenal tissues of rats administered orally with Zamicastat (30 mg/Kg) or vehicle, or in human neuroblastoma (SK‐N‐SH) cells. Zamicastat pharmacodynamic characterization in vivo was performed in Wistar rats by evaluating NE and DA levels in tissues using HPLC with electrochemical detection at 9h post‐administration. Additionally, systolic and diastolic blood pressure, heart rate and activity were measured continually by telemetry in SHR rats (a model of essential hypertension) and their controls, Wistar Kyoto (WKY). Zamicastat was found to be a reversible noncompetitive inhibitor of DβH towards tyramine, with a Ki value of 43 (21; 66) nM and either noncompetitive or uncompetitive towards ascorbic acid. Zamicastat (30 mg/Kg, p.o.) fully inhibits adrenal DβH activity from 2 to 8h recovering to baseline values after 24h. This is accompanied with a decrease in NE and an increase in DA in peripheral tissues, namely left ventricle and kidney but not significantly in the central nervous system (brain stem or prefrontal‐cortex). Zamicastat at 3, 30 and 100 mg/Kg displayed a dose‐dependent decrease on systolic and diastolic blood pressure with no effect in heart rate or overall activity when evaluated in SHR rats by telemetry. In conclusion, Zamicastat is a DβH inhibitor displaying binding affinity for both the free enzyme and the complex enzyme‐substrate, that when orally administered at 30 mg/Kg to rats acts peripherally and significantly reduces sympathetic activity by decreasing NE levels and consequently systolic and diastolic blood pressure in an animal model of essential hypertension.
Protocols for Reducing Autofluorescence and Improving Fluorescent Nuclear Staining in Mouse Testis
Authors: Roy Arpan , Sekulovski Nikola , MacLean James , Cheatwood Joseph L.
Published: April 2020

Previously, we had encountered significant autofluorescence (AF) in Leydig cells in the testis. Leydig autofluorescence often represents a barrier to effectively analyzing and depicting the expression of proteins by testicular Sertoli cells. Biological AF is an intrinsic property of cells and tissues, and intrinsic autofluorescence of some tissues can serve as a useful diagnostic indicator in certain disease situations. However, it is often an obstacle to immunofluorescence‐based analyses by masking and interfering with specific fluorescent signals. Hence, the need to reduce tissue AF is imperative for a successful visualization of co‐localizing proteins. Therefore, we sought to utilize the fat‐soluble diazo dye Sudan Black B (SBB) to reduce undesirable autofluorescence in our samples. Following treatment of the testis tissue sections with Sudan Black B for 10–15 min, the intrinsic fluorescence was significantly masked, allowing specific staining of Sertoli and Leydig cells with antibodies against SOX9 and DDX4. Additionally, we sought to use an alternative fluorescent nuclear stain, as 4′,6‐diamidino‐2‐phenylindole (DAPI) produces insufficient labeling in the nuclear of testis cells fixed with Bouin’s solution, which is used more commonly than formaldehyde solutions in testis research. As a result, we have developed protocols to significantly 1) treat tissue with SBB to reduce autofluorescence, and 2) improve fluorescent nuclear staining in mouse testis, without changing fixative solutions. These protocols can be used separately or together and can be altered to fit optimal experimental parameters. The method developed in this study will improve the visualization of multiple AF signals.
The Complete Molecular Analysis of PTP1B Function
Authors: Pelletier Kristiane Torgeson , Page Rebecca , Peti Wolfgang
Published: April 2020

Protein function originates from a cooperation of structural rigidity, dynamics at different timescales and allostery. Recently, we showed that PTP1B, a drug target for diabetes and cancer that catalyzes the dephosphorylation of numerous substrates in essential signaling pathways, uses both conformational and dynamic allostery to communicate between its allosteric and active sites. Further, we also discovered that the rigidity of the catalytically essential WPD loop is indispensable for PTP1B activity, demonstrating that both conformational rigidity and dynamics are essential for controlling protein activity. To determine if additional pockets distal from the active site may also influence PTP1B activity, we integrated bioinformatics analysis, activity assays, X‐ray crystallography and a broad variety of different NMR dynamics measurements. Our data revealed the presence of additional sites, distinct from the established allosteric pocket, which can potently alter PTP1B activity when perturbed. Our data suggest that that are a variety of ways to modulate the catalytic activity of PTP1B, and, in turn, may mitigate disorders due to PTP1B dysregulation, such as cancer.

Support or Funding Information

T32 GM008804
Sigma‐1 Receptors Promote Lymphatic Endothelial Barrier Function
Authors: Abdelmaboud Salma S. , Motawe Zeinab Y. , Breslin Jerome W.
Published: April 2020

The lymphatic endothelium plays important roles in lymphatic contractile activity, lymph transport and maintaining a barrier between the lymph and interstitial compartments and its dysfunction is a key factor in the pathogenesis of lymphatic diseases such as lymphedema. Sigma‐1 (σ1) receptors are chaperone proteins located in the endoplasmic reticulum were recently identified to promote collecting lymphatic dilation through endothelial production of nitric oxide. However, the role of σ1 in other functions of lymphatic endothelial cells are not clear. The aim of the current study was to test the hypothesis that σ1 promotes barrier function of lymphatic endothelium. We confirmed expression of σ1 in adult and juvenile human dermal lymphatic endothelial cells (HDLEC) using the ProteinSimple Western (WES) system and immunofluorescence microscopy. To test whether activation of σ1 promotes lymphatic endothelial barrier function, we applied the selective agonist PRE‐084 to cultured monolayers of HDLEC and determined the transendothelial electrical resistance (TER) as an index of barrier function using electrical cell‐substrate impedance sensing. The contribution of basal σ1 activity to HDLEC TER was also assessed either with the σ1 antagonist BD1047 (200 nM) or by silencing σ1 expression with siRNA. Because σ1 has been previously described to be located in the ER‐mitochondrial membrane interface, we also evaluated its role in endothelial bioenergetics using the Agilent Seahorse system glycolytic rate assay. Lastly, we tested the ability of PRE‐084 to counteract IL‐1β‐induced barrier dysfunction in HDLEC monolayers. The results show that the activation of σ1 receptor with PRE‐084 increases HDLEC TER in a concentration‐dependent manner, with 50, 100, 150, and 200 μM PRE‐084 eliciting significant elevations in TER (100 μM was chosen for subsequent experiments). Blockade of σ1 either with the antagonist BD‐1047 or reducing σ1 expression with siRNA resulted in decreased HDLEC TER. PRE‐084 also enhanced basal and compensatory glycolysis as well as proton efflux rate in HDLEC. Moreover, PRE‐084 partially counteracted the IL‐1β‐induced TER drop indicating its potential anti‐inflammatory impact on lymphatic endothelial cells. Collectively our results suggest that σ1 contributes to basal lymphatic endothelial barrier function, potentially through its ability to enhance glycolytic ATP production. Our work also highlights the therapeutic potential of σ1 agonists for preventing lymphatic barrier dysfunction caused by inflammatory mediators.

Support or Funding Information

This work was supported by NIH Grant R01GM120774.
Hypertension Upregulates Cerebral Microarteriolar Expression of p47phox and Contractions to Angiotensin II in Mice
Authors: Wilcox Christopher Stuart , Li Lingli , Lai En Yin , Welch William
Published: April 2020

Background

The cerebral circulation requires mechanisms to protect it against vasoconstriction since prolonged reductions in cerebral blood flow predispose to stroke or cognitive decline. However, although cerebral microarterioles (CAs) are normally resilient to angiotensin II (Ang II) contractions, prolonged Ang II causes oxidative stress and enhanced contractility in many vascular beds, but direct effects on CAs are not known. In contrast, the renal circulation requires robust responses to adjust renal function to the body’s needs. We reported that contractions of renal afferent arterioles (Affs) are enhanced by superoxide from p47phox/NOX2 or from reduced superoxide dismutase (SOD) expression but diminished by H2O2 from POLDIP2/NOX4. We tested the hypothesis that hypertension itself enhances vasoconstriction of CAs via p47phox/NOX2 or POLDIP2/NOX4 and contrasted Ang II contractions in CAs with Affs.

Methods

Mice fed high salt were infused with Ang II (1000 ng ·Kg−1 · min−1 sc) or implanted with DOCA (vs sham procedures) for 4 weeks. The MAP of these mice was increased >25 mmHg. Intraparenchymal CAs were isolated from within the frontal cortex and Affs from the outer renal cortex. Vessels were perfused at 40 mmHg and tested with graded Ang II (10−12 to 10−6 M) vs vehicle.

Results

CAs from hypertensive mice had > 50% enhanced expression of p47phox and > 50% reduced expression of SOD1 and 3 but unchanged POLDIP2 and NOX4. CAs from sham mice failed to contract even with the highest concentration of Ang II (10−6 mol · l−1; change in diameter 0 ± 0%) whereas Affs contracted strongly. Ang II contractions were evoked by Ang II in CAs from hypertensive mice infused with Ang II (−7 ± 2%; P < 0.01) or implanted with DOCA (−18 ± 3%; P < 0.01) whereas contractions in Affs were unchanged. However, preventing hypertension with hydralazine, hydrochlorothiazide and reserpine reduced Ang II contractions in Ang II infused mice (−2 ± 0 vs −7 ± 1; P < 0.01) or DOCA mice (−4 ± 1 vs −18 ± 3; P < 0.01). Enhanced Ang II contractions of CAs were prevented in Ang II infused p47phox −/− mice (−1 ± 1 vs −7 ± 3%; P < 0.05) but were maintained in Ang II infused POLDIP2 +/− mice (−7 ± 1 vs −7 ± 3%; NS).

Conclusion

The protection of CAs from Ang II contractions breaks down during hypertension per se because of enhanced expression of p47phox/NOX2 and reduced expression of SODs rather than POLDIP2/NOX4 highlighting targets for cerebral protection from hypertensive damage or dementia.
RELATIONSHIP BETWEEN BODY WEIGHT AND LENGTH IN NEWBORNS AND INDICATORS OF AUTHONOMIC NERVOUS SYSTEM IN CHILDREN
Authors: Belova Mariia , Filippova Irina , Saperova Elena
Published: April 2020

The purpose of this work is to study the relationship between body weight and length in newborns and indicators of autonomic nervous system in children. To study the features of the autonomic regulation of heart rhythm, we studied children aged from four to seven years (125 boys and 114 girls). Five‐minute ECG were recorded during rest period. In four‐year‐old boys, we have found positive correlation between body weight at birth and SI («stress index») (r = −0.257, p <0.05), AMo (r = −0.169, p <0.05), heart rate (r = −0.002, p <0.05). In girls of the same age, multiple regression analysis showed a pronounced effect of body weight at birth on SI (r = 0.506, p <0.016) and AMo (r = 0.525, p <0.01). In boys of five years old, a significant correlation was found between body weight at birth and the average value of RR intervals (r = 0.647, p <0.001), Mo (r = 0.598, p <0.001), AMo (r = −0.442, p <0.01), SI (r = −0.465, p <0,05), heart rate (r = −0.666, p <0.001). For boys of five years old, significant correlation was observed between the length of the body of newborns and the average value of RR‐intervals (r = 0.583, p <0.001), Mo (r = 0.518, p <0.002), MxDMn (r = 0.441, p <0.01), AMo (r = −0.539, p <0.001), SI (r = −0.606, p <0.001), heart rate (r = −0.600, p <0.001). In girls of five years of age, a significant positive relationship was found between body weight at birth and SI (r = −0.173, p <0.05), between the length of the body at birth and SI (r = −0.166, p <0.05). The results of multiple regression analysis allow us to come to the conclusion about a more pronounced effect of body length at birth on indicators of autonomic nervous system in children. Body weight and body length of newborns may be use as cofactors when analyzing the influence of environmental factors on the functional state of the cardiovascular system.
Hypoxia increases endothelin‐1 production via Hif1α in adipocytes
Authors: Rivera-Gonzalez Osvaldo , Stapel Jennifer , Speed Joshua S.
Published: April 2020

Severe obesity causes a state of adipose tissue hypoxia and low blood flow. Both human and rodent visceral adipose tissue have been shown to have low oxygen tension (~15mmHg PO2) and increased levels of hypoxia inducible factor alpha (Hif1α), a prominent transcription factor in response to hypoxic conditions. Hif1α has been shown to increase endothelin‐1 (ET‐1) levels at the transcriptional level in endothelial cells. Obesity is associated with high circulating levels of ET‐1, which plays an important role in energy metabolism and insulin signaling when acting on the ET‐1 type A and B receptors on adipocytes. Therefore, the goal of this study was to determine whether hypoxia increases ET‐1 expression in adipocytes via Hif1α and inhibits insulin signaling. We utilized 3T3‐L1 murine fibroblast (passage 3–4) differentiated into adipocyte like cells. Exposure of 3T3‐L1 adipocytes to hypoxia (5% O2) for 24 hours led to a significant 3‐fold increase in ET‐1 mRNA compared to normoxic control cells (21% O2). Associated with increased ET‐1, hypoxia reduced insulin mediated glucose uptake and glycerol release compared to normoxic cells (148.8±16.4 vs. 237.6±24.4 glucose, 83.17±0.6 vs. 93.22±2.8 glycerol percent of control). In a separate experiment, 6‐hour exposure of 3T3‐L1 adipocytes caused a significant increase in ET‐1 and Hif1α mRNA (25.5±2.5 vs. 77.5±7.09 ET‐1, 2117±235.5 vs. 5015±370.2 Hif1α copies/50ng of RNA). Inhibition of Hif1a with either IDF‐11774 (1,25, and 50uM) or GN 44028 (1,10,20 uM) resulted in a significant attenuation in ET‐1 mRNA in response to hypoxia (77.5±7.09 vs. 22.35±3.7 ET‐1, 5015±370.2 vs. 3569±223.5 Hif1α Copies/50ng of cDNA) in a dose dependent manner. These data suggest that a hypoxia promotes ET‐1 production in adipocytes via Hif1α and is associated with diminished insulin sensitivity, suggesting a potential mechanism by which adipose tissue hypoxia promotes insulin resistance in obese individuals.

Support or Funding Information

R00HL127178, P206M104357
Trypanosoma Cruzi Induces Phospho‐proteome Profile in Primary Colon Epithelial Cells During Early Infection
Authors: Rayford Kayla JaMayha , Suman Shankar , Cooley Ayorinde , Rachakonda Girish , Villalta Fernando , Pratap Siddharth , Lima Maria Fatima
Published: April 2020

Trypanosoma cruzi, an obligate intracellular protozoan parasite, is the etiological agent of Chagas Disease. Due to globalization, the disease is now present in all economically advanced countries of the world. About 30% of T. cruzi infected individuals will develop cardiac, neurological, and/or gastrointestinal pathology, particularly megacolon. Megacolon, one of Chagas disease major pathologies, is accompanied by gastrointestinal motility disorders that have been attributed to alterations in the number of interstitial cells of Cajal and enteric nervous system defects. The objective of this study is to begin to define early molecular mechanisms that contribute to chagasic megacolon that can be exploited for development of molecular intervention strategies. In order to do this, we challenged primary colon epithelial cells (HCoEpiC) with invasive T. cruzi trypomastigotes at different time points to decipher changes in levels of phosphorylated proteins. Using a proteome profiler Human phospho‐kinase array, we show that T. cruzi induced a unique phospho‐proteome profile. We validated the phospho‐kinase array data via immunoblotting and immunofluorescence. Modulators of host immune response such JAK2, STAT1, and NF‐kB showed increased activation. Increased phosphorylated levels of kinases such, as p‐Src and p‐Fyn and the Heat Shock protein (p‐HSP27) chaperone illustrate dysregulation of intracellular signaling. We found that p‐JNK and p‐AKT exhibited increased phosphorylation and their downstream effectors p‐CREB and p‐c‐Jun showed increased nuclear colocalization. Early infection also induced increase thrompsopondin‐1 expression, a known fibrogenic protein. We generated a biological interaction network using Ingenuity Pathway Analysis (IPA) to further characterize protein‐protein interactions within the phospho‐proteome profile. In conclusion, we demonstrate a unique phospho‐proteome profile induced by T. cruzi during early infection of HCoEpiC and illustrate biological networks and signal transduction pathways that are activated during infection.

Support or Funding Information

This work was supported by NIH grants SC1AI127352, 5U54MD007586 (The RCMI Program in Health Disparities Research), and 5R25GM059994‐19 (RISE).
Health Professions Student Perceptions of the Anatomage Virtual Dissection Table and Digital Technology
Authors: Kar Rekha , Skaggs Steven , Wang Hangzhang , Nation Haley , Sakaguchi Alan Y.
Published: April 2020

3D virtual technologies are increasingly used in contemporary anatomy curricula with ongoing debate about their benefits and impact on learning. One such technology, the Anatomage Virtual Dissection Table is a touch‐pad instrument capable of displaying four life‐size fully segmented male and female bodies which can be virtually dissected layer‐by‐layer to display all organ systems. Little data is available describing how best to incorporate the Table into anatomy instruction either as a stand‐alone tool or in conjunction with other teaching modalities. In 2019, seven Anatomage Tables were used in their first large scale deployment in two different anatomy courses at UT Health San Antonio for 44 Occupational Therapy Doctorate (OTD) and 48 Physician Assistant Studies (PAS) students . To gauge student impressions about digital technology, cadaver dissection and the Anatomage Table, voluntary, anonymous pre‐ and post‐course surveys were conducted in each course. Survey results indicated that 97.8% (95% confidence interval [CI], 91.9–99.9%) of the OTD and PAS students viewed digital technology favorably; 51% previously used digital anatomy resources, but few had used the Table. 57.6% (CI=47.4–67.2%) chose the University of Texas Health San Antonio program because it offered cadaver dissection while 86.4% (CI=77.1–92.4%) were also eager to use digital anatomy resources as well. In the post‐course survey, the Anatomage Table was praised for image size and realism, availability of male and female specimens, high resolution regional anatomy, but some features (touch and rotation functions) were difficult to operate for some students. 65.9% (CI=55.5–75%) felt that the Table is a good learning tool to supplement cadaver dissection. 69.3% (CI=59.0–78.0%) felt the Table helped them better understand structures on the cadaver. Most felt that they benefited from the Table as a supplement to their cadaver dissection. A minority of students (12.5%, CI=7–21.2%) of preferred the Anatomage Table over cadaver dissection, while 73.9% (CI=63.8–82%) preferred the latter. These results provide insights highlighting the benefits and limitations of the Table especially when used in conjunction with cadaver dissection. The Table’s benefits may be even more apparent in programs where dissection of human cadavers is unavailable.
K48 Linked Ubiquitin Chains Affect Proteasomal Unfolding Ability via Interactions with Rpn10
Authors: Boscia Joseph Anthony , Hughes Danielle J. , Kraut Daniel Adam
Published: April 2020

The ubiquitin proteasome system plays a crucial role in homeostasis by preferentially degrading proteins that are damaged, misfolded, or no longer necessary in regulatory processes. Substrates are recognized and polyubiquitinated by E1, E2, and E3 enzymes, which leads to recruitment and degradation by the proteasome. However, the proteasome does not always completely degrade its target and will release a protein fragment if unfolding does not occur. We previously showed that the proteasome’s unfolding ability is dependent on the ubiquitin chains’ interactions with proteasomal ubiquitin receptors, and that different chain architectures (linear K63 chains vs branched K48 and K63‐containing chains) had different receptor dependencies. To investigate the interaction of substrates bearing linear K48‐linked chains, we turned to the E3 ligase Ubr1, which recognizes N‐end rule substrates. A model unfolding ability substrate containing an N‐terminal arginine was degraded by wild‐type and ubiquitin‐receptor mutant proteins. The results indicate that although ubiquitin receptors Rpn1, Rpn10, and Rpn13 are all important for degradation of this substrate, Rpn10 has the greatest effect on the proteasome’s unfolding ability.

Support or Funding Information

This material is based upon work supported by the National Science Foundation under Grant No. 1515229 to DAK.
Synaptic circuit restoration and functional recovery in the mouse visual cortex after ischemic injury and direct in vivo reprogramming of astrocytes into neurons
Authors: Chubykin Alexander , Tang Yu , Wu Qiuyu , Ryu Esther , Pei Zifei , Chen Yuchen , Chen Gong
Published: April 2020

Ischemic injury caused by stroke is one of the leading causes of severe morbidity and mortality. Neuronal loss, proliferation of reactive astrocytes and the formation of a glial scar represent the three critical problems preventing the functional recovery. Direct reprogramming of astrocytes into neurons in vivo using NeuroD1‐containing AAV virus represents a new promising gene therapy capable of replenishing the neuronal population and reducing gliosis. We have used NeuroD1‐containing AAV virus to convert astrocytes into neurons in the mouse primary visual cortex (V1) following focal ischemic injury. We have demonstrated that this in vivo reprogramming can restore visual evoked potentials (VEPs) and individual neuronal responses impaired after ischemic injury in V1. We have used Channelrhodopsin Assisted Circuit Mapping (CRACM) to demonstrate that this functional restoration is accompanied by the integration of the newly reprogrammed neurons into the visual microcircuit and reestablishment of the specific interlaminar long‐range connections. Our work suggests that in vivo NeuroD1‐based viral therapy can convert reactive astrocytes into neurons and restore visual function lost after local ischemia

Support or Funding Information

Purdue Research Foundation
LDN‐212320 improves neuroprotection and synaptic plasticity in the hippocampus in an inflammatory pain model
Authors: Alotaibi Ghallab , Rahman Shafiqur
Published: April 2020

The astroglial glutamate transporter‐1 (GLT‐1) in the hippocampus is critically involved in acute and chronic nociceptive pain. We have previously shown that 3‐[[(2‐Methylphenyl) methyl] thio]‐6‐(2‐pyridinyl)‐pyridazine (LDN‐212320), a GLT‐1 activator, in the hippocampus attenuates acute and chronic nociceptive pain. However, the molecular mechanisms of GLT‐1 modulation in the hippocampus in chronic pain‐induced cognitive deficits and anxiety‐like behaviors remain unknown. Therefore, we have examined the effects of LDN‐212320, a GLT‐1 activator, in complete Freund’s adjuvant (CFA)‐induced cognitive deficits and anxiety‐like behaviors in mice. Furthermore, we have evaluated CFA‐induced impairment of spatial, working, and recognition memory using Y‐maze and object‐place recognition test. In addition, we have measured chronic pain‐induced anxiety‐like behaviors using elevated plus maze and marble burying test. We have also determined the effects of LDN‐212320 on cAMP response element‐binding protein (pCREB) and brain‐derived neurotrophic factor (BDNF) expressions in the hippocampus during CFA‐induced cognitive deficits and anxiety‐like behaviors using Western blot analysis and immunofluorescence assay. Pretreatment of LDN‐212320 (20 mg/kg) significantly attenuated CFA‐induced impairment of spatial, working, and recognition memory. Furthermore, LDN‐212320 (20 mg/kg) significantly reduced CFA‐induced anxiety‐like behaviors. Additionally, LDN‐212320 (20 mg/kg) significantly reversed CFA‐induced decreased pCREB and BDNF expressions in the hippocampus. Overall, these results suggest that GLT‐1 activator improves neuroprotection and synaptic plasticity by restoring pCREB and BDNF expressions in the hippocampus. Therefore, LDN‐212320 may have clinical potential for the prevention and treatment of chronic pain‐associated with cognitive deficits and anxiety‐like behaviors.
The Use of Biometrics and Rubric Analysis to Assess How Students View, Cognitively Process, and Respond to 3D Physical Molecular Models in Undergraduate Biochemistry
Authors: Terrell Cassidy R. , Freeman Sienna , Calvert Jenifer , Randolph Adriane , Cortes Kimberly Linenberger
Published: April 2020

Among the STEM disciplines biochemistry curricula present a particular challenge to students with abstract concepts that can lead to rampant and resistant misconceptions impeding learning and therefore impact retention. Moreover, research has shown two‐dimensional representations not only fail to effectively convey biochemical concepts, but also propagate misconceptions. In particular, research has shown students have significant difficulty understanding enzyme‐substrate relationships. One way instructors have suggested to support students while learning this content is through the use of 3D physical models of the enzyme and substrate. However, little research has been conducted on how the models impact the learning process and how to best design these models and corresponding activities. This presentation will describe an iterative design process based on biometric, observational and rubric data analysis. The biometric data is collected using electroencephalographic (EEG) and eye tracking tools and voice recordings from simulated learning environments where biochemistry students completed a serine protease activity with corresponding physical models. These results offer insights into how students view and cognitively process activities using physical models with respect to how the instructor intends students to learn with and use these tools. In addition, observational and rubric analyses of student responses to activities from the simulated learning environment and real classroom sessions provide further data for the iterative activity design process.

Support or Funding Information

This project is supported by the National Science Foundation under award number IUSE 1711402/1711425 to University of Minnesota, Rochester and Kennesaw State University.
Comparison of effectiveness of case‐based teaching and traditional teaching method in preparing students for board exam USMLE Step 1
Authors: Nayakanti Abraham R.J. , Arja Sireesha Bala , Arja Sateesh Babu
Published: April 2020

The introduction of an interactive student‐centred and self‐directed approach in medical education has changed the way students learn. Scientific literature demonstrated the effectiveness of group discussion for greater understanding and retention of materials, when compared to traditional teaching. Teaching and learning in an active way, develop problem‐solving, critical thinking, and reasoning skills will help the students to become better doctors in the future. This study aims to find out the effectiveness of learning in terms of students reaching milestones of learning objectives in Histology part of Human structure and function course in an integrated curriculum with both case‐based learning and traditional teaching methods.

This is a randomized experimental study. Students are divided into two small groups. Each group had 7 students. One group (Group A) received the case‐based discussion on the topic of esophagus and another group( Group B) received the traditional teaching method on the same topic. For the other topic on kidney of urinary system Group A received the traditional method of teaching and Group B received the case‐based teaching. Students performance in assessments for these topics were measured using knowledge‐based questions of clinical vignettes. The questions used were same as USMLE style.

We analyzed the performance of students by using two‐tailed t‐test. The results were better with with case‐based discussions as the students have already gone through simulated situations.

Support or Funding Information

American Association for Anatomists
Melanocytes in the Leptomeningeal Region of Fetal Sheep Cerebral Cortex: Response to Exposure to Bacteria
Authors: Fohn Alexis , Wood Charles E.
Published: April 2020

Over the past several decades, there has been growing evidence in support of the in utero colonization hypothesis, stating that womb is not sterile and that low levels of bacteria may be found in fetal tissues without harming the fetus. Exposure of the fetus to bacteria would likely stimulate an immune response. Melanin, produced in the brain by several cell types, may play a role in the inflammatory response as it does in the epidermis. Melanocytes are phagocytic cells that synthesize melanin and are thought to function in response to inflammation. We hypothesize that we can find melanocytes in fetal brain tissues, and that their numbers are increased in fetuses exposed to bacteria. We assessed tissues from 31 fetal sheep that were catheterized and exposed to experimental manipulation (n=23) or were non‐catheterized (naïve; n=8). Identified histologically using Fontana‐Masson stain and quantified as number of cells in a 40x field (10 fields/animal), we found melanocytes in the leptomeningeal region of all fetuses. There was a higher presence of (leptomeningeal) melanocytes in catheterized animals compared to non‐catheterized animals (25±4 vs 3±1 cells/field) per 32,000 um2. We conclude the leptomeningeal melanocytes in the fetal cerebral cortex are part of the fetal innate immune response to exposure of the fetus to pathogen‐associated molecular patterns.

Support or Funding Information

NIH Grant HD033053