bluuebunny/crossref_metadata_2025 huggingface dataset:
Microglia Prevent the Loss of Visual Function in the Aging Retina by Maintaining the Health of Retinal Pigment Epithelial Cells
Authors: Karg Margarete M. , Moorefield May , Hoffmann Emma , Shu Daisy , Krasniqi Drenushe , Philipose Hannah , Ksander Bruce R. , Saint‐Geniez Magali
Published: May 2022

Even in healthy individuals, aging leads to the progressive deterioration of visual function that coincides with increasing cellular dysfunction in retinal neurons and retinal pigment epithelial (RPE) cells. Interestingly, aging also coincides with the migration of microglia from the inner to the outer retina where they reside in the subretinal space between photoreceptor outer segments and the apical surface of RPE. Healthy young microglia act as the immune sentinels of the retina, and thus are protective and maintain retinal homeostasis. However, age alters cellular functions and the role of aging subretinal microglia is not known. Here we investigated whether aged microglia residing in the subretinal space are either preventing or promoting loss of visual function.

Microglia were depleted in 33‐month‐old C57/BL6 mice fed for 6 weeks with a chow containing PLX5622, a small molecule inhibitor of colony‐stimulating factor‐1 receptor (Csf1r) which is expressed by microglia and is required for their proliferation, differentiation, and survival. As a negative control, age‐matched mice received a control diet. Following microglia depletion, the optomotor reflex (OMR) was used to assess visual acuity and contrast sensitivity and electroretinogram (ERG) assessed retinal function. Morphological analysis was determined by immunohistochemical staining and TEM.

To distinguish microglia from infiltrating macrophages, retinas from old mice were stained with P2RY12, a microglia‐specific marker. The vast majority of cells residing between the photoreceptor outer segments and RPE layer in old mice were P2RY12+ microglia. These cells displayed a highly amoeboid and activated morphology and were filled with autofluorescence droplets similar to lipofuscin. PLX5622 treatment depleted up to 90% of the microglia. Microglia preserved visual function, and in the absence of microglia, there was a significant loss of contrast sensitivity and ERG c‐wave, as compared with untreated controls. Loss of microglia coincided with a loss of RPE cells and increased RPE swelling indicating that microglia support RPE in aged animals. TEM indicates that subretinal microglia actively phagocytize shed photoreceptor outer segments, thereby compensating for the known age‐related decline of RPE phagocytosis.

We conclude that microglia in aging mice migrate to the subretinal space of the outer retina to support and maintain RPE cell functions that preserves vision.
Empagliflozin inhibits intermittent hypoxia‐induced TRAF3IP2/NF‐κB/HIF‐1α/IL‐6‐dependent human aortic smooth muscle cell proliferation
Authors: Higashi Yusuke , Dashek Ryan , Russell Jacob J. , Rector R. Scott , Chandrasekar Bysani
Published: May 2022

Chronic intermittent hypoxia (IH), a characteristic feature of obstructive sleep apnea and chronic obstructive pulmonary disease, contributes to cardiovascular diseases, including atherosclerosis, through persistent oxidative stress and inflammation. During atherogenesis, vascular smooth muscle cells acquire a proinflammatory and a proliferative phenotype. TRAF3IP2 (TRAF3 Interacting Protein 2) is an oxidative stress‐responsive proinflammatory adapter molecule and plays a causal role in a preclinical model of atherosclerosis. Therefore, we tested the hypothesis that IH stimulates human aortic smooth muscle cell (SMC) proliferation via TRAF3IP2, and this effect is inhibited by the SGLT2 (Sodium/Glucose Cotransporter 2) inhibitor empagliflozin. Results show that IH upregulates TRAF3IP2 expression, TRAF3IP2‐dependent superoxide, hydrogen peroxide and nitric oxide production, HIF‐1α and NF‐κB activation, IL‐6 expression, and SMC proliferation. These effects were inhibited by TRAF3IP2, NOX2, NOX4, p65 or IL6R knockdown, or pre‐exposure to potent and specific iNOS inhibitor AMT or 1400W, the JAK inhibitor Tofacitinib, the STAT3‐specific inhibitor HO‐3867, or the gp130 inhibitor SC144. Importantly, SMC express SGLT2, and pretreatment with empagliflozin abrogated IH‐induced TRAF3IP2 induction, nitroxidative stress, HIF‐1α and NF‐κB activation, IL‐6 expression, and SMC proliferation. In summary, these results suggest the therapeutic potential of empagliflozin in IH‐associated inflammatory vascular proliferative diseases.
Role of the Dopamine Subtype 1 Receptor (D1R) Modulation of the Ih Current in Rhythmic Spinal Mammalian Motor Networks
Authors: Lee Grace , Diaz‐Rios Manuel
Published: May 2022

Restless Legs Syndrome (RLS) is a chronic sensorimotor disorder that has been recently associated with decreased expression of inhibitory Gi‐coupled adenosine A1 receptors (A1Rs), leading to increased dopamine release (hyperdopaminergic signaling) within the central nervous system. Previous studies have shown that A1Rs specifically inhibit the Gs‐coupled dopamine D1 receptor (D1R) subtype through a functional A1R‐D1R heteromer receptor complex. Furthermore, D1Rs control spinal motor neuron (MN) excitability by increasing cyclic adenosine monophosphate (cAMP) concentration and opening hyperpolarization‐activated cyclic nucleotide‐gated non‐selective cation channel (HCN). This finding raises the possibility of increased HCN activity underlying some of the motor dysfunctions associated with periodic leg movements (PLMS) during sleep in RLS. Thus, this study aims to elucidate the importance of the D1 receptor subtype on modulating the hyperpolarization‐activated cation current (Ih) in spinal mammalian motor networks. I hypothesized that blocking Ih in the presence of a D1R agonist (enhanced dopaminergic modulation) would slow locomotor activity and disrupt MN bursting stability. Neonatal (P0‐P5; n = 7) mouse spinal cord preparations were used to measure MN bursting activity patterns including amplitude, duration, and cycle period of extracellular ventral root (L2 and L5) recordings via suction electrodes. Treatment conditions include serotonin (9‐15 μM 5‐HT) and N‐methyl‐D‐aspartate (6 μM NMDA) with and without a D1R agonist (10 μM SKF38393) to assess the effect of D1R on rhythmic bursting. Subsequently, an Ih blocker (1 μM ZD7288) was added to observe the importance of Ih in modulating MN burst activity. For both L2 and L5 roots, blocking the Ih current led to increases in burst amplitude, duration, and cycle period in respect to the control 5‐HT/NMDA condition. Our results propose that D1Rs play a significant role in modulating Ih by increasing cAMP levels and opening HCN1 channels at a more depolarized membrane potential. Future experiments involve: 1) using TM5 transmembrane disrupting peptide to disrupt A1R‐D1R heteromers in spinal MNs (and use TM7 as a negative control) to confirm if dopamine modulation of Ih activity is linked to the A1R‐D1R heteromer complexes and 2) applying adenyl cyclase activators and blockers to verify that the modulation of the Ih current is through the D1R‐mediated Gs second messenger pathway.
Histological Influences of Food Restriction on Brown Adipose Tissue in Young OLETF Rats
Authors: Nishihira Misuzu , Nguyen Son T. , Nagamori Keita , Fujita Naoto , Urakawa Susumu
Published: May 2022

Whitening of brown adipose tissue (BAT) promotes obesity and causes obesity‐related health problems. It has been suggested that food restriction can prevent BAT whitening and inhibit the decline of BAT function with obesity. However, details of the histological feature remain unknown. The purpose of the present study was to investigate the influences of food restriction during childhood on histological features of BAT in Otsuka Long‐Evans Tokushima fatty (OLETF) rats.

Male OLETF rats and Long‐Evans Tokushima Otsuka (LETO) rats were used as models of obesity and non‐obesity, respectively. OLETF rats were divided into the food restriction and ad libitum feeding groups. OLETF rats with food restriction were received the daily average amount of food consumed by same‐age LETO rats. The others were fed ad libitum with standard laboratory rodent chow from 4‐ to 20‐week‐old. At 20‐week‐old, the BAT was collected from the interscapular region and the sections were stained with hematoxylin and eosin for histological observation. This study was approved by the Institutional Animal Care and Use Committee of Hiroshima University (A19‐163) and conducted in accordance with the Hiroshima University Regulations for Animal Experimentation. All experiments complied with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals.

Body weight of OLETF rats ad libitum feeding was significantly higher than those of LETO rats and OLETF rats with food restriction at 20‐week‐old. Enlarged unilocular adipocytes were often located in the peripheral area of the adipose lobule, the number appeared to be higher in OLETF rats ad libitum feeding than in LETO rats. Although the number of whitened unilocular adipocytes located in the central area was significantly lower in OLETF rats with food restriction than in OLETF rats ad libitum feeding, the number of enlarged unilocular adipocytes located in the peripheral area was a bit less in OLETF rats with food restriction.

These results suggest that BAT whitening in young OLETF rats is started from the peripheral area of the adipose lobule, adipocytes located in the central area of the lobule translate from multilocular to unilocular with the progression of hyperphagia‐induced obesity.
Human Cytomegalovirus (HCMV) Tegument Protein pUL35 in the establishment of latency in models of quiescent infection
Authors: Anguiano Ayax , Kulesza Caroline
Published: May 2022

Human Cytomegalovirus (HCMV) is a double stranded DNA, beta herpes virus that primarily infects those with transplants, advanced HIV/AIDS, cancer, newborns, and those with a compromised immune system. This virus is the leading cause of virus‐induced birth defects and can lead to death. HCMV is able to establish a latent phase of virus replication, in which the host is not producing infectious virus, but still retains the complete genome. It can remain in the host for its lifetime until replication is reactivated with the appropriate signal, where the virus can again cause disease. The HCMV tegument is composed of several cellular proteins and mRNAs, and it is unclear if these proteins play any role in the establishment of latency in the host. The tegument protein pUL35 has been shown to have an effect on viral transcription, but the role of this protein in latency is still unclear. The purpose of this study is to determine the role of tegument protein pUL35 in a model of latent infection. Recombinant HCMV was constructed in order to create a deletion of pUL35. Cell models of quiescent and lytic infection were then infected with recombinant virus and measured E, IE, and L gene expression along with genome copy number to determine latent or lytic replication. There are currently no vaccines for prevention and the antiviral therapy that is available has been effective where it can be used, but resistance can develop. The exploration of tegument proteins in latent models of infection could lead to the development of vaccines to target specific viral proteins.
Substrate Stiffness Modulates Normal and Diabetic Coronary Smooth Muscle Cell Stiffness
Authors: McCallinhart Patricia E. , Trask Aaron J.
Published: May 2022

We have previously reported that coronary resistance microvessels (CRMs) from 16‐week‐old type 2 diabetic (db/db) mice were less stiff compared to normal heterozygous Db/db controls, a finding that was driven by less stiff coronary vascular smooth muscle cells (VSMCs). Because the surrounding extracellular matrix (ECM) substrates in vivo may impact cellular properties such as elastic modulus (a measure of stiffness), the aim of this study was to test the hypothesis that modulating substrate stiffness correspondingly modulates the elastic modulus of normal and diabetic coronary VSMCs in vitro. In this study, we plated low‐passage coronary VSMCs isolated from Db/db and db/db mice onto 60 mm polyacrylamide dishes (Matrigen) that were of varying stiffnesses – 4 kPa, 25 kPa, and 50 kPa (n=4 per group). Cells were serum starved for 24 hours, and then cell stiffness was measured by atomic force microscopy (AFM). We observed a stepwise increase in cellular elastic modulus with each increase in polyacrylamide substrate stiffness in both the normal and diabetic coronary VSMCs (Db/db 4 kPa = 3.39±0.48 kPa, Db/db 25 kPa = 5.46±0.78 kPa, Db/db 50 kPa = 7.13±0.29 kPa; db/db 4 kPa = 2.49±0.23, db/db 25 kPa = 5.00±0.52 kPa, db/db 50 kPa = 5.58±0.16 kPa, p<0.0001). Similar to what we have previously reported on cells grown on plastic, diabetic coronary VSMCs were consistently less stiff compared to normal at each substrate stiffness. We did not observe statistically significant differences in cellular filamentous (F) actin, globular (G) actin, or the F/G actin ratio on the varying polyacrylamide substrate stiffness for neither Db/db nor db/db coronary VSMCs. However, there was a preliminary trend for lower F actin (Db/db: 1141.00 ± 472.20 AU vs. db/db: 422.50 ± 240.30 AU at 4 kPa, p=0.24, n=2 per group) and F/G actin ratio (Db/db: 4.01 ± 0.92 AU vs. db/db: 2.55 ± 0.35 AU at 4 kPa, p=0.21, n=2 per group) in the db/db VSMCs, corroborating our past data demonstrating that db/db coronary VSMCs are less stiff compared to normal Db/db coronary VSMCs. Overall, these data show that substrate stiffness correspondingly modulates normal and diabetic coronary VSMC stiffness.
Accessory Muscle Support of Breathing in Young Dystrophin‐deficient mdx Mice
Authors: Slyne Aoife D. , O'Halloran Ken D. , Burns David P.
Published: May 2022

Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder that occurs due to the absence of the structural protein dystrophin and secondary consequences. DMD is characterized by severe and progressive skeletal muscle weakness, which extends to the respiratory muscles. In young dystrophin‐deficient mdx mice, peak inspiratory pressure‐generating capacity is preserved despite diaphragm muscle weakness and reduced electromyogram (EMG) activitya. Our overarching hypothesis is that accessory muscle compensation limits ventilatory deficit in early dystrophic disease. Four‐month‐old male wild‐type (n=39) and mdx (n=41) mice were studied. Breathing was assessed in conscious mice using whole body plethysmography during normoxia and chemochallenge (10% O2/6% CO2). Thoracic oesophageal pressure and obligatory (diaphragm and external intercostal) respiratory muscle EMG activities were recorded in urethane (1.7g/kg i.p.) anaesthetised spontaneously breathing mice during baseline and sustained tracheal occlusion (maximal muscle activation). Diaphragm and scalene (accessory respiratory muscle) force output were examined ex vivo. Data were statistically compared using unpaired Student’s t test or two‐way ANOVA with Bonferroni post hoc test. Peak diaphragm force ex vivo (P < 0.0001) and baseline diaphragm EMG activity (P = 0.01) were significantly lower in mdx compared with wild‐type. Minute ventilation was equivalent in age‐matched wild‐type and mdx mice during baseline and chemochallenge (P = 0.1). Peak diaphragm (P < 0.0001) and external intercostal (P = 0.01) EMG activities during obstruction were significantly lower in mdx mice compared to wild‐type, while peak inspiratory pressure was equivalent (P = 0.4). Interestingly, peak scalene muscle force ex vivo was preserved in 4‐month‐old mdx mice (P = 0.8). Despite a decline in diaphragm muscle performance (decreased EMG activity and mechanical weakness) at baseline, ventilatory capacity is preserved in mdx mice. Furthermore, despite lower peak electrical activation of the obligatory muscles of breathing and diaphragm muscle functional deficit, peak inspiratory pressure is preserved in 4‐month‐old mdx mice, confirming our previous findings in 2‐month‐old micea. Our finding of preserved scalene muscle force suggests that compensation may be provided by accessory muscles to support ventilatory and non‐ventilatory behaviours in early dystrophic disease. Current studies are examining the EMG activity of accessory respiratory muscles (e.g. scalene, sternomastoid, cleidomastoid and trapezius) in early and advanced dystrophic disease in mdx mice.

References a Burns DP, Murphy KH, Lucking EF, O'Halloran KD. Inspiratory pressure‐generating capacity is preserved during ventilatory and non‐ventilatory behaviours in young dystrophic mdx mice despite profound diaphragm muscle weakness. J. Physiol. 2019;597(3):831‐848.
Mass Spectrometry‐based Proteomics in Deciphering Hormonal Regulation of Ubiquitin Linkages
Authors: Vemana Hari Priya , Abo‐Ali Ehab M. , Abzalimov Rinat , Dukhande Vikas V.
Published: May 2022

Hormones such as insulin and glucagon regulate metabolic organ liver. Ubiquitin (Ub) is a 76‐amino acid protein that can be attached on a lysine of the substrate protein. Ub posttranslational modification (PTM) is unique in its diversity as many possible mono‐ and multiple Ub molecules can be attached to protein substrates leading to homotypic and mixed linkages. The type of linkages added determine the functional outcomes such as protein degradation or localization changes. Hormones regulate protein PTM and metabolic disorders such as diabetes involve dysregulation of hormonal signaling. We hypothesized that hormones regulate Ub linkage profile and dysfunction of this regulation is a part of diabetes pathophysiology. We studied the effect of insulin and glucagon on protein ubiquitination in HepG2 cells. We utilized Tandem Ubiquitin Binding Entities (TUBE) and immunoprecipitation strategies for linkage‐specific ubiquitin protein enrichment and further analyzed them by western blotting and proteomics. Our preliminary data indicates differential regulation by hormones of the protein Ub linkage profile. Significant leads obtained from proteomic studies will be further characterized. In addition, we aim to study the ubiquitin linkage profiles in tissues obtained from rodent diabetes models. Our study has potential to find key targets involved in hormonal action and their dysfunction in diabetes.
Yo‐Yo Diet Has Adverse Effects on Cardiac Function and Insulin Sensitivity in Female Fischer Rats
Authors: de Souza Aline , Ji Hong , Korolowicz Kyle , Rodriguez Olga , Albanese Chris , Ecelbarger Carolyn , Sandberg Kathryn
Published: May 2022

Introduction

We have shown in female rats on a severe food restricted (sFR) diet that rapid body weight (BW) loss is associated with long term cardiovascular dysfunction months after BW has returned to normal levels due to refeeding. To further investigate the long term adverse cardiovascular effects of a sFR diet, we studied a model of BW cycling in which BW is rapidly lost, gained back, then lost again, since BW cycling is common in individuals who voluntarily diet (e.g., yo‐yo dieting) or those exposed involuntarily to inadequate caloric consumption (e.g., due to poverty).

Methods

Three‐month‐old female Fischer rats were divided into 2 groups and maintained on a control (CT; regular chow ad libitum for duration of study, n=8) or sFR (60% reduction of daily food intake, n=8) diet for 2 weeks. The sFR rats then received regular chow ad libitum for 3 weeks. This cycle of 2 weeks sFR diet plus 3 weeks of refeeding (c‐sFR) was repeated twice more. Cardiac and renal function were assessed using ultrasound and insulin sensitivity was evaluated using the homeostatic model assessment of insulin resistance (HOMA‐IR) index.

Results

During the 1st c‐sFR, rats lost 20% of their BW after 14 days on the sFR diet; however, their BW rapidly returned to CT levels during the 3‐week refeeding period. The 2nd and 3rd c‐sFR led to similar BW losses (20% and 19%, respectively). No differences were observed in mean arterial pressure (MAP) and heart rate (HR) after the 3rd c‐sFR compared to CT rats. However, the c‐sFR rats (n=4) showed 20‐40% reductions in renal artery flow (mm/s: CT, 295±12 vs c‐sFR, 235±6; p<0.05), cardiac output (mL/min: CT, 43±2 vs c‐sFR, 31±3; p<0.05) and stroke volume (mL: CT, 153±4 vs c‐sFR, 89±10; p<0.05) while the HOMA‐IR index was 2.6‐fold higher (AU: CT, 3±0.8 vs c‐sFR, 8±1.9; p<0.05) compared to CT rats (n=4).

Conclusion

Despite normalization of BW, MAP and HR after 3 c‐sFR periods, c‐sFR rats had reduced heart and kidney function and higher insulin resistance when compared to CT rats. This study suggests individuals who engage in yo‐yo dieting or have experienced BW cycling due to inadequate food intake from other causes (e.g., very low food security, war, natural disasters) are at increased risk for developing cardio‐metabolic disease.
Hyperpolarization‐Activated Current Shows Excitatory Effect on Fictive Locomotion in the Neonatal Mouse Under Hyperdopaminergic Conditions
Authors: Raymond Abigail , Diaz‐Rios Manuel
Published: May 2022

Restless Leg Syndrome (RLS) is a chronic sensorimotor disorder that has recently been linked to a downregulation in adenosine A1 receptors (A1R), leading to decreased formation of adenosine‐dopamine heteromers and a disinhibited or hyperdopaminergic transmission pathway within the central nervous system. Dopamine D1 receptor (D1R) activation has been shown to modulate HCN1 channels via its stimulatory effect on cAMP production to increase the hyperpolarization‐activated cation current (Ih). Ih has been related to the regulation of bursting activity in rhythmic motor networks, specifically in controlling the firing frequency of the system. Our study aims to establish the importance of Ih in mammalian locomotion under hyperdopaminergic conditions mimicking those seen in RLS patients. We hypothesized that blocking Ih would increase the cycle period and destabilize the rhythm.

Methods

We isolated the spinal cords of neonatal (P0‐P5; n = 6) Swiss Webster mice via ventral laminectomy and recorded the extracellular activity of ventral roots L2 and L5 using suction electrodes to assess burst amplitude, burst duration, and cycle period. Fictive locomotion was induced using serotonin (5HT; 9‐15μM) and N‐methyl‐D‐aspartate (NMDA; 6 μM) before adding dopamine (DA; 50μM) to determine the effect of DA on the system. We then used ZD7288 (1μM) block HCN1 channels to investigate the role of Ih in motor‐like activity.

Results

Blocking Ih caused a significant increase in all three parameters relative to the 5HT/NMDA control in the L2 roots, but only cycle period was significantly increased relative to the high dopamine condition. There were no significant changes in any of the parameters in the L5 roots.

Conclusions

Our results suggest that Ih primarily plays an excitatory role in locomotor regulation under hyperdopaminergic conditions, such as those seen in RLS. The differential effects between roots may suggest that one is more susceptible to the blockade of Ih by ZD7288; however, given the large degree of variability in the L5 data, a larger sample size is needed to confirm this hypothesis. Future experiments include collecting more data on L5 to confirm if there are differential effects, as well as pharmacological approaches to confirm that D1R is upregulating Ih via the cAMP pathway.
Green Tea Alters Bile Acid Micellar Solubility of Raloxifene
Authors: Oyanna Victoria O. , Graf Tyler N. , Oberlies Nicholas H. , Clarke John D.
Published: May 2022

Green tea is a popular botanical natural product, and its consumption has been associated with pharmacokinetic natural product‐drug interactions (NP‐DIs). Unpublished clinical data show that acute and chronic oral administration of green tea decreased the systemic exposure of raloxifene in healthy subjects. These clinical data indicate an intestinal interaction, although the mechanism for reduced absorption and systemic exposure is not known. NP‐mediated modulation of drug solubility in the intestinal lumen is an area of increasing interest and concern. Intestinal bile acids can enhance the absorption of hydrophobic compounds by micellar solubilization, and raloxifene is a Biopharmaceutic Drug Disposition Classification System (BDDCS) class 2 drug with poor water solubility. We hypothesize that green tea reduces intestinal absorption of raloxifene through decreased micellar solubility. To investigate this interaction, bile acid mixed micelles were prepared and incubated with raloxifene, green tea extract (GTE), (‐)‐epigallocatechin gallate EGCG, and a combination of raloxifene with GTE or EGCG. The size of the micelles was determined by dynamic light scattering and raloxifene quantification was determined by UPLC‐MS/MS. Raloxifene did not affect micelle diameter (micelle alone: 63.2 ± 5.6 nm, micelle with raloxifene 76.0 ± 5.8). In contrast, micelle diameter increased after incubation with GTE (416 ± 150.3 nm), EGCG (300.9 ± 173 nm), raloxifene + GTE (451 ± 164.2 nm), and raloxifene + EGCG (294.8 ± 303 nm). The micellar solubility of raloxifene was decreased by ~80% after incubation with GTE, whereas EGCG produced inconsistent results. These data suggest GTE decreased the micellar solubility of raloxifene and may play a role in the observed green tea‐raloxifene intestinal interaction.
Activation of APJ Receptors by CMF‐019, but not Apelin, Causes Endothelium‐Dependent Relaxation of Spontaneously Hypertensive Rat Coronary Arteries
Authors: Anto Santo K. , Sun Chengwen , O'Rourke Stephen T.
Published: May 2022

Hypertension is a major risk factor for coronary artery disease, but the pathophysiological mechanisms linking these diseases are not completely understood. Apelin is a novel vasoactive adipokine. Receptors for apelin (APJ receptors) are G‐protein‐coupled receptors and are widely expressed throughout the cardiovascular system. APJ receptors can also signal via G‐protein‐independent pathways, including G‐protein‐coupled‐receptor kinase 2 (GRK2), which inhibits nitric oxide synthase (eNOS) activity and nitric oxide (NO) production in endothelial cells. Apelin causes endothelium‐dependent, NO‐mediated relaxation of coronary arteries from normotensive animals, but the effects of activating APJ receptor signaling pathways in hypertensive coronary arteries are unknown. Since endothelium‐dependent relaxations are often blunted in arteries from hypertensive animals, we tested the hypothesis that apelin‐induced relaxation is impaired in coronary arteries from spontaneously hypertensive rats (SHR). Moreover, we hypothesized that endothelium‐dependent relaxations in response to CMF‐019, an APJ receptor G‐protein biased agonist with little effect on GRK2, would be unaffected in SHR coronary arteries. Western blot and RT‐qPCR analysis demonstrated that APJ receptor protein and mRNA expression did not differ between normotensive WKY (Wistar‐Kyoto) and SHR coronary arteries. eNOS protein expression was also similar between SHR and WKY coronary arteries, but GRK2 expression was significantly increased in SHR coronary endothelial cells, as compared to WKY. In myograph studies, apelin (10‐9‐10‐6 M) caused endothelium‐dependent relaxation of isolated WKY coronary arterial rings (pD2= 7.00 ± 0.11; Emax= 54 ± 4% relaxation; n=6), but had no effect in arteries from SHR. By contrast, CMF‐019 (10‐9‐10‐6 M)‐induced relaxation did not differ between normotensive and hypertensive coronary arteries. The relaxation response to CMF‐019 was abolished by the APJ receptor antagonist, F13A (10‐7 M), by removal of the endothelium, and by inhibition of eNOS with nitro‐ L‐arginine (3 x 10‐5 M). Moreover, the GRK2 inhibitor, CMPD 101 (3 x 10‐5 M), partially restored the relaxation response to apelin in SHR coronary arteries (pD2= 6.56 ± 0.22; Emax= 46 ± 6% relaxation; n=6). Immunoblot analysis of SHR coronary endothelial cells demonstrated that CMF‐019 (10‐7 M) significantly increased eNOS phosphorylation whereas apelin (10‐7 M) had no effect. We conclude that apelin failed to cause relaxation in hypertensive coronary arteries, likely due to impaired eNOS activity resulting from APJ receptor signaling via the GRK2 pathway and decreased eNOS phosphorylation. APJ receptor activation by the biased agonist, CMF‐019, caused similar endothelium‐dependent relaxations in WKY and SHR coronary arteries. Thus, APJ receptor‐biased agonists, such as CMF‐019, may be more effective than apelin in causing vasodilation of hypertensive coronary arteries.
Coring vs scrolls: Inclusion of the muscularis layer on tissue scrolls of the urinary bladder leads to inaccurate miRNA expression
Authors: Clark Samuel , Varvil Mara , dos Santos Andrea Pires
Published: May 2022

Formalin fixation with paraffin embedding (FFPE) is a method used to preserve tissues. FFPE tissues can be sampled through tissue cores or scrolls. In the study of urothelial carcinomas (UC), the use of a biopsy needle to acquire cores has the advantage of sampling the targeted superficial epithelial cell layer. In contrast, tissue scrolls are collected using a microtome, which includes surrounding tissues such as the muscularis layer. While both methods allow sample collection to extract small molecules, such as miRNAs, there is a lack of literature comparing these methods. MiRNAs are non‐coding RNAs responsible for gene regulation, altered in cancer. UC is the most common malignant bladder tumor in dogs, with 20,000 cases annually. The diagnosis of UC is challenging due to the small sample size of biopsies acquired by endoscopy. This project aimed to compare miRNA expression between the FFPE collection methods in UC (n=3) and healthy bladder (n=1) from dogs to understand the effect of including tissues surrounding a lesion in scrolls. MiRNA was extracted using a commercial kit, and the expression was achieved through RT‐qPCR. Cores targeting the epithelia and the muscularis layer were compared to their corresponding scrolls. MiR‐214, miR‐152, miR‐181a, and miR‐39 were differentially expressed when comparing cores with scrolls and the muscularis layer, while miR‐1842 and miR‐143 were not. Comparison between methods within individual samples also showed differential expression for all miRNAs. These results show that the inclusion of the muscularis layer results in misleading miRNA expression and verifies cores as a more accurate technique in miRNA extraction and expression, supporting further miRNA biomarker research.
S1P controls endothelial sphingolipid homeostasis via ORMDL
Authors: Sasset Linda , Chowdhury Kamrul H. , Manzo Onorina L. , Rubinelli Luisa , Konrad Csaba , Maschek J. A. , Manfredi Giovanni , Holland William L. , Di Lorenzo Annarita
Published: May 2022

Sphingolipids (SL) are both membrane building blocks and potent signaling molecules regulating a variety of cellular functions, in both physiological and pathological conditions. Sphingosine‐1‐phosphate (S1P) is a secreted bioactive sphingolipid acting through G‐protein coupled receptors namely S1PR1 to S1PR5, with major impact on cardiovascular and immune systems. Current drugs targeting S1PRs have been approved by the FDA to treat autoimmune conditions. However, these drugs manifest cardiovascular‐adverse effects, suggesting that knowledge gaps on S1P functions remain, limiting our ability to exploit this pathway therapeutically. Disruption of sphingolipid homeostasis and signaling has been implicated in cancer, diabetes, cardiovascular and autoimmune diseases. Yet, under normal physiology, sphingolipid levels are tightly‐regulated. Mechanisms governing cellular sensing of SL, and according regulation of sphingolipid biosynthesis, remain largely unknown. Serine palmitoyltransferase (SPT), which catalyzes the first and rate limiting step of sphingolipid de novo biosynthesis, is functionally antagonized by Orosomucoid like 1, 2 and 3 proteins (ORMDLs). Here, we identify S1P as the key sphingolipid sensed by cells via S1PRs, which stabilizes ORMDLs to negatively regulate SPT and maintain SL homeostasis. These findings reveal the S1PR/ORMDLs axis as the sensor‐effector unit regulating SPT activity accordingly. Mechanistically, the hydroxylation of ORMDLs at Pro137 allows a constitutive degradation of ORMDLs via ubiquitin‐proteasome pathway, therefore preserving SPT activity at steady state. The increase of S1P‐S1PR signaling stabilizes ORMDLs, which downregulates SPT activity to maintain SL within a physiological range. The disruption of the S1PR/ORMDL axis results in ceramide accrual and endothelial dysfunction, which is an early event in the onset of cardio‐ and cerebrovascular diseases. It is likely that the disruption of S1P‐ORMDL‐SPT signaling might be implicated also in the pathogenesis of other conditions such as diabetes, aging, cancer, and cardiomyopathy, and neurodegeneration characterized by deranged SL metabolism.
Silymarin‐mediated Natural Product–Drug Interactions involving Enteric Organic Anion Transporting Polypeptides
Authors: Bechtold Baron , Graf Tyler , Oberlies Nick , Clarke John
Published: May 2022

Organic anion transporting polypeptides (OATPs in humans; Oatps in rodents) represent a large family of uptake transporters important to drug disposition. Several factors can disrupt transport activity of OATPs/Oatps, including inhibition by natural products found in the diet and in dietary supplements. Silymarin, an extract of Silybum marianum (L.) Gaertn. seeds, is a widely consumed dietary supplement for hepatobiliary disorders that is reported to inhibit OATPs/Oatps. Although there is potential for high intestinal concentrations of flavonolignans after oral consumption, very little research has explored the risk of an enteric interactions between silymarin and OATP/Oatp substrates. Therefore, we hypothesize that clinically relevant doses of silymarin will inhibit enteric OATP/Oatp activity and pose a risk for decreased systemic drug concentrations. IC­50 data for the individual flavonolignans were collected using cells overexpressing OATPs. OATP2B1, the major OATP expressed in human enterocytes, was inhibited by silymarin (IC50: 3.9 µM). Further analysis of individual flavonolignans, which are the major constituents in silymarin revealed IC50 values below 10 µM (isosilybin A: 2.7 µM, isosilybin B: 9.4 µM, silychristin: 8.2 µM, silydianin: 5.5 µM). FDA guidance provides metrics for predicting the risk of an intestinal in vivo interaction from in vitro data (R value ≥ 11) based on the proportionality between the maximal extracellular inhibitor concentration and the IC50 data. For flavonolignans from silymarin, maximal intestinal concentrations are estimated to be considerably greater than the experimentally derived IC50 data. Consequently, silymarin doses as low as 140 mg produced R values for flavonolignans that exceeded the FDA threshold. These data suggest enteric OATPs/Oatps may be susceptible to silymarin‐mediated inhibition and potentially decrease systemic drug concentrations.
Learning Anatomy in a developing country under COVID‐19 lockdown measures: How did the learners perceive it?
Authors: Mbagwu Smart I. , Edem Edem E. , Akunna Gabriel G. , Makanjuola Victor O. , Adegbilero‐Iwari Oluwaseun , Ogbo Felix , Enye Linus A.
Published: May 2022

Introduction

Acknowledging the fundamental role anatomy education plays in medical training and taking into consideration the nascence of online anatomy education in Nigeria, we evaluated the current and future perceptions of medical and allied‐health students towards online anatomy education during the COVID‐19 lockdown in Nigeria.

Methods

Google Form questionnaires were distributed via different social and academic platforms to medical and allied‐health students in Nigeria between September 28 –December 17, 2020, using a purposive sampling. 954 students responded to questions on what they felt about the online anatomy teaching including how it could affect their academic performance in anatomy. Additionally, they were asked to give their opinion of the future of e‐learning in anatomy in Nigeria. The data collected from the study were analyzed using descriptive and inferential techniques.

Results

Out of a total of 954 students who participated in the study, 947 valid responses were recorded. From the responses, most of the students (77.6%) had computer/IT skills. Unexpectedly, many of

the students (60%) did not favor online learning approaches as effective tools for instructions in the anatomical sciences while more than a half of the students (55%) did not find the online classes in anatomy interesting. On the other hand, a majority (84%) believed that physical classes would have improved their performance in anatomy. Almost all the students (91.5%) believed anatomy educators in Nigeria need advanced skills for online education and that anatomy online teaching needs more advanced technology to be implemented in Nigeria(94.2%).

Conclusion

After evaluating online anatomy education experience during the COVID‐19 lockdown in Nigeria from medical and allied‐health students’ position, it was observed that there was an inclusive negative perception about the suitability and effectiveness of online education with regards to the anatomical sciences in Nigerian medical/health training. Majority of the students indicated that there is need for the enhancement of online teaching and quality training of the anatomy educators. Addressing the challenges that were presented during online anatomy education at the heat of the pandemic is recommended. This will incline science education towards the development and implementation of a workable and sustainable online education model in anatomical sciences for Nigeria.
Role of key protein residues involved in DinB DNA damage specificity: an in silico study
Authors: Sampoli Benitez Benedetta A. , Tan Rodger , Tauro Tracy
Published: May 2022

Y‐family DNA polymerases are a class of error‐prone polymerases able to perform DNA translesion synthesis. E. coli DNA Polymerase IV (also called DinB from the name of the gene) is a Y‐family DNA polymerase implicated in stress‐induced mutagenesis. As other DNA polymerases, DinB has the general architecture of a right hand, with palm, thumb, and fingers subdomains. In addition, Y‐family polymerases have an extra domain, namely the “little finger” or polymerase‐associated domain (PAD) and have a more solvent accessible active‐site than their high‐fidelity replicative counterparts. Kinetics studies have shown that DinB is particularly efficient at bypassing damaged guanosines, such as the N2‐furfuryl‐dG, and even more efficient in lesion bypass on damaged DNA than in replication of undamaged DNA. Hydrogen exchange experiments have suggested that the protein undergoes a conformational change in the presence of certain DNA damages on the template strand, but not others, implying a possible mechanism of bypass discrimination. However, the exact nature of such movement is not well characterized. Using molecular dynamics (MD) simulations we explored different DNA/protein/incoming nucleotides combinations to dissect the protein conformational change. Specifically, we analyzed a lesion that the protein can bypass (N2‐furfuryl‐dG) and one that instead the protein does not bypass well (O6‐Methylated dG). Our MD simulations revealed that there are some key residues in the fingers domain that are important switches for the conformational change. For example, R35 assumes a characteristic position when the protein is in a catalytically competent state, while it blocks the active site when the protein is not able to bypass the lesion. This result is in agreement with previous studies which showed that mutations of the R35 residue alter the protein specificity. Other areas of DinB which seem to undergo large movement reside in the PAD domain. This domain, which makes contact with the DNA major groove, has been implicated in DNA damage specificity, so it makes sense that residues on this domain assume different conformations, depending on the type of lesions present on the templating strand. A discussion of possible roles for some of the residues on the PAD will be presented.
Secondary Right Renal Artery Occurrence: A Case Report on Anatomic Variation
Authors: Shin Yunjoo , Kuperus Joshua , Woznicki Alexander , Sarsfield Kristin , Mueller Erin , Patel Jhanvi , Barremkala Malli
Published: May 2022

In this case study, we analyzed the anomalous occurrence of a secondary right renal artery of similar diameter to the main right renal artery in the abdomen. This secondary renal artery was present inferior to the main renal artery in a female cadaver aged 46 years. Previous studies have found that secondary and tertiary renal arteries have a population incidence of 20‐30% (Mir et al., 2008), with the majority of additional renal arteries having a markedly smaller diameter than the main artery (Bordei, Sapte, Iliescu, 2003). There is some discrepancy as to whether this occurs more frequently in males or females, with certain studies indicating greater incidence in males (Satyapal et al., 2001) and others in females (Bordei, Sapte, Iliescu, 2003). Additional renal arteries are hypothesized to be remnants of embryological blood supply to the kidneys, and they may occur when there is a delay in fetal migration of the kidney into the abdomen (Jeon, Lee, Yoon, 2014). Dissection of 37 cadavers (average age = 51 years old) found one cadaver (2.7%) (Female; 46 years old) with a secondary renal artery. Our study builds upon current literature surrounding the frequency and placement of additional renal arteries. Research on anomalous renal artery presence is clinically significant because understanding vascular variations to the kidney is necessary for effective procedures such as renal vascular surgeries, interventional radiological procedures, and kidney transplants (Budhiraga, Rastogi, Asthana, 2010).
Lipid Raft Disruption Alters Human Follicle Stimulating Hormone Receptor Signaling
Authors: Godek Rachel , Cohen Brian
Published: May 2022

Over 6.7 million people struggle with infertility each year. Studying signaling by reproductive hormones in fertility can allow us to gain a better understanding of the signaling pathways that must function correctly for proper fertility. Some infertility is due to incorrect human follicle stimulating hormone receptor (hFSHR) function. When follicle stimulating hormone (FSH) binds to hFSHR this begins a signaling cascade where the end product is the maturation of sperm by Sertoli cells in men, and egg development and production of estrogen through stimulation of granulosa cells in women. It has been determined that hFSHR is localized to microdomains of the cell membrane called lipid rafts which are characterized by higher concentrations of sphingolipids and cholesterol. This composition and structure makes them less fluid than non‐raft membranes, and it is believed that lipid rafts regulate signaling of many proteins that reside in the rafts, including hFSHR. It was hypothesized that if the lipid rafts in cell membranes were removed then hFSHR signaling would be altered. To remove lipid rafts HEK293 cells stably expressing hFSHR were treated with sphingolipid synthesis inhibitors (Myriocin or Fumonisin B1) or a cholesterol withdrawing drug (Methyl‐β‐cyclodextrin). Removing either the sphingolipids or cholesterol results in disruption of the lipid rafts. hFSHR signaling from the cells with disrupted lipid rafts were compared to wild type hFSHR signaling by western blot. Two pathways were investigated: cAMP production and the activation of p44/42 MAP kinase (ERK1/2). cAMP production was measured indirectly by detecting phosphorylated cAMP Response Element Binding protein (p‐CREB) and p44/42 MAPK signaling was measured by directly detecting the phosphorylation of the kinase (p‐p44/42). It was found that treatment with the lipid raft disrupting agents resulted in increased basal cAMP production (as measured by pCREB activation). However, time‐dependent hFSH stimulation was decreased compared to cells with intact lipid rafts. In contrast, p‐p44 signaling in the drug treated cells was not altered compared to untreated cells. These results suggest that G protein mediated activation of adenylyl cyclase to produce cAMP is lipid raft dependent while p44/42 MAPK activation is not. Further understanding of how lipid raft residency allows for functional hFSHR signaling would allow for the development of new treatments and pharmaceuticals for men and women struggling with infertility.
SK Channel Dysfunction in the Hypothalamic Paraventricular Nucleus Contributes to Sympathoexcitation in Dahl Salt‐Sensitive Rats
Authors: Larson Robert A. , Chen Xinqian , Gu Mingjun , Shan Zhiying , Chen Qinghui
Published: May 2022

Salt‐sensitive hypertension is characterized by marked activation of the sympathetic nervous system. Previous studies have consistently demonstrated that increased excitability or activity of pre‐sympathetic neurons in the hypothalamic paraventricular nucleus (PVN) contribute to the increased blood pressure and sympathetic nerve activity (SNA) in salt‐sensitive hypertension; however, the underlying mechanisms have not been fully elucidated. Small conductance calcium‐activated potassium (SK) channels act as negative feedback regulators of neuronal excitability in PVN neurons, and inhibition of SK channel function in the PVN is known to augment SNA and arterial blood pressure. In the present study, we tested the hypothesis that reduced SK channel function in the PVN contributes to sympathoexcitation in Dahl salt‐sensitive rats fed a high salt (HS, 4%) diet compared to a normal salt (NS, 0.4%) diet. Sympathoexcitatory responses to bilateral microinjection of the SK channel blocker apamin into the PVN were assessed in anesthetized 12–14‐week‐old male Dahl salt‐sensitive rats fed a high salt (n=7) or a normal salt (n=6) diet for 4 weeks. Baseline mean arterial pressure (MAP) was significantly elevated in rats fed a HS diet compared to NS (HS 129±4 vs. NS 114±4 mmHg, p<0.05), however baseline heart rate (HR) was not different between groups (HS 411±10 vs. NS 385±13 beats/min, p=0.07). Bilateral microinjection of apamin (12.5pmol, 50nL) into the PVN significantly increased splanchnic SNA in both HS and NS rats compared to baseline, however responses were significantly attenuated in HS rats compared to NS (HS 97±22% vs. NS 249±57%, p<0.01). In contrast, increases in MAP in response to PVN microinjection of apamin were similar between HS and NS rats (HS 24±4 vs. NS 29±4 mmHg, p=0.25), whereas HR responses were variable but not significantly different between groups (HS ‐8.5±9 vs. NS 7.5±7 beats/min, p=0.11). Next, we utilized qPCR to assess mRNA expression of SK1, SK2, and SK3 channels in PVN tissue from HS (n=5) and NS (n=6) rats. Interestingly, there was no significant difference in mRNA levels of either SK1, SK2, or SK3 channels between HS and NS fed rats. In summary, increases in splanchnic SNA due to PVN SK channel blockade with apamin were significantly attenuated in Dahl salt‐sensitive rats fed a HS diet, whereas there was no difference in SK channel expression between rats fed a HS vs. NS diet. These results suggest that dysfunction of SK channels in the PVN, but not decreased expression, contributes to sympathoexcitation Dahl salt‐sensitive rats fed a HS diet.