bluuebunny/crossref_metadata_2025_split huggingface dataset:
An Evolutionarily Conserved Residue in ABCG2 Regulates the Transport Cycle and Inhibitor Activity
Authors: Gose Tomoka , Das Sourav , Wang Yao , Lynch John , Fukuda Yu , Wills Medb , Baril Stefanie , Shelat Anang , Schuetz John D. 
Published: May 2022

ABCG2 is a medically significant ATP‐binding cassette (ABC) transporter restricting the cellular penetration of more than 200 toxins and drugs, as well as promoting physiological detoxification and multidrug resistance in cancers. Although ABCG2 cryo‐EM structural studies reveal an overlap between substrate and inhibitor binding sites in the ABCG2 binding pocket, it is unknown how much these substrate and inhibitor interactions will translate into functional knowledge that is exploitable in live cells.We generated multiple single point‐mutations in the residues of the ABCG2 binding pocket to determine if there is a relationship between binding (defined by thermal shift stabilization) and transport. Unexpectedly, the relationship between substrate‐induced thermal stabilization and transport was poor and an alanine substitution in a highly conserved asparagine residue in ABCG2 markedly impaired substrate‐induced thermal stabilization, but paradoxically enhanced substrate transport. To determine if the substitution altered ABCG2 conformational response to substrate binding, we used live cells and performed copper phenanthroline induced disulfide cross‐linking analysis on cells expressing the appropriate cysteine substituted ABCG2 (1). Consistent with the enhanced transport, the substitution promoted a substrate‐induced conformational change from an inward‐ to an outward‐facing ATP‐bound state. Molecular dynamics (MD) simulations using an ABCG2 structure (PDB: 6VXH) suggested a mechanism for this enhanced transport by the alanine mutant: disruption of a water‐bridge interaction with the asparagine residue coupled with an alteration in contacts with certain residues in the binding pocket. We next investigated if this alanine substitution affected inhibitor binding and transport inhibition. The substitution strongly reduced inhibitor‐induced thermal stabilization and transport inhibition. The disulfide cross‐linking analysis revealed that the substitution abolished the ability of many inhibitors to block the transition to an outward‐facing state. For these inhibitors, MD simulations revealed that the substitution disrupted a hydrogen bond interaction with the asparagine residue and impacted inhibitor interactions with multiple residues. Overall these changes indicate that inhibitors have mostly lost their ability to act like “wedges” to lock ABCG2 into an inward‐facing conformation.These findings reveal that an evolutionarily conserved asparagine residue, in the binding pocket, appears to have a key role in regulating the conformational dynamics of ABCG2.Reference:(1) Orlando, B. J. and Liao, M., ABCG2 transports anticancer drugs via a closed‐to‐open switch. Nat. Commun., 11, 2264 (2020)
The role of primary cilia in pulmonary hypertension
Authors: Solymosi Philip , Perret Paul‐Lennard , Gallo Kathleen , Sinn Katharina , Klepetko Walter , Kwapiszewska Grazyna , Preissner Robert , Kuebler Wolfgang M. 
Published: May 2022

IntroductionVascular remodeling in pulmonary hypertension (PH) is characterized by endothelial dysfunction and smooth muscle cell proliferation and hypertrophy ultimately causing right heart failure and death. Up to now, the molecular mechanisms of PH pathogenesis have not been fully resolved, preventing the development of novel therapies against PH.Based on increased expression and activity levels in both patients and animal models with PH, respectively, PDGF‐BB, TGF‐β and mTOR signaling have been proposed to play critical roles as drivers of lung vascular remodeling. Among other effects, PDGF‐BB, TGF‐β and mTOR signaling cause shortening of the primary cilium – an antenna‐like organelle that functions as a flow‐sensor and signaling hub. Loss or shortening of cilia is characteristically associated with cell proliferation and promotes mTOR signaling, thus potentially establishing a positive feedback loop. Here, we hypothesized that PH is associated with a loss of primary cilia in pulmonary artery endothelial (PAECs) and smooth muscle cells (PASMCs) which in turn drives cell proliferation and thus, remodeling in the pulmonary arterial wall.MethodsPulmonary artery tissue from PH‐patients and non‐PH donors was fixed and stained for acetylated α‐tubulin to determine the number of primary cilia. PAECs and PASMCs from PH patients and non‐PH donors were exposed in vitro to three different characteristic stimuli or mediators, respectively, of PH, namely PDGF‐BB, TGF‐β1, or hypoxia (1%). Cells were fixed and stained for measurement of primary cilia length. IFT88 ‐ an essential structural protein of the primary cilium ‐ was knocked down by siRNA in PAECs and PASMCs to assess the effect of cilium loss on migration and proliferation. Mice with an endothelial‐specific deletion of IFT88 (Cdh5‐CreERT2+, IFT88fl/fl) were housed for 5 weeks in normoxia or hypoxia (10% O2) to test for the role of endothelial primary cilia in the development of PH.ResultsIn pulmonary arteries of PH patients, the number of primary cilia per area was reduced by 80% as compared to healthy donor lungs. Analogously, PASMCs from PH patients had shorter cilia. Following IFT88 knock‐down, PAECs and PASMCs showed increased migration and proliferation as compared to control cells. In both PAECs and PASMCs, stimulation with PDGF‐BB, TGF‐β1, or hypoxia shortened primary cilia as compared to controls. Mice lacking primary cilia in endothelial cells showed a significant increase in right ventricular systolic pressure after 5 weeks of hypoxia as compared to control mice.ConclusionsHere, we demonstrate that PH is associated with a loss of cilia in vivo and reduced cilium length in vitro. Primary cilium loss promotes proliferation and migration of PAECs and PASMCs in vitro, as well as the development of PH in vivo. Cilium loss may as such be an important propagator of vascular remodeling in PH and may present a target for novel therapeutic interventions.
The Magnitude of Long‐Term Facilitation is Influenced by Modifications to Mild Intermittent Hypoxia Protocols in Participants with OSA
Authors: Panza Gino , Kissine Dylan , Puri Shipra , Lin Ho‐Sheng , Matieka Jason 
Published: May 2022

IntroductionMild intermittent hypoxia (MIH) with sustained hypercapnia initiates long‐term facilitation (LTF) of minute ventilation in humans. To date the impact of different protocols administered acutely, and repeatedly for a select number of days, on the magnitude of LTF in humans with obstructive sleep apnea (OSA) has not been investigated. Thus, the primary aim of this study was to investigate the impact of two different MIH protocols administered over 10 days on the magnitude of LTF.MethodsTwenty‐seven males with OSA participated in the investigation. Both protocols were characterized by 12 episodes of hypoxia of similar intensity (PETO2 ≍ 50 mmHg). However, one protocol (P1) was comprised of 2‐minute hypoxic episodes and the other protocol (P2) was comprised of 4‐minute hypoxic episodes. In addition, during P1 PETCO2 was maintained 2 mmHg above baseline throughout the protocol, while PETCO2 was sustained 4 mmHg above baseline during the completion of P2. During the completion of both protocols, minute ventilation was measured initially under conditions of normoxia and hypercapnia (i.e. 2 mmHg or 4 mmHg above baseline) prior to exposure to MIH. Following exposure to MIH, minute ventilation was recorded for an additional 30 minutes (i.e. end recovery period). Data was averaged for the initial 2 days and final 2 days of the protocol. Absolute measures of minute ventilation, and measures standardized to baseline measures on the initial days, were compared between protocols.ResultsThirteen participants completed P1and 14 participants completed P2. Absolute measures showed that minute ventilation during end‐recovery was greater than baseline on the initial and final days of both protocols (P < 0.01). However, minute ventilation during end‐recovery on the initial and final days was greater after the completion of P2 compared to P1 (P < 0.01). This difference remained after measures of minute ventilation were standardized relative to baseline (P < 0.05). Minute ventilation during end‐recovery on the final days was also greater compared to the initial days after completion of P2 (P < 0.05). This was not the case following completion of P1 (P > 0.3). This difference remained after the data was standardized to baseline measures (P < 0.05).ConclusionOur results showed that LTF of minute ventilation can be initiated using two different protocols that varied in regard to episode duration and the degree to which PETCO2is maintained above baseline. We also showed that increases in episode duration and sustained PETCO2 results in a greater magnitude of LTF on a given day. Moreover, these modifications appear to enhance the magnitude of LTF following repeated daily exposure.
A humanized anti‐cocaine mAb blocks the cardiovascular effects of cocaine in rats
Authors: Koch Sheryl E. , Marckel Jordan , Rubinstein Jack , Norman Andrew B. 
Published: May 2022

Cocaine use and abuse is associated with significant morbidity and mortality. Further, its use has significant effects on cardiovascular function in humans and has been associated with development of chest pain, myocardial ischemia and in rare cases cardiac death. Currently, the FDA has no approved pharmacotherapy for cocaine abuse. We have developed and generated a humanized anti‐cocaine monoclonal antibody (mAb), h2E2, which dramatically decreases brain cocaine concentrations by sequestering cocaine in the plasma. As h2E2 proceeds into clinical trials, a significant question remains as to whether the increased plasma cocaine concentrations, increased circulation time of cocaine, and the decrease in cocaine excretion into the urine affects cardiac function. Therefore, the objective of this study was to determine the dose, duration and effects of cocaine administration in the presence and absence of mAb h2E2 on echocardiographic variables in an in vivo rat model.Echocardiography was performed on Sprague‐Dawley rats with indwelling catheters using the Vevo 2100 ultrasound machine with a 20mHz probe. Baseline echocardiographic long axis M‐mode images were collected prior to an i.v. cocaine push and at subsequent time points thereafter. Initial studies determined the dose‐dependent effect of cocaine on cardiac parameters including ejection fraction (EF), heart rate (HR) and cardiac output (CO). The most significant increase in EF was observed with 1.68mg/kg cocaineHCl, which was then used in the subsequent experiments.As noted, cocaine administration increased EF, with inversely proportional changes in left ventricular systolic volume. In addition, cocaine produced an immediate decrease in HR, which did not fully return to baseline over the 45 minute time course, likely due to the anesthesia, as the saline control group demonstrated comparable effects. Similarly, cocaine caused an immediate decrease in CO, which normalized over time.Pre‐administration of a single equimolar dose of h2E2 significantly blunted the effect of cocaine on EF, including both the initial drop as well as the subsequent increase. At one week and one month post h2E2, there was no significant blunting of the increased in cocaine‐induced inotropy. However, the initial decrease in EF was still blocked after one week. The initial cocaine‐induced drop in HR was blocked by h2E2, even at 1 week post‐injection. h2E2 similarly blocked the increase in CO, however this effect was not detectable after 1 week or 1 month.In conclusion, there were no significant adverse cardiac effects noted with h2E2 administration and co‐administration with cocaine resulted in significant blunting of both cocaine’s inotropic and chronotropic effects. Elevated plasma cocaine concentrations are pharmacologically inert in the presence of h2E2.
Slc2a5 (GLUT5) upregulation in hyperuricemia drives risk for fructose induced NAFLD
Authors: Hoque Kazi M. , Halperin Kuhns Victoria L. , Woodward Owen M. 
Published: May 2022

Epidemiological and observational studies have found a strong correlation between hyperuricemia (clinically elevated serum urate levels) and the onset of metabolic and systemic diseases. To investigate a potential causal role for urate in metabolic disease, we created a humanized mouse model utilizing a loss of function mutation in a critical intestinal urate transport protein, ABCG2. Male animals from this model were hyperuricemic and displayed fat deposition in the liver and significantly elevated serum glucose and insulin levels, hallmarks of metabolic dysfunction, and similar to what is often observed in high fructose diet models of NAFLD (Nonalcoholic fatty liver disease). However, the relationship between hyperuricemia, liver pathogenesis, and altered intestinal fructose uptake is largely unexplored.Using the Q140K+/+ hyperuricemia model we observed higher gene expression of Slc2a5 as well as increased abundance of its protein product, the apical fructose transporter GLUT5, in enterocytes. In addition, ex‐vivo intestinal fructose transport studies revealed a corresponding significant increase of fructose transport across the intestinal tissue of Q140K+/+ mice (16.497 ±5.955 vs. 34.684 ±5.31 µM/cm2). We hypothesized that HNF4a, the critical transcription factor that regulates the expression of many gut transport proteins, and has potential binding sites in the human and mouse Slc2a5 promoter region, might be sensitive to urate levels. Treatment of the human intestinal Caco‐2 cell monolayers with urate increased the protein abundance of both GLUT5 and HNF4a in a dose‐dependent manner, potentially explaining the observed increased mRNA levels of Slc2a5 in our model. We next explored the possibility that the GLUT5 abundance was further altered due to secondary urate effects on the critical transporter scaffold NEDD4 like E3 ubiquitin‐protein ligase (NEDD4L). Nedd4L gene expression was decreased, and its negative regulator, Sgk2, was significantly increased in intestinal tissue from the hyperuricemic model. Follow‐up experiments demonstrated SGK2 protein abundance is increased in Caco‐2 cells by extracellular urate. Further experiments are needed to confirm if hyperuricemia induced up‐regulation of SGK2, and subsequent down‐regulation of NEDD4L would prevent GLUT5 ubiquitination and increase its residence and abundance at the apical membrane.In conclusion, we find that hyperuricemia induced via common variants in the key intestinal urate transporter, ABCG2, can cause NAFLD and metabolic phenotypes indistinguishable from high fructose diet models of NAFLD. Here we demonstrate that urate may significantly upregulate the capacity for fructose reabsorption through the upregulation of Slc2a5 transcription and GLUT5 abundance, a finding that suggests human carriers of ABCG2 variants are not only at higher risk for hyperuricemia and gout but also the development of metabolic syndrome.
Morphometric Aspects ot the Alba and Lunate Lineas and Correlations with the Physiological Diastasis of the Rectus Abdominis Muscles: Prospective Study
Authors: Barros Mirna D. , Leite Eduarda C. R. , Uehara Elisa Y. T. , Mendes Carlos J. L. 
Published: May 2022

IntroductionThe anatomical study of the aponeuroses of the anterolateral abdominal wall (AAW) is described, but the AAW aspects remain without clarification. The decussation of the myoaponeurotic of the external oblique (EO), internal oblique (IO) and transversus abdominis (TA) muscles form the abdominal rectus muscle sheath. Theses muscles are not tangential in their respective lateral margins, then they form the semilunar linea (LS), and on the medial margin of the abdominal rectus muscle they form the alba linea. Even having few citations on the width of the LS and the LA, the literature presents clinical aspects, classifications and proposals for the correction of the pathological width of this line: the diastasis. The measurements of the LA and the LS need reliability for the establishment of criteria for normality or for disorder.ObjectiveThe aim of this study was to measure the width of the LA and LS, the length of the LA, the distance from the umbilicus (CU) to the anterior margin of the lumbar vertebra (VL) in the plane passing through the CU and to establish whether there is a correlation between the measurements of the LS, LA and the distance from the CU to the anterior margin of the VL.MethodTrough the tridimensional reconstruction of computerized tomographies of the AAW in adults, measurements were made of the distances to the LA and LS in five levels from the xifoid process to the pubic symphysis using Osirix MD software.ResultsA total of fifteen individuals were studied as the pilot sample for the calculation of the sample, with the standard deviation for the average and 95%IC. The calculation led to the evaluation of 29 individuals (15 men and 14 women). The analyzed outcomes were: 1. Dpx_sp_reta; 2. Dpx_sp_pela_LA; 3. Dcu+VL; 4. DLA_NSu1; 5. DLA_NSu2; 6. DLA_NSu3; 7. DLS_dir_NSu3; 8. DLS_esq_NSu3; 9. DLA_NU; 10. DLS_dir_NU; 11. DLS_esq_NU; 12. DLA_Niu1; 13. DLS_dir_Niu1; 14. DLS_esq_Niu1; 15. DLA_Niu2; 16. DLS_dir_Niu2; 17. DLS_esq_Niu2. Considering that the DLA – distance from alba linea, DLS – distance from semilunar linea, sp – pubic symphysis; vL – lumbar vertebra; NSu – supraumbilical level; Niu – infraumbilical level. The maximum and average measurements were established for each outcome for men and women and the measurements (DLA_NSu1) e (DLA_Niu1) presented significant differences between men and women.ConclusionThe results may lead to a relevant discussion on the concept and the classifications of diastasis, producing a deeper knowledge of the morphological variations of the abdominal wall and the normal and unnormal functional aspects of these muscles and fáscias. This anthopometric anatomical parameter will fill in a blank with relevant data for the determinations of the criteria of normality, anatomical variation or disorder.
Assessment of Skeletal Muscle Oxygenation during Exercise in Patients with Peripheral Artery Disease Using Near Infrared Spectroscopy: Impact of Skin Color and Adipose Tissue Thickness
Authors: Pae Byung Joon , Hirai Daniel , Monroe Jacob , Motaganahalli Raghu , Song Qifan , Roseguini Bruno 
Published: May 2022

IntroductionNear infrared spectroscopy (NIRS) is a potentially valuable tool to examine disease severity and the effects of therapeutic interventions on microvascular leg oxygenation in patients with peripheral artery disease (PAD). However, multiple factors can confound the interpretation of NIRS signals and therefore limit its clinical applicability, including adipose tissue thickness (ATT) and skin melanin content. We tested the hypothesis that the changes in the concentration of oxygenated [oxy‐Hb+Mb] and deoxygenated [deoxy‐Hb+Mb] hemoglobin + myoglobin would be reduced in the calf of black PAD patients during exercise when compared to white patients. Similarly, we hypothesized that patients with a thicker calf ATT would exhibit a lower change in oxy[Hb+Mb] and deoxy[Hb+Mb] during exercise relative to patients with low ATT.MethodsTwenty‐four participants with a history of exertional calf pain (18 males, 6 females, 67±7 years; ankle‐brachial index: 0.6±0.1) were asked to complete two graded tests on the treadmill. The protocol consisted of a constant walking speed of 2 mph and progressive increases in grade of 2% every 2 min. The NIRS device was secured to the skin over the medial gastrocnemius muscle of the most symptomatic leg. Oxy[Hb+Mb] and deoxy[Hb+Mb] were measured continuously at rest and during exercise. The final 20‐s averages of stages 1, 2 and peak exercise were utilized for analysis. Magnetic resonance imaging of the calf was used to measure ATT. The average ATT among all individuals (7±3 mm) was used to divide participants into ‘low’ (n=12) or ‘high’ (n=12) ATT groups. A three‐way repeated measures analysis of variance was used to evaluate the effects of skin color, ATT, and stage of the treadmill test (i.e. stage 1, stage 2, peak walking time) on the changes from baseline in calf oxy[Hb+Mb] and deoxy[Hb+Mb].ResultsSeven participants were black and the remaining 17 were white. The average ATT at the medial gastrocnemius site was 4±1 mm in the low ATT group and 9±2 mm in the high ATT group. A significant interaction was observed between skin color, ATT, and exercise stage for the change from baseline in oxy[Hb+Mb] (p=0.049). Post‐hoc testing revealed that among white participants, the change in oxy[Hb+Mb] during exercise was significantly lower in the high ATT group when compared to the low ATT group (p=0.039). Among black participants, the change in calf oxy[Hb+Mb] was similar between high and low ATT (p=0.57). There was a trend for a main effect of ATT on the changes in deoxy[Hb+Mb] during exercise (p=0.1), but the main effects of skin color (p=0.2) and the interaction between skin color, ATT, and exercise stage (p=0.5) were not significant.ConclusionsIn conclusion, the changes in NIRS‐derived calf oxy[Hb+Mb] during exercise are impacted by both skin color and ATT in patients with symptomatic PAD.
Optimizing Newborn Primate Endocast Reconstruction in 3DSlicer
Authors: Bryson E. R. , King Scot E. E. , McGovern‐Lind Brenna R. , Proffitt Kathryn A. , Smith Timothy D. , DeLeon Valerie B. 
Published: May 2022

Natural and artificial endocasts have been used for decades to enable researchers to visualize the endocranial space and approximate the morphology of internal soft tissue structures. Producing an endocast of the newborn primate skull is made difficult by the large fontanelle spaces between growing bones. As there is no definitive endocranial boundary, these spaces have the potential to introduce error into both manual and semi‐automatic methods of endocast reconstruction.This study examines three methods of capturing endocranial volume (EV) from the endocasts of multiple species of newborn primates. For each specimen an EV was produced using three methods: a) manual, b) semi‐automatic and c) “patched” semi‐automatic segmentation. Manual segmentation is considered the “gold standard” of endocast reconstruction, however it lacks the efficiency of semi‐automatic methods. The “patched” method proposed here is a novel approach to fontanelle closure that is accomplished using the “Baffle Planner” module in 3DSlicer, an open‐access post‐processing software that is freely accessible to all researchers. The interosseous patches constructed from user‐defined landmarks are placed around the ossifying edges of the fontanelle and reflect the curvature of adjacent bones.In this study we provide guidance for optimizing the parameters of newborn primate endocast reconstruction in open‐access software. Our study found both quantitative and qualitative differences between the models produced by these methods suggesting the chosen method of reconstruction affects the resulting EV measurement. We conclude that a patched semi‐automatic approach with minimal baffles appears to reflect the anatomy with sufficient accuracy, while increasing efficiency and repeatability.