bluuebunny/crossref_metadata_2025_split huggingface dataset:
Investigating the Mechanistic Strategies of the Two‐component FMN‐dependent Alkanesulfonate Monooxygenase Systems
Authors: Somai Shruti , Ellis Holly R. 
Published: May 2022

The two‐component alkanesulfonate monooxygenase systems, consisting of a flavin reductase (SsuE and MsuE) and an alkanesulfonate monooxygenase (SsuD and MsuD), enable bacterial organisms to utilize a broad range of alkanesulfonates when sulfur is limiting. The flavin reductases supply reduced flavin to their partner monooxygenase for the desulfonation of sulfonated compounds through the formation of a flavin oxygenating intermediate. Commonly, flavin monooxygenases have been proposed to utilize a C4a‐(hydro)peroxyflavin as the oxygenating intermediate. However, unlike other flavin monooxygenase enzymes, this intermediate has not been spectrally observed in the alkanesulfonate monooxygenase enzymes. Recent studies reported the formation of a flavin‐N5‐oxide which forms as an intermediate during turnover or as the final product in some flavin monooxygenases. It is hypothesized that the alkanesulfonate monooxygenases could employ a flavin‐N5‐adduct based on amino acid sequence alignments and structural similarities with enzymes employing similar intermediates.SsuD and MsuD share comparable structural properties but have different specificities for alkanesulfonate substrates. Although the substrate preference is critical for catalytic competence, SsuD and MsuD likely utilize similar catalytic steps for desulfonation. Previous studies identified various polar and nonpolar amino acid residues that were critical for the formation and stabilization of the flavin‐N5‐oxide. The conserved nonpolar amino acids are proposed to control the interaction of the flavin‐N5 with molecular oxygen, whereas the polar amino acids are involved in stabilizing the superoxide anion involved in the formation of the flavin‐N5 oxygenating intermediate. Some of these amino acids were also found to be conserved in the alkanesulfonate monooxygenases and their role was further evaluated through site‐directed mutagenesis. The V108T and T109A SsuD variants had similar activity to wild‐type SsuD; however, the N108L SsuD variant had no measurable activity. These results support the role of Asn in stabilizing the proposed flavin‐N5 oxygenating intermediate. Since a flavin‐N5‐oxide has been identified as an intermediate or as the final product in some two‐component monooxygenases, HPLC and mass spectrometric analyses were performed to determine if the alkanesulfonate monooxygenases form the flavin‐N5‐oxide. To provide evidence for kinetic steps and identify flavin‐N5 reaction intermediates, stopped‐flow kinetic experiments were performed with wild‐type and variants of SsuD and MsuD. The findings from this study provide insight into the mechanistic features of the alkanesulfonate monooxygenases as well as their role in the overall global sulfur cycle.
Enriching Anatomy Learning with Virtual Reality Clinical Scenarios
Authors: Hasan Farah Z. , McBride Jennifer , Mitchell Josh P. , Anwarzi Deewa , Sonnadara Ranil R. , Wainman Bruce C. 
Published: May 2022

IntroductionThe use of virtual reality (VR) to simply display anatomical specimens typically fails to exploit the potential of VR to enhance the learning environment. For example, anatomy education normally occurs within the confines of a laboratory which is devoid of relevant context, while VR is uniquely capable of providing a clinically‐relevant space that may enhance the anatomy learning experience. However, it is also possible that an enriched VR environment may inhibit learning, perhaps as a result of increased cognitive load. In general, well‐designed educational materials create schemas and organize the acquired knowledge in a way that promotes deeper learning, retention, and easy retrieval in other settings. Efforts must be made, in instructional design, to reduce extraneous and increase germane cognitive load, but how exactly this is done in VR remains an open question.ObjectiveThe purpose of this study is to evaluate the educational efficacy of a VR module which includes a clinical scenario presented in an immersive, contextually‐relevant virtual environment.HypothesisAlthough learning in a VR enriched environment with a clinical scenario can increase extraneous cognitive load, we hypothesize that it will ultimately facilitate memorization of anatomical structures, when compared with using 3D‐printed physical models or an interactive, 2D environment.MethodsParticipants with no prior knowledge of pelvic anatomy will be randomly assigned to one of three groups to learn human pelvic anatomy in an identical module presented in VR, an interactive 2D computer‐based module, or physical environment consisting of 3D‐printed models. Prior to the learning phase, participants will complete the Mental Rotation Test (MRT), as well as the Titmus Fly and Titmus Circles tests to assess spatial visualization ability and stereoscopic vision, respectively. They will then complete a pre‐test assessment where they will be asked to identify anatomical structures labeled on a 3D‐printed model. Participants will then be given 10 minutes to learn and memorize pelvic anatomy using their assigned anatomy modality. This will be followed by a post‐test assessment with another set of labeled structures to identify. Finally, participants will complete a survey to share feedback on the learning experience and will complete the Simulation Task Load Index (SIM‐TLX) questionnaire to assess cognitive load.ConclusionThe impact of contextually‐relevant enriched virtual environments and clinical scenarios for VR‐based anatomy education have yet to be explored. The findings from this study will provide valuable insight to inform the design of future VR learning tools that not only reduce or limit factors which can impair learning, but also suggest factors within the learning environment which have the potential to improve anatomy learning.
Aortic Stiffness is Negatively Associated with Microvascular Glycocalyx Thickness and Microvascular Perfusion among Older Adults: Role of Carotid Pressure Pulsatility
Authors: Gimblet Colin , Armstrong Matthew , Nuckols Virginia , Stroud Amy K. , Guerrero Mariah , Pierce Gary 
Published: May 2022

IntroductionThe glycocalyx layer is essential to the maintenance of a healthy microvascular endothelium and diminishes with advancing age. Similarly, the aorta stiffens with aging and increases the risk of cardiovascular disease and target organ damage, possibly through pressure pulsatility‐associated microvascular damage. However, whether aortic stiffness and/or pressure pulsatility are associated with microvascular endothelial glycocalyx structure/function among older adults is unknown. Therefore, the objective of this analysis was to investigate the potential relation between aortic stiffness and pressure pulsatility with glycocalyx thickness and microvascular perfusion in older adults. We hypothesized that increased aortic stiffness and pressure pulsatility would be associated with less glycocalyx thickness and microvascular perfusion in older adults.MethodsSublingual microvessels were assessed using intravital microscopy in older adults (n=24; age 61 ± 6y, 46% women). Perfused boundary region (PBR) and red blood cell (RBC) filling %, indirect indices of glycocalyx thickness (larger PBR=smaller thickness) and microvascular perfusion respectively, were determined in vessels between 5‐25 µm RBC column width. Aortic stiffness was quantified by carotid‐femoral pulse wave velocity (cfPWV) and carotid pressure pulsatility as carotid pulse pressure (PP)/mean arterial pressure (MAP).ResultsAdjusting for age, sex and MAP, cfPWV was associated with larger PBR 5‐25 (β: 0.08; p=0.001) and lower RBC filling % (β: ‐0.02, p=0.002). Higher carotid pressure pulsatility was associated with lower RBC filling % (β: ‐0.12; p=0.03), but this relation was attenuated after adjusting for sex (p=0.11). Upon stratification by sex, higher carotid pressure pulsatility was associated with larger PBR 5‐25 (r=0.66, P=0.03) and lower RBC filling % (r=‐0.73, P=0.01) in women, but not men (P=0.94 and P=0.77 respectively).ConclusionHigher aortic stiffness was negatively associated with microvascular glycocalyx thickness and microvascular perfusion among older adults. Additionally, carotid pressure pulsatility was negatively associated with glycocalyx thickness and microvascular perfusion in women only. The mechanisms that contribute to sex differences in the relation between pressure pulsatility and glycocalyx structure/function in older adults require further study.
Nonselective Cation Channels Maintain Alveolar Fluid Clearance After Exposure to Toxins from Respiratory Pathogens
Authors: Galarza Paez Lilian D. , Cespedes Victoria E. , Moseley Auriel , Yue Qiang , Lucas Rudolf , Eaton Douglas 
Published: May 2022

Normal gas exchange in the alveoli is facilitated by a thin fluid layer. This layer is carefully regulated by two ion channels: highly selective channels (HSC or ENaC) and nonselective cation channels (NSC). Previous research found that both channels contribute about equally to alveolar fluid clearance (AFC). Since the molecular composition of NSC is a recent discovery, not much is known about NSC’s response to various respiratory pathogens. NSC are composed of an alpha‐ENaC subunit and at least one acid‐sensing ion channel 1a (ASIC1a) subunit. Our study aims are to understand how NSC contribute to alveolar epithelial permeability and how the lungs respond to toxins from respiratory pathogens. We hypothesize that AFC will be lower in the absence of ASIC1a (ASIC1 KO) when exposed to toxins from respiratory pathogens: Lipopolysaccharide (LPS) from E. coli and Pneumolysin (PLY) from P. pneumoniae. Additionally, we hypothesize that ASIC1 KO animals will have increased baseline alveolar epithelium permeability. All animal experiments were approved by Emory’s IACUC.We introduced pathogen toxins via intratracheal instillation to ASIC1 KO (KO) and wildtype (WT) mouse models (both on a C57Bl6 background). After a four‐hour incubation period, we euthanized the mice by isoflurane overdose. We determined AFC by instilling their lungs with 0.7ml of a 2.0% Evans Blue (EB) or Fluorescein isothiocyanate–dextran (FITC) solution via the trachea. We took samples at t=0 and t=30 minutes after instillation. We determined the EB/ FITC concentration of both samples using spectrophotometry and calculated AFC from the change in dye concentration.Our preliminary findings suggest that LPS and PLY inhibit all AFC associated with HSC leaving only AFC associated with NSC. The AFC of LPS exposed KO animals is low compared to AFC for LPS exposed WT animals (6.04 ± 3.02, n= 15 vs 15.8 ± 5.61, n= 11, p< 0.001). Similarly, AFC of PLY exposed KO mice is lower than AFC for PLY exposed WT mice (2.78 ± 0.96, n= 10 vs 12.4 ± 4.36, n= 4, p< 0.001). In the toxin exposed KO groups, neither HSC nor NSC are fully functioning or contributing to AFC. We are still collecting data on the permeability of the alveolar epithelium.There is evidence to suggest that the regulation of HSC and NSC are drastically different. Given the critical nature of the alveoli fluid layer, it would be logical that NSC channels serve as a compensatory mechanism when HSC are inhibited by pathogens. These findings can help guide treatment for alveolar flooding caused by toxins from different respiratory pathogens.
Unraveling the labyrinth of our balance system: Visualizing bony otolith organ structure through novel 3D modeling
Authors: Smith Christopher M. , Curthoys Ian , Mukherjee Payal , Wong Christopher , Laitman Jeffrey T. 
Published: May 2022

The organs of balance, the semicircular canals (SCCs) and otolith organs (utricle and saccule) of the inner ear labyrinth, are essential to basic human movement and function. While the SCCs (both external and internal structure) are well visualized and understood, the seemingly vague bony structure of the utricle and saccule remains largely unexplored. Consequently, relationships between this bony structure and the internal otolith organs themselves remain unknown. As a result, a comprehensive understanding of the morphology of our balance organs and relationships to function has eluded us.As a first step to understand the relationships of the utricle and saccule (that is the membranous otolith organs responsible for detecting linear motion) to their external bony coverings, we have developed a morphometrically‐based modelling system to visualize bony otolith organ structure. The indeterminate shape of this capsule has precluded a standard metric approach to capture such relationships. Our method combines osmium tetroxide contrast enhanced micro‐computed tomography (μCT) imaging of human temporal bones (n=13), digital visualizations, and landmark‐based 3D shape analyses of bony and membranous structure. This was done in order to: 1) test the hypothesis that the bony form surrounding the otolith organs covaries with internal membranous anatomy (including the utricle and saccule); and 2) pinpoint specific arrangements of landmarks on the bone that best reflect the internal structure of the utricle and saccule. This approach allows us to extract the bony otolith organ structure from an otherwise nebulous bony form.Tests of integration on 3D shape data between bony and membranous tissue demonstrate a significant association between the hard and soft tissues of the otolith organs (p<0.05). Relative orientation of the otolith organs also significantly correlates with surrounding bony structure (p<0.005). These data support our initial hypothesis and inform the creation of our 3D visualization of human otolith organ structure, establishing the configuration of the otolith organs to bony structure of the labyrinth. Furthermore, this model enhances our understanding of spatial relationships between the otolith organs and cranial structure and provides the foundation to assess the evolution of a prime sensory system essential for providing a gravitational frame of reference for all vertebrate life.
Evaluation of a Cardiovascular Therapeutics Case‐Based Learning Activity
Authors: Guillory Ashley N. , Marciano Veronica 
Published: May 2022

Physician Assistant students at the University of Texas Medical Branch often struggle with learning how patient characteristics affect cardiovascular therapeutic treatment choice. We implemented a case‐based learning (CBL) activity in the cardiovascular therapeutics module to address this problem.The pre‐work for the CBL was the module lectures. Students completed an individual pre‐activity quiz then worked in small groups to discuss six cardiovascular cases. Each case included questions related to choosing the most appropriate treatments based on patient characteristics such as age or comorbidities, as well as the pharmacological characteristics of the drugs. Groups recorded their answers in a worksheet that was submitted for grading. The CBL concluded with an individual post‐activity quiz. Optional pre‐ and post‐session surveys evaluated their level of confidence in their knowledge as well as their satisfaction with the CBL.Prior to the CBL, 74% of survey respondents indicated they lacked confidence in their knowledge of cardiovascular therapeutics. Following the activity, only 26% of respondents indicated they still lacked confidence in their knowledge of these topics. Students enjoyed working and discussing in their groups, having realistic complex patient cases, figuring out how patient characteristics alter medication choice, and thought the cases helped guide their studying for the subsequent exam. Ideas for improvement of the CBL included utilizing smaller learning groups, reducing the number of cases, adding additional time to the activity, and adding a large group discussion to review the case answers.Overall, the response to the CBL was largely positive and the feedback will be useful for improving the session when it is next deployed. This type of activity can be used to reinforce material for other pharmacology topics students find difficult.
Hepatocyte β‐catenin is regulated by endothelial cell‐derived Wnt2 and Wnt9b to govern liver metabolic zonation and regeneration
Authors: Hu Shikai , Poddar Minakshi , Singh Sucha , Bell Aaron , Monga Satdarshan 
Published: May 2022

BackgroundWnt/β‐catenin signaling pathway regulates hepatic metabolic zonation (MZ) and liver regeneration (LR) after surgical or toxicant injury. Central vein endothelial cells (CVECs) are the main source of Wnts regulating MZ, while liver sinusoidal ECs (LSECs) are the major source of Wnts regulating LR after 70% partial hepatectomy (PH). Of the 19 Wnt genes, Wnt2 and Wnt9b were reported to be expressed in CVECs and the same 2 Wnts were also upregulated in LSECs isolated from regenerating livers at 12h post PH. However, conclusive evidence of the specific roles of Wnt2 and Wnt9b in regulating MZ and LR, is lacking.MethodsHere, we report generation of Wnt2‐floxed mice and generation of Wnt2‐Wnt9b‐double‐floxed mice, which were then bred to lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1)‐Cre mice to delete Wnt2, Wnt9b or both from both CVECs and LSECs referred to as EC knockouts (KO). We characterize EC‐Wnt2‐KO, EC‐Wnt9b‐KO, and EC‐Wnt2‐9b‐double KO (DKO), and assess for baseline phenotype, MZ, and LR after PH.ResultsUsing reporter mice, we first reaffirmed that Lyve1‐Cre induces recombination of floxed alleles in both CVECs and LSECs. Liver weight to body weight ratio (LW/BW) was lower in EC‐Wnt2‐9b‐DKO mice. Subsets of these mice showed an abnormal increase in one liver lobe size and mass at the expense of other lobes, along with mild periportal fibrosis. Male but not female EC‐Wnt2‐KO and EC‐Wnt9b‐KO mice had partial loss of pericentral expression of glutamine synthetase (GS), cytochrome P450 2e1 (Cyp2e1), and cytochrome P450 1a2 (Cyp1a2). Both male and female EC‐Wnt2‐9b‐DKOs lacked GS, Cyp2e1 and Cyp1a2, phenocopying EC‐Wntless‐KO mice in which all Wnts secretion from ECs were inhibited. After PH, EC‐Wnt2‐KO and EC‐Wnt9b‐KO mice showed decreased expression of Cyclin D1, Ki67 and incorporation of BrdU at 40h, which was even lower in EC‐Wnt2‐9b‐DKOs. The subset of EC‐Wnt2‐9b‐DKO mice with abnormal lobar morphology developed multiple necrotic foci infiltrated with immune cells, had a detrimental delay of LR and exhibited increased mortality after PH.ConclusionWnt2 and Wnt9b from CVECs play additive roles in controlling β‐catenin pericentral target gene expression. Additionally, Wnt2 and Wnt9b from LSECs play additive roles in regulating β‐catenin activation during LR after PH. Further studies are needed to address how and why ECs are the chief source of these two Wnts to regulate MZ and LR.
Regulation of BK channel expression by Paraoxonase 3
Authors: Shi Shujie , Montalbetti Nicolas , Kleyman Thomas 
Published: May 2022

Paraoxonase 3 (PON3) is a molecular chaperone that is primarily expressed in the aldosterone‐sensitive distal nephron (ASDN) where the fine tuning of Na+ and K+ homeostasis occurs. The flow‐induced K+ secretion in the distal nephron is mediated by large conductance Ca2+‐activated K+ (BK) channels in intercalated cells (ICs). As PON3 co‐localizes with BK in the ICs and Pon3 KO mice have altered blood [K+] at baseline, we hypothesize that PON3 has a role in regulating BK expression and functional activity.To determine whether the functional expression of BK channels is regulated by PON3, the pore‐forming α subunits of mouse BK (αBK) was expressed in HEK293 cells with or without mouse PON3. We performed co‐immunoprecipitation (co‐IP) to examine whether PON3 interacts with αBK. We also examined the effects of PON3 on αBK expression and BK channel activity using biochemical and electrophysiological approaches.When expressed in HEK293 cells, PON3 was pulled down by the αBK‐IP while αBK was detected in the PON3‐pulldown, only in cells expressing both proteins. Our data suggest that there are direct or indirect interactions between PON3 and αBK, the pore‐forming subunit of BK channels. We performed whole cell patch clamp to measure BK channel activity in HEK293 cells. we found that IBTX‐sensitive whole cell K+ currents were reduced approximately 50% by PON3. Consistently, both whole cell and surface abundance of αBK was significantly lower in cells co‐expressing PON3. Together, our data suggest that PON3 negatively regulates the expression and activity of BK channels.In conclusion, we identified the BK channel as a novel target of the chaperone PON3. Future studies will determine mechanisms by which PON3 regulates BK channel functional expression as well as physiological consequences of this regulatory interaction in mouse kidney.
Home‐Based Leg Heat Therapy Does Not Improve Vascular Function in Patients with Symptomatic Peripheral Artery Disease
Authors: Monroe Jacob C. , Parker Jason G. , Perkins Susan M. , Han Yan , Joon Pae Byung , Klein Janet , Motaganahalli Raghu L. , Roseguini Bruno T. 
Published: May 2022

INTRODUCTIONPatients with peripheral artery disease (PAD) suffer from severe exercise intolerance and accelerated rates of functional decline, resulting, in part, from significant endothelial cell dysfunction and the consequent impairment in leg blood flow. Unfortunately, few non‐invasive therapeutic interventions have been shown to ameliorate the microvascular dysfunction in patients with PAD. Repeated exposure to heat therapy (HT) improves endothelial function in sedentary young individuals, but the effects of this therapy on vascular function in patients with PAD remain poorly defined. The objective of the present study was to test the hypothesis that 8 weeks of home‐based leg HT would improve leg vascular function in patients with symptomatic PAD.METHODSThirty‐four patients with symptomatic PAD (66 ± 8 years, ankle‐brachial index: 0.68 ± 0.12) were randomized into one of two groups: home‐based leg HT (HT, n=18) or a control treatment (CON, n=16). Patients in both groups were provided with identical water‐circulating trousers and a portable heating pump and were asked to apply the therapy daily (7 days/week, 90 min per session) for 8 consecutive weeks. The pump issued to participants circulated water through the trousers at 43ºC in the HT group and 33ºC in the control group. Laser‐Doppler flowmetry was used to measure red blood cell flux, an index of skin blood flow, during local skin heating to 39°C for 40 min, followed by 43ºC for 20 min. Cutaneous vascular conductance (CVC) was calculated as red blood cell flux divided by mean arterial pressure and expressed as a percentage of maximal vasodilation (%CVCmax). Calf skeletal muscle perfusion was assessed using arterial spin labeling magnetic resonance imaging (MRI) following circulatory occlusion of the thigh for 5 min.RESULTSFive participants were either withdrawn prior to study completion or failed to complete the 8‐week follow‐up visit. The remaining 29 participants were included in the final analysis (CON: n=14, HT: n=15). Two participants from CON and 4 from HT were unable to undergo the MRI scans. At baseline, local skin heating to 39ºC increased %CVCmax to 55.8 ± 12.9% in CON and 52.5 ± 18.7% in the HT group. The change from baseline to 8 weeks in %CVCmax was similar between CON and HT (CON: ‐2.6 ± 13.9%, HT: ‐0.7 ± 16.3%, p=0.74). Peak calf blood flow during reactive hyperemia was comparable between groups at baseline (CON: 48.0 ± 24.6 ml/min/100g, HT: 43.8 ± 20.4 ml/min/100g, p=0.76). The change from baseline to 8 weeks in peak calf perfusion was also similar between CON and HT (CON: ‐4.8±21.9 ml/min/100g, HT: ‐2.8±11.0 ml/min/100g, p=0.84).CONCLUSIONEight weeks of home‐based leg HT does not improve leg vascular function in patients with symptomatic PAD.