bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ferric Chelate Reductase Knockdown in Drosophila S2 Cells
Authors: Murphy Laura Grace , Holst Jessica , Kane Gregory , Gorman Maureen J. , Ragan Emily J. 
Published: April 2020

Although the mechanisms of cellular iron uptake in mammals are largely understood, there is still little known about the mechanism of iron transport in insects. Not only is iron essential, it can also be toxic and therefore needs to be well regulated to maintain balance between necessity and toxicity. In mammals, a duodenal cytochrome b acts as a ferric reductase to allow for uptake of iron into intestinal cells. We identified a cytochrome b561 family member in Drosophila, CG8399, that is expressed in Drosophila S2 cells. CG8399 is similar to ferric chelate reductase‐1 in humans as both contain reeler, domon and cytochrome b561 domains. We are testing the hypothesis that there is reduction of iron by CG8399, which allows the transport of iron into the cells. To test this hypothesis, we used RNA interference (RNAi) to knockdown the CG8399 mRNA. We quantified the RNAi knockdown caused by RNAi using real time PCR and compared iron concentrations between untreated and treated cells using a ferrozine‐based iron content assay. A lower concentration of iron in CG8399 RNAi‐treated cells would support our hypothesis.Support or Funding InformationNSF IOS‐1656407
Designing a Measurement Methodology for the Upper Aerodigestive Tract of Phoca vitulina
Authors: Nourbakhsh Hirad , Adams Arlo , Vogl Wayne , Dawson Camilla , Raverty Stephen , Haulena Martin , Skoretz Stacey A. 
Published: April 2020

In North America, wild harbor seal neonates and pups (Phoca vitulina) are among the most common marine mammals admitted to rescue centers. Nutritional support is typically via gavage as most are admitted prior to weaning. In order to advance seal feeding and swallowing rehabilitation, we require a comprehensive understanding of their upper aerodigestive tract (UAT) as it applies to swallowing physiology. Anatomical studies of the harbor seal UAT are limited, and standardized UAT measurements for neonates and pups have not yet been determined. Our objective is to design a systematic method to measure their UAT structures and then obtain quantitative values. During post mortem examination, we photographed UAT structures using scalars. Structures included: tongue, vocal folds, cricoid cartilage, trachea, and epiglottis. A priori, we developed a manual outlining measurement start and endpoints using ImageJ (NIH, 2010). Following measurements, we summarized results with means (standard deviations, [SD]) and medians (interquartile ranges, [IQR]) as appropriate and explored correlations (Pearson’s correlation coefficient, one‐tailed) between UAT structures and other body measurements. We used IBM SPSS analytical software version 26 (IBM Corporation, 2019) for analyses with significance of p<.05. Our specimens included 14 deceased harbor seal neonates or pups (9 males, 5 females) aged between three days and 2 months old at the time of death. Causes of death were either: spontaneous, congenital, or due to infectious, metabolic or malnutrition disorders, or by euthanasia after a humane endpoint was reached. On admission to rehabilitation, body measurement ranges included: 46.0 to 57.0 (body length [cm]), 36.0 to 59.0 (axillary girth [cm]) 5.8 to 12.6 (weight [kg]). Post‐mortem UAT measurements (mm, mean ± SD) included: 1) tongue tip to epiglottis base: 82.7 ± 6.9, 2) vocal fold length: 13.9 ± 2.0, 3) tracheal diameter: 10.3 ± 1.4, and 4) epiglottic length: 20.0 ± 2.8. Correlations between seal body length and UAT dimensions were not statistically significant. In conclusion, we have developed a systematic method to measure harbor seal neonate and pup UAT structures. To the best of our knowledge, these measurements, as they relate to swallowing, have not been previously reported. These findings expand the seal macroscopic anatomy knowledge base and contribute to cross‐species conceptual convergences between human and seal swallowing physiology and its rehabilitation.Support or Funding InformationSupported by a UBC Faculty of Medicine Summer Student Research Program 2019 award to Hirad Nourbakhsh, UBC Faculty of Medicine Start‐up grant to Stacey A. Skoretz and by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant to A. Wayne Vogl (RGPIN‐2018‐03727).
Impact of Ovariectomy on Arterial Stiffness
Authors: Wong Tristen J. , Ogola Benard O. , Kilanowski-Doroh Isabella M. , Harris Nicholas R. , Clark Gabrielle L. , Miller Kristin S. , Lindsey Sarah H. 
Published: April 2020

Arterial stiffness predicts cardiovascular mortality independent of blood pressure, and women have lower arterial stiffness than men until menopause. Therefore, the lower prevalence of cardiovascular disease in women may be attributed to the impact of estrogen on vascular compliance. The current study evaluated the impact of ovariectomy (OVX) on systolic blood pressure and carotid artery stiffness. We hypothesized that loss of estrogen via OVX would increase blood pressure and induce arterial stiffness. Female mice underwent OVX at 8–9 weeks of age (n=9) or were left ovary‐intact (n=7). Blood pressure was measured via tail cuff plethysmography twice a week, and pulse wave velocity was obtained via high resolution ultrasound. At 15–17 weeks of age, carotids were excised and subjected to biaxial pressure myography under passive conditions to measure large artery distensibility. OVX induced a significant increase in body weight (21.1 ± 0.7 vs. 23.0 ± 0.6 g, unpaired t‐test, p=0.046) and decrease in uterine weight (107 ± 9 vs. 31 ± 13 mg, unpaired t‐test, p=0.0005). Despite similar systolic blood pressures (139 ± 6 vs. 139 ± 4 mmHg; unpaired t‐test, p=0.98), pulse wave velocity was significantly increased by OVX (1.25 ± 0.07 vs. 1.67 ± 0.09 m/s, unpaired t‐test, p=0.02). Pressure myography experiments showed a significant interaction between pressure and distensibility (repeated measures two‐way ANOVA, interaction p=0.0007) with OVX vessels having decreased distensibility at higher pressures. In comparison with carotid cross‐sections from intact mice, OVX vessels had larger external diameters (453 ± 11 vs 495 ± 14 μm, unpaired t‐test, p=0.04) but similar internal diameters (216 ± 17 vs 237 ± 11 μm, unpaired t‐test, p=0.31) . In conclusion, OVX induced an increase in pulse wave velocity without altering blood pressure. The increase in elastic artery external diameter without changes in internal diameter suggests outward vascular remodeling. Additional studies will assess other aspects of vascular geometry as well as changes in components of the extracellular matrix.Support or Funding InformationNIH HL133619
Anatomy Education Across the Health Professions: A Comparison of Student Perspectives on Body Donation
Authors: Noel Geoffroy P.J.C. , Claveria Alexandra , Bachour Dona , Dube Joseph , Antonacci Rosetta , Ventura Nicole 
Published: April 2020

Cadaver‐based learning provides students with the opportunity to not only acquire the visual and tactile experiences necessary to properly apply anatomical knowledge but more importantly, an opportunity for students to reflect upon fundamental values of humanism, such as respect, empathy and compassion. Differences in students' perspectives across the health professions on body donation and the use of body donors as a component of their education has not yet been explored. The goal of the study was to qualitatively analyze the commonalities and differences in student perspectives on body donation across varying health professional programs including nursing, physical and occupational therapy and medicine. One‐page reflections written by nursing (N=37), physical and occupational therapy (N=49) and medical students (N=66) regarding their experiences in the McGill University anatomy laboratory were collected and qualitatively analyzed by deductive coding methods using Atlas.ti software. All of the data were thematically coded using a deductive approach, by 2 independent coders, according to the themes and sub‐themes described by Stephens et al., 2019 (How does Donor Dissection Influence Medical Students’ Perceptions of Ethics? A Cross‐Sectional and Longitudinal Qualitative Study, ASE 2019). IRB# A06‐B42‐17A. Despite the students having different curricula, there were only a few differences in what the nursing, physical and occupational therapy and medical students discussed in their reflections about body donation and cadaveric‐based learning, suggesting that the anatomy laboratory had similar effects on the different users. The nursing students were not reflective about the idea that working with different body donors provided them with unique learning experiences in the anatomy laboratory. In addition, the physical and occupational therapy students did not discuss the idea of maintaining the body donors' privacy by keeping parts of their bodies covered during the anatomy lab. The majority of the students from all three healthcare profession groups have mentioned that, as a result of their positive anatomy lab experience, they would like to give future generations the same privilege they had by donating their bodies to science. However, there were two nursing students that have concluded that they have decided not to donate their bodies to science. This qualitative study is the first of its kind because it directly compares student perspectives on body donation and cadaveric‐based learning, amongst three different health profession groups: nursing, physical and occupational therapy and medicine. This new information can help design interprofessional activities, which require mutual respect of another profession’s perspective, such commemorative services.Support or Funding InformationThis work was generously supported by the McGill University Faculty of Medicine Research Bursary Program.
Very Low‐Density Lipoprotein and a Sphingosine‐1‐Phosphate Receptor Agonist Regulate Aldosterone Production via Lipin‐1 in Human Adrenocortical Cells
Authors: Chowdhury Shinjini , Choudhary Vivek , Choudhary Mrunal , Chen Xunsheng , Bollag Wendy B. 
Published: April 2020

Aldosterone plays an important role in blood pressure homeostasis, and hyperaldosteronism can result in hypertension. Aldosterone is considered to be a link between hypertension and obesity; obese individuals have high serum levels of very low‐density lipoprotein (VLDL). VLDL has been shown to stimulate aldosterone production in multiple zona glomerulosa cell models via phospholipase D (PLD), an enzyme that hydrolyzes phosphatidylcholine to phosphatidic acid (PA), a lipid second messenger. In addition, sphingosine‐1‐phosphate (S1P), a bioactive sphingolipid also elevated in obesity, has been reported to be a novel stimulator of aldosterone secretion and PLD activity. The mechanisms underlying the action of these non‐conventional secretagogues of aldosterone remain to be elucidated. Angiotensin II, a classical aldosterone secretagogue, not only activates PLD but also elevates the expression of lipin‐1, an enzyme that converts PA to another lipid second messenger, diacylglycerol (DAG), in human adrenocortical carcinoma (HAC15) cells. However, it is unclear which of the two lipid signals, PA or DAG, underlies PLD’s role in aldosterone production. Herein, we used HAC15 cells to determine whether VLDL and an S1P1 receptor (S1PR1) agonist (SEW2871) stimulate aldosterone production by increasing steroidogenic gene expression via lipin‐1‐mediated metabolism of PA to DAG. We overexpressed lipin‐1 using an adenovirus or inhibited it using propranolol followed by treatment with or without VLDL or SEW2871 for 24 h. The expression of steroidogenic genes and aldosterone secretion were monitored using qRT‐PCR and radioimmunoassay. We demonstrated that lipin‐1 overexpression enhanced the VLDL‐stimulated 55‐fold increase in CYP11B2 expression by 75% while lipin‐1 inhibition decreased the VLDL‐stimulated 21‐fold increase in CYP11B2 expression by 66%. A parallel trend was observed with aldosterone secretion levels: VLDL‐stimulated increase in aldosterone production was enhanced by lipin‐1 overexpression (182%) and was decreased by propranolol (80%). Similar results were obtained with SEW2871. Our results are, therefore, suggestive of DAG being the key lipid signal since lipin‐1 regulates VLDL‐ and S1PR1 agonist‐stimulated expression of steroidogenic genes and ultimately, aldosterone production. Our study warrants further investigation into these steroidogenic signaling pathways which can lead to the identification of novel therapeutic targets such as lipin‐1, or its downstream pathways, to potentially treat obesity‐associated hypertension.Support or Funding InformationThis work was supported in part by the Augusta University Adrenal Center. WBB was also supported by VA Merit Award #BX001357.
Characterization of Biofilms Produced by Bacillus Wild Isolates
Authors: Wacker Sarah A. , Torres Ryan , Lara-Garcia Juan , Bendesky Andrés 
Published: April 2020

Many bacteria form complex multicellular communities known as biofilms. Biofilms are a common source of human infections and are associated with chronic infections and with infections resulting from biomedical implants. Bacillus subtilis has been used as a model organism to study biofilms, leading to important insights into how biofilms are regulated and form. However, most research has examined biofilm formation in a single laboratory strain of B. subtilis. We sought to examine differences in biofilm formation between twenty wild isolates that had been published by others to belong to the species B. subtilis. We characterized the biofilm phenotypes of the strains on several types of media and also sequenced the whole genome of each of the strains. We found a wide array of biofilm phenotypes with most being more intense than biofilms of the laboratory strain. Furthermore, biofilm phenotypes and intensity of the biofilm were media‐dependent. Our genetic results indicated that nine of the twenty strains are not actually Bacillus subtilis, but are likely to be a related species, Bacillus velezencis. Interestingly, biofilm strength did not correlate with species. Among the B. subtilis strains, several coding genetic variations were identified in genes important for biofilm formation. Together our study provides insight into variation in biofilm formation among related bacteria and creates a list of possible variations that can be tested for their impact on biofilm formation.Support or Funding InformationResearch was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number R15GM135861. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Differential Augmentation of Thrombin Generation by Andexanet Alfa in Lymphoma Patients
Authors: Siddiqui Fakiha , Antic Darko , Bontekoe Emily , Hoppensteadt Debra , Gerotziafas Grigoris , Tafur Alfonso , Fareed Jawed 
Published: April 2020

IntroductionThe prevalence of thrombosis in lymphoma patients is reportedly high and ranges from 3–10%. Vascular malfunction and inflammatory processes further contribute to the thrombotic activation processes in these patients. Andexanet alfa is an antidote for factor Xa inhibitors and its usage has been reported with thrombotic complications. This study was designed to compare effect of andexanet alfa (AA) on the thrombin generation potential and its relevance to the generation of thrombin.Materials and MethodCitrated blood samples from 78 patients with confirmed diagnosis of non‐Hodgkin lymphoma (NHL), Hodgkin lymphoma (HL) and Chronic lymphocytic leukemia/Small lymphocytic lymphoma (CLL/SLL) were collected from the Clinic of Hematology Unit, University of Belgrade, Belgrade, Serbia. 50 samples of normal human plasma (NHP) was obtained from George King Biomedical (Overland park, KS). NHP was prepared for referencing purposes. Individual samples were supplemented with andexanet alfa at 100 ug/ml. Thrombin generation studies were carried out using a commercially available a kinetic fluorogenic substrate method (calibrated automated thrombogram; CAT). Thrombin generation parameters such as peak thrombin (PT), lag time (LT) and area under the curve (AUC) were compiled. Results were compiled in terms of mean ±SD.ResultsOn a cumulative basis, lymphoma patients showed an increase in LT (2.9 1.15) in comparison to NHP (2.06 .02) which decreases with AA (1.94 0.1). The PT levels were decreased (120.34 44.3) in comparison to NHP (159.20 4.8) and increases with AA (153.70 37.2). The AUC was also decreased (668.85 284.3) in comparison to NHP (756.46 34.5) which increases with AA (758.32 226.4). When the lymphoma patients were sub‐grouped, peak thrombin levels were similar in HL (128.16), NHL (113.99) and CLL (129.77) were decreased compared to normal human plasma (159.2) which increases with AA, in HL (180.75), NHL (154.16) and CLL (142.46) compared to AA control (153.7). The AUC was increased in the HL (769), decreased in NHL (642.4) and was comparable for CLL (715.4) compared to normal human plasma (756.46). However, with AA the AUC values increases for HL (938.31) and decreases for NHL (744.52) and CLL (725.26) in comparison with AA control (758.32). Variations in lag time were noted in the three groups.ConclusionLymphoma patients represent a heterogenous group of patients in which both the hypercoagulable state and inflammatory responses simultaneously occur. Thrombin is constantly generated in some of these patients leading to the formation of fibrin. The observed decrease in thrombin generation potential in these patients may be due to the consumption of prothrombin and other coagulation factors. Differential increase in thrombin generation in post andexxa supplemented samples suggest that the use of this agent may potentially be associated with thrombotic complications.
Adenosine Protects Zebrafish Photoreceptors from Apoptosis and Damage in a Dose‐Dependent Manner in a Light‐Induced Retinal Degeneration Model of Age‐Related Macular Degeneration
Authors: Khan Amanda S. , Stella Salvatore L. 
Published: April 2020

PurposeAdenosine has been shown to be neuroprotective in the CNS and in retinal ganglion cells in the inner retina. However, the neuroprotective effects of adenosine on outer retinal neurons, in particular photoreceptors, is not known. Age‐related macular degeneration (AMD) is a multifactorial disease characterized by the impairment or loss of central vision due to macular dropout and loss of photoreceptors. Adenosine has been shown to inhibit photoreceptor excitability by suppressing Ca2+ influx through voltage‐gated Ca2+ channels, which could lead to cell death. The purpose of this study was to see if adenosine would reduce photoreceptor damage and death in a light‐induced retinal degeneration (LIRD) model in pigmented zebrafish.MethodsAdult wild‐type and Tg(nrd:GFP) zebrafish (used to GFP label rod photoreceptors) were dark‐adapted for 24 hours, followed by intravitreal injections of ~ 0.5 ml of 0.1 mM, 1 mM, or 5 mM (final conc: 1.67 to 167 mM) of adenosine in both eyes. Controls were injected with saline in both eyes. All zebrafish were exposed to full spectrum high‐intensity light (≥ 20,000 lux) for four days and collected at 0 and 96 hours post dark‐adaptation. Immunohistochemical analysis was performed on vertical retinal sections in order to visualize cones, rods, and the outer nuclear layer of the retina. Retinal damage was assessed using confocal microscopy with a TUNEL assay and cell specific markers.ResultsPhotoreceptors in control retinas collected at 96 hours exhibited severe truncation or loss of the outer segments, and swelling of the cell body. Additionally, there was an increase in TUNEL‐positive cells and a significant decrease in rod and cone photoreceptors in the control retinas. Photoreceptors from retinas treated with higher concentrations of adenosine (1 mM and 5 mM) lacked swelling, exhibited less cell death, reduced truncation of the outer segments, and had cell morphology similar to retinas collected prior to light exposure. Photoreceptors from retinas treated with 0.1 mM adenosine exhibited no significant differences in cell death in comparison to control retinas at 96 hours, and had similar levels of outer segment truncation and cell body morphology. However, there was a significant reduction in the number of cones and rods lost in the 0.1 mM adenosine‐treated retinas at 96 hours in comparison to control retinas at 96 hours.ConclusionThese findings show that adenosine can prevent light‐induced structural damage to rod and cone photoreceptors, prevent photoreceptor death and loss, and provide evidence of a potential novel therapeutic target for the treatment of the “dry” form of AMD.Support or Funding InformationHMC CURE Zebrafish Pilot Grant from Penn State University College of Medicine, Pennsylvania Department of Health Tobacco CURE Fund
The FSGS protein Actinin‐4 (ACTN4) is expressed in Thick Ascending Limbs and interacts with NKCC2 and ALMS1 to regulate NKCC2 trafficking.
Authors: Maskey Dipak , Liao Tang-Dong , Ares Gustavo R , Ortiz Pablo A. 
Published: April 2020

Thick ascending limb (TAL) of the loop of Henle reabsorbs 30% NaCl via the apical Na/K/2Cl cotransporter (NKCC2). Maintenance of steady‐state surface NKCC2 levels is results from a balance between endocytosis, exocytosis and recycling. These trafficking steps are controlled by signaling and protein‐protein interactions. Very little is known about how NKCC2 endocytosis is controlled and only a handful of proteins that mediate NKCC2 internalization and trafficking are known. To identify new NKCC2 interacting partners we immunoprecipitated NKCC2 from TAL lysates using a C‐terminus antibody and identified interacting proteins by mass spectrometry. We identified Actinin‐4 (ACTN4), a protein studied in podocytes, that when mutated causes Focal segmental glomerulo‐sclerosis (FSGS) in humans. We hypothesized that in addition to podocytes, ACTN4 is expressed along the nephron and is part of protein complex that binds apical NKCC2 and other proteins such as Alstrom Syndrome 1 (ALMS1) and promotes its endocytosis. To begin studying these mechanisms we used a targeted proteomics screen to identify new binding partners for ACTN4 in the TAL. We confirmed that glutathione‐S‐transferase (GST) ACTN4 (full length) pulled down NKCC2 and ALMS1 from TAL lysates identified by mass spectrometry (LC‐MS) or Western blot. GST‐ALMS1 also pulled down ACTN4, identified by LC‐MS. Because there are no studies on the role of ACTN4 in NaCl reabsorption along the nephron, we studied its localization in the kidney. ACTN4 was abundant in glomeruli but also localized in cells along the nephron in a punctate vesicular pattern. ACTN4 was expressed in TALs and partially co‐localized with NKCC2 the subapical space. To study if ACTN4 mediates NKCC2 trafficking, we generated adenoviruses expressing ACTN4 shRNA. In vivo transduction of the rat renal medulla decreased ACTN4 expression in TALs by 66±9% (p<0.04). Silencing ACTN4 in rat TALs increased the surface to total NKCC2 ratio by 49±16% (p<0.01) but did not affect total NKCC2, suggesting that ACTN4 regulates NKCC2 trafficking. To study the relationship of ACTN4 with ALMS1 and NKCC2, we generated nephron‐specific ALMS1 knockout mice and measured surface NKCC2 and ACTN4. Deletion of ALMS1 increased surface to total NKCC2 ratio by 55±19% (p<0.05), but also decreased ACTN4 expression by 45±9% (p<0.05). We conclude that ACTN4 binds NKCC2 and ALMS1 and it is required for proper NKCC2 trafficking. It is likely that a large protein complex containing ACTN4 and ALMS1 mediates NKCC2 endocytosis to control renal salt reabsorption.Support or Funding InformationNIH