bluuebunny/crossref_metadata_2025_split huggingface dataset:
The chloride channel Bestrophin‐1 acts in the Malpighian tubule and hindgut of Drosophila melanogaster to maintain salt and water balance
Authors: Rodan Aylin , Pleinis John , Jonusaite Sima , Hudac Jacob , Goodwin Austin , Zhang Feng , Sosa-Pagan Jason , Streeter Forest , Rothenfluh Adrian 
Published: April 2020

ObjectiveThe Malpighian tubule and hindgut are iono‐ and osmoregulatory epithelia in insects. The tubule secretes KCl‐rich fluid, and ions and water are subsequently reabsorbed in the hindgut. We characterized the role of the chloride channel, Bestrophin‐1 (Best1), in the tubule and hindgut of Drosophila melanogaster.MethodsWe studied flies carrying a germline mutation in Best1, Best1c04106, as well as Best1c04106 flies with genomic rescue, and flies with tissue‐specific knockdown (quantified by qRT‐PCR) or rescue of Best1. Malpighian tubule ion flux was measured ex vivo, and immunostaining was performed using antibodies to HA‐tagged Bestrophin‐1. Flies were assayed for lethality on normal vs. 0.3 M‐supplemented NaCl (high‐salt) diet and after water restriction. Excretion was quantified by counting excretory drops. Hemolymph and excretory drop osmolality were measured using a vapor pressure osmometer. Water weight was determined by measuring flies’ weight before and after baking at 60 °C.ResultsBest1c04106 mutant Malpighian tubules had decreased fluid secretion and transepithelial ion flux that was rescued by expression of wild‐type Best1 in the K+‐secreting principal cell. Principal cell Best1 knockdown decreased transcript levels by 80% and decreased fluid secretion and ion flux. We previously showed that NKCC is required for principal cell K+ secretion. Here, we immunolocalized both Best1 and NKCC to the principal cell basolateral membrane. There was no further decrease in ion transport in Best1 knockdown tubules when NKCC was mutated, suggesting that Best1 allows Cl− entering through the NKCC to recycle across the basolateral membrane. Best1c04106 mutant flies also had increased lethality on high‐salt diet. Hemolymph osmolality increased in control flies fed high‐salt diet, but Best1c04106 mutants had decreased osmolality on both normal and high salt diet, and increased number and osmolality of excretory drops. As a consequence, Best1c04106 mutant flies were dehydrated and died when water restricted. The Best1c04106 mutant phenotypes were rescued by genomic rescue (which recapitulates normal Best1 expression throughout the body), or targeted rescue in the Malpighian tubule and hindgut.ConclusionBest1 is required for normal ion transport in the Drosophila Malpighian tubule, and in the tubule and hindgut for iono‐ and osmoregulation, where ion reabsorption through Best1 in high‐salt conditions allows an increase in hemolymph osmolality to prevent osmotic water loss and dehydration.Support or Funding InformationNIH DK110358
Effects of Lipid Oxidation Products Found in Used Restaurant Fryer Oils on Adipocyte Differentiation and Lipid Metabolism
Authors: Arowolo Folagbayi , Bengtson Marah E. , Andhole Neha G. , Firmino Sofia N. , Shanmuganayagam Dhanansayan 
Published: April 2020

Incidence of chronic disease such as obesity, metabolic syndrome and diabetes are rapidly increasing globally, and disproportionately in Western countries. A common theme in the initiation and progression of many chronic diseases, is the exacerbating role of metabolic deregulation associated with observed in obesity and metabolic syndrome. Here, metabolic deregulation and inflammation driven by adipose tissue is a notable culprit. It is suspected that endogenous oxidation of stored and membrane lipids produce lipid oxidation products (LOPs) that alter cellular function and signaling. However, what is neglected is in Western countries, that large amount of LOPs, produced exogenously by Western cooking practoces (e.g., deep frying, microwave cooking), are consumed in large quantities and subsequently stored in adipose. The effects of these LOPs on adipose biology is has not been not well studied. In this study, we examined the effects of a number of LOPs, including 2,4‐decadienal (24DD), found in locally‐sourced used restaurant fryer oil on adipocyte differentiation and lipid metabolism. During induction and subsequent maturation of pre‐adipocytes (3T3‐L1), the cells were cultured in 0, 1, 10, 100, 500 and 1000μM 24DD. Eight days post‐induction, lipid accumulation in mature adipocytes was determined. Lipid accumulation was markedly reduced in cells treated with 100, 500 and 1000μM 24DD, in a dose‐dependent manner. This data suggests that 24DD inhibits adipogenesis. Analysis of changes in gene‐expression are underway to elucidate mechanisms of effect.Support or Funding InformationThe research was supported by the Biomedical & Genomic Research Group Discretionary Fund, University of Wisconsin‐Madison.
CRISPR Knockout of CUL5 increases cell growth and affects the expression of Estrogen Receptor‐α in T47D breast cancer cells
Authors: Bonema Sarah C. 
Published: April 2020

The CUL5/VACM‐1 protein is a component of the CRL5 E3 ligase complex responsible for tagging proteins with ubiquitin for degradation in the proteasome. Previous work has shown CUL5/VACM‐1 dependent E3 ligase decreases cellular proliferation, and lack of regulation in this ubiquitin proteasome pathway can lead to uncontrolled cell growth and tumor formation. The CRISPR/Cas9 system, which is a bacterial immune system used to edit genomic DNA, was independently designed in the lab to target and knockout the function of CUL5/VACM‐1 in T47D breast cancer cells and human umbilical vein endothelial cells (HUVEC). AlamarBlue® growth assays and Matrigel® growth imaging demonstrate that in the absence of CUL5/VACM‐1 cellular proliferation significantly increases. Subsequent immunocytochemistry analysis of estrogen receptor‐α (ERα) expression in T47D cells showed that in the absence of CUL5/VACM‐1 ERα expression was increased. Finally, q‐PCR OpenArray targeted gene expression analysis of control and CRISPR treated cells showed changes in gene regulation across signal transduction pathways. These results make CUL5/VACM‐1 a potential potent target for drug design and development in breast cancer treatment.Support or Funding InformationThis work was supported by the A. Paul Schaap Fund, Hope College, Chemistry Department.
Exploring the Effect of Growth Conditions on the Volatile Metabolome of Chromobacterium vaccinii using GC×GC‐TOF‐MS
Authors: Ramirez Diana , Higgins Keppler Emily A. , Bean Heather D. 
Published: April 2020

Chromobacterium spp. have been found to grow in a variety of environments and have an extensive metabolic flexibility. Due to this flexibility, studies have shown that Chromobacterium spp. produce an array of properties that can positively impact medicine, industry, and agriculture. C. vaccinii, isolated from cranberry bog soil, has been found to inhibit the growth of multiple pathogenic plant fungi through the production of volatile compounds. Fungal inhibition has been observed when the fungi is co‐cultured with C. vaccinii in separate petri dishes but with shared gas exchange. The same fungal inhibition is not observed when the fungus is exposed to C. vaccinii metabolites produced in liquid media. Based on this observation, we hypothesize that the volatiles produced in liquid media are not the same as the volatiles produced in agar. Experiments are being conducted to study the influence of growth conditions on C. vaccinii volatile metabolites. C. vaccinii will be cultured in liquid and semi‐solid media, the volatiles will be collected using thin film solid phase microextraction (TF‐SPME) and analyzed with two‐dimensional gas chromatography – time‐of‐flight mass spectroscopy (GC×GC‐TOFMS). The goal is to determine the differences between the volatiles produced in the different types of media. These results will lead to a better understanding of this natural occurring soil bacteria.Support or Funding InformationNSF LEAP Scholars Program
Sex differences in ET‐1 levels in primary human endothelial cells
Authors: Kuczmarski Andrew V. , Hobson Joshua C. , Patik Jordan C. , Edwards David G. , Wenner Megan M. 
Published: April 2020

Endothelin‐1 (ET‐1) is produced and secreted primarily by endothelial cells and acts as a potent vasoconstrictor. Our laboratory has previously shown that ET‐1 contributes to impaired vascular endothelial function in postmenopausal women (PMW). However, compared to PMW, ET‐1 mediated vasoconstriction is greater in men of a similar age. Although sex differences in ET‐1 receptor expression and/or responses may partially explain these differences, it is unknown whether ET‐1 levels in endothelial cells differ between PMW and men of similar age. The purpose of this study was to test the hypothesis that PMW have lower endothelial cell ET‐1 levels compared to men of similar age. Endothelial cells were harvested from the antecubital vein of healthy PMW (n=9, 59±3 yrs) and age‐matched men (n=7, 58±6 yrs) by threading a guide wire through an 18g IV catheter. The guide wire was rinsed in dissociation buffer to remove endothelial cells, which were then fixed in 4% paraformaldehyde, washed in phosphate buffered saline, pipetted onto coverglass, dried at 60°C, and stored at −80°C until staining. Immunocytochemistry was used to stain for ET‐1 (Endothelin‐1 Monoclonal Antibody (TR.ET.48.5), Thermo Fisher Scientific, Cat# MA3‐005), vascular endothelial cadherin (VE‐Cadherin polyclonal antibody, Thermo Fisher Scientific, Cat# 36‐1900) to identify endothelial cells, and 4′,6‐diamidino‐2‐phenylindole (DAPI, nuclear stain). Fluorescence intensity was measured in 30 cells on a Zeiss Axio imager using consistent laser intensity and exposure times. ET‐1 intensity was normalized to human umbilical vein endothelial cells, consistent with published literature. Results are reported as mean ± standard deviation. PMW and men were well match based on BMI (PMW; 25±5 kg/m2 vs. Men; 25±3), mean arterial blood pressure (PMW; 88±8 vs. Men; 93±9 mmHg), total cholesterol (PMW; 193±45 vs. Men; 192±40 mg/dL), LDL cholesterol (PMW; 103±39 vs. Men; 111±34 mg/dL), and fasting plasma glucose (PMW; 88±4 vs. Men; 92±6 mg/dL) all P>0.05. ET‐1 intensity levels were significantly lower in PMW (0.57±0.12 a.u) compared to Men (0.98±0.27 a.u, P =0.001). These preliminary findings suggest differences in endothelial ET‐1 levels between PMW and age‐matched men. Moreover, these data provide further evidence of the sexual dimorphisms in the ET‐1 system, and add insight into mechanisms underlying sex differences in vascular function.Support or Funding InformationNIH R01 HL 146558, HL‐113514, P20 GM 113125, AHA 16SDG30700015, and University of Delaware Research Foundation.
The Sphenoidal Synchondroses: Implications for the Midfacial Growth of Anthropoid Primates
Authors: Wood Brody , Mano Nanami , Oladipupo Lanre , Corbin Hayley , Vinyard Christopher , DeLeon Valerie B. , Smith Timothy D. 
Published: April 2020

The basicranium of anthropoid primates is more flexed than in lemurs and lorises (strepsirrhines), which has implications for orientation of facial growth. Differential growth among cranial synchondroses is one suggested mechanism for variation in basicranial development. In this study, we measured length of bony elements of the cranial base using Amira software in a cross‐sectional age sample (late prenatal to juvenile), which included three monkey species and four species of lemurs and bushbabies (n = 59). In addition, we made preliminary measurements of the anteroposterior length of the spheno‐occipital synchondrosis (SOS), intrasphenoidal synchondrosis (ISS), and prespheno‐septal synchondrosis (PSept) using newborn samples. In order to assess differences in growth potential among sync
hondroses, the diameter of the proliferating (PZ) or hypertrophic (HZ) zones were also measured. Our other work has indicated that measurements of ossified elements of the cranial base reveal disproportionately more growth of presphenoid (PS) relatively to basisphenoid (BS) in monkeys. But in strepsirrhines, these bones scale similarly to one another when plotted against increasing cranial or midfacial length. When expressed as a ratio of the length of the PZ or HZ to total anteroposterior diameter of the synchondrosis, growth is relatively similar in ISS and SOS among all species studied. However, in anthropoids the length of the PZ in PSept is notably (more than two‐fold) greater than in any other synchondrosis. In strepsirrhines, length of the PZ in PSept is relatively similar to the same zone of other synchondroses. This suggests that the rapid increase in length of the presphenoid in anthropoids is mostly occurring anteriorly, at the midfacial interface. These results, taken at face value, might suggest that anthropoids have a greater magnitude of growth in the septal cartilage compared to strepsirrhines. However, our previous work established that growth at PSept in more multidirectional (radial) than in most other mammals. Instead, our findings emphasize different patterns of sphenoid growth among primates, and offer a tissue‐level explanation for the relatively large PS segment in anthropoid primates. In addition, these findings indicate suggest that growth of the sphenoid is more highly integrated with PS than BS and more interdependent with facial growth in monkeys than in strepsirrhine primates.Support or Funding InformationNSF grant # BCS‐1830894, BCS‐1830919, BCS‐0959438
Formulation of CRISPR Edited Cells to Evaluate Glypican‐Targeting in Glioblastoma
Authors: Pitcher Morgan , Zewdu Tehut , Yusuf Diamond , Karmacharya Trishala , Farrell Nicholas , Katner Samantha 
Published: April 2020

Heparan sulfate proteoglycans (HSPGs) are ubiquitous protein/carbohydrate complexes that are found on all cell surfaces. They play a role in various biological pathways such as cellular growth and vascularization, processes that are exploited by cancer. Heparanase is an enzyme that modifies HSPGs which can alter the structure of the proteoglycan and change its bioactivity. In glioblastoma, these enzymes are overexpressed and are thought to contribute to the rapid and aggressive growth of cancer. HSPGs, particularly glypicans, are known to be overexpressed in glioblastoma. Here, we generated double and single knockouts of glypican‐3 (GPC3) and glypican‐1 (GPC1) in glioblastoma cells using CRISPR editing with specific sgRNA. Sanger sequencing and western blot determination were utilized to verify the knockout. We then compared these knockouts to unedited glioblastoma cells to understand the importance of glypicans in glioblastoma as well as investigate their sensitivity to platinum‐based chemotherapeutics. The further exploration of targeting HSPGs in general could allow for tailored chemotherapeutic treatments to patients with glioblastoma. This may pave the way for new molecular targets and more effective treatments for glial tumors in the future.Support or Funding InformationThe President's Commission for the Status of Women for funding my research. My collaborates Dr. Karin Forsberg Nilsson at Uppsala University, Sweden for donating the GL261 cells. Dr. Nicholas P. Farrell at the Massey Cancer Center, Virginia Commonwealth University, Richmond, VA for collaborating with me.Dr. Samantha Katner for her continuous support, encouragement, guidance and great mentorship
Loss of Caveolin‐1 Increases Macromolecule Transport Across Lymphatic Endothelium
Authors: Baranwal Gaurav M. , Upchurch Bradley D. , Cromer Walter E. , Smithhart Matt C. , Vadlamani Suman S. , Clark Mary-Catherine C. , Blais Stephanie N. , Zawieja David C. , Dongaonkar Ranjeet M. , Rutkowski Joseph M. 
Published: April 2020

Transport of fluid and macromolecules from the interstitium into lymphatic vessels is necessary for maintaining tissue homeostasis. Lymphatic capillary structure suggests that passive, paracellular transport would be the predominate route of macromolecule entry into lymphatics. Active transcellular transport, however, was recently identified to be equally important to solute flux across lymphatic endothelium in vitro. To specifically test if loss of caveolar transport impacted lymphatic transport, lymphatic conductance and permeability were measured in mice with a global deletion of caveolin‐1 (“CavKO”), the scaffolding protein necessary for caveolae formation, and in mice with a lymphatic endothelial cell‐specific deletion of caveolin‐1 (Lyve1‐Cre x Cav1flox/flox; “LyCav”). In each mouse line, lymphatic capillary architecture was largely unchanged. Using a quantitative microlymphangiography model, lymphatic conductance (mm3 fluid uptake/mm3 tissue/time) was measured to be significantly higher in CavKO mice. Increased lymphatic conductance was also measured in CavKO mice following nitric oxide synthase inhibition and in LyCav mice with lymphatic‐specific loss of caveolae. The permeability to 70kDa dextran was significantly increased in monolayers of lymphatic endothelial cells isolated from CavKO mice. These findings confirm that macromolecule transport is increased with loss of caveolar trafficking. Interestingly, macromolecule transport within the lymphatic system, measured by transit rate of dextran to a sentinel lymph node, was significantly reduced in CavKO and LyCav mice and isolated vessel studies identified increased permeability of CavKO collecting lymphatic vessels resulting in macromolecule loss from the lumen. These studies identify lymphatic caveolar biology as a key regulator of active lymphatic transport functions.Support or Funding InformationThis research was supported by the Texas A&M University College of Medicine and Department of Medical Physiology.
Disentangling The Heterogeneity Of The Midfacial Epithelium To Elucidate The Pathogenesis Of Cleft Lip/Palate
Authors: Llabata Marta Losa , Faure Louis , Chacon Brandon , Adameyko Igor , Selleri Licia 
Published: April 2020

Craniofacial development results from a tightly regulated choreography involving patterning, morphogenesis, and growth of multiple embryonic tissues and cell types. Clefts of the lip and/or palate (CL/P) are maiming disruptions of facial shape and represent the most common human craniofacial birth defect. During mammalian facial development, the maxillary prominence extends medially to contact the two nasal prominences (medial and lateral) at a three‐way anatomical seam named lambdoidal junction (λ), wherein the three prominences coalesce. When fusion of the facial prominences is impaired, orofacial clefting ensues. We have previously reported an essential role for the cephalic epithelium at the λ to enable normal fusion of the murine embryonic facial prominences. Using the mouse as a model, I am currently studying the spatial and temporal characteristics of these epithelial subpopulations using single‐cell RNAseq (scRNAseq) on microdissected facial prominences prior and after fusion occurs. By this approach, I have identified the transcriptional profiles that characterize the different epithelial subpopulations at the λ. Employing different bioimaging and fate mapping techniques, I am characterizing the cell behaviors and trajectories of selected λ epithelial subpopulations. In parallel, I am also conducting these analyses directly comparing control and mutant mouse embryos obtained from available genetic mutant lines with orofacial clefting (Pbx1/2 compound mutants). These studies have identified key changes in both transcriptomic signatures and cell behaviors in mutant mouse embryos, providing a deeper understanding of the molecular mechanisms underlying the pathogenesis of CL/P. Altogether, these approaches are helping me to elucidate the spatiotemporal dynamics of the cell subpopulations that comprise the λ epithelium in vivo, and the perturbed landscapes that cause disfiguring anomalies of the facial anatomical features in mammals.Support or Funding InformationThis work was funded by the National Institute of Health [RO1 grant DE024745 to L.S] and by the American Association of Anatomists [Postdoctoral Fellowship to M.L.].
Toxicokinetic of Sublethal Concentrations of Nitrite in Goldfish (Carassius auratus)
Authors: Martinez Melody Anaiz , Mancha Fabiola , Gomes Ana , Huertas Mar 
Published: April 2020

Increased agriculture and industrial activity have elevated the concentration of nitrogenous compounds in the terrestrial and aquatic ecosystems. Nitrogenous compounds can naturally occur in aquatic ecosystems, concentrations are controlled and maintained through the nitrogen cycle by bacterial denitrification/nitrification. As large quantities of nitrogenous compounds drain into streams and rivers it can dramatically alter the natural nitrogenous concentrations in aquatic ecosystems. Nitrite is an intermediate compound in the nitrogen cycle, and its concentrations can range from 1 mM to 0.5 mM in freshwater and marine ecosystems. Exposure to elevated nitrite concentrations can disrupt physiological and endocrine processes such as ion and hormone regulation, respiratory, and cardiovascular activity. However, there is a lack of research in the effects of nitrite on sensitive organs such as the olfactory epithelium. Fish olfactory systems is highly sensitive and can detect odors in the picomolar concentration. To detect odors, this specialized sensitive system must be continuously exposed to the environment, making it highly susceptible to environmental toxins. We hypothesize olfactory tissues will be more sensitive to long term exposure to sub lethal concentrations of nitrite. Thus, the goal of this study is to determine the chronic effects of nitrite and its accumulation in the olfactory system and vital organs in goldfish (Carassius auratus). To test our hypothesis, goldfish were exposed to nitrite concentrations (0 mM, 10.0 μM, 100.0 μM, and 1000.0 μM) for 69 days. The nose, brain, gill, liver, kidney, and blood samples were collected on the last day of exposure. Samples were flash frozen (20°C) for biochemical analysis and subsamples were fixed in Bouin or 4% Paraformaldehyde (PFA). After 18hr, subsamples were transferred to ethanol or sucrose, respectively. Sub‐samples of flash frozen brains were dissected, hypothalamus samples were collected for RNA analysis. Notable browning or darkening of blood was seen in goldfish from 100.0 μM, and 1000.0 μM treatments, indicating irreversible oxidation of hemoglobin known as methemoglobinemia. Biochemical analysis showed nitrite accumulated in the nose, brain, and gill tissues (1.0 μM ±0.3, 8.1 μM ±1.4, and 11.1 μM ±0.4, respectively) in 100.0 μM treatment. Nitrite concentrations in these tissues increased as treatment concentration increased. RNA samples were prepared for qPCR analysis to measure gene expression related to appetite. Preliminary histological analysis of nose, gill, liver, and kidney tissues shows structural changes in tissues. The biochemical results indicate environmental concentrations of nitrite accumulate in vital tissues. Furthermore, findings indicate nitrite is entering and accumulating in the brain. These results suggest, nitrite is potentially acting as a neurotoxin.Support or Funding InformationResearch support: Texas State University start‐up funding to Dr. Mar Huertas.