bluuebunny/crossref_metadata_2025_split huggingface dataset:
Urinary Concentrating Defect Contributes to Polyuria in a Mouse Model of Cystinosis
Authors: Amlal Hassane , Dong Fei , Kao Winston 
Published: April 2020

Cystinosis is a rare inherited disorder caused by mutations in the CTNS gene, which encodes the lysosomal cystine transporter, cystinosin. Both humans and cystinosin deficient mice exhibit significant polyuria, which is attributed to proximal tubule fluid reabsorption defect. However, whether cystinosis is also associated with impaired urinary concentrating ability of the kidney remains elusive.We have developed a new mouse model of cystinosis using an insertional mutation generated by CRISPR gene editing techniques of fertilized eggs of C57/BL6 mice. Homozygous mutant (Cyst−/−) mice exhibited cysteine crystals in the cornea within 4 weeks of age, as compared to Wild‐type (WT) mice. Wild‐type, Cyst−/− and heterozygous (Cyst+/−) mice were placed in metabolic cages with free access to rodent chow and distilled water and tested for renal phenotype at 5, 12 and 20 months. To assay the activity of the proximal tubule, the animals were given a single injection of acetazolamide (ACTZ) in a 24hour period, or subjected to metabolic acidosis using 280 mM NH4Cl loading for 5 days. The diuretic effect of ACTZ and the adaptation to chronic metabolic acidosis in terms of urinary ammonium (NH4+) excretion level were similar between all three genotypes at 5 and 12 months of age. Further, the baseline data for water balance and urine osmolality were also similar among the three genotypes at 5 and 12 months of age. However, at 20 months, Cyst−/− mice exhibited a steady‐state significant polyuria, polydipsia and reduced urine osmolality, as compared to WT or Cyst+/− mice. Immunoblotting and immunofluorescence studies showed that the impaired water balance correlated with a significant reduction in AQP2 protein abundance throughout the kidney regions in Cyst−/− vs. WT or Cyst+/− mice. AQP1 and NKCC2 expression was not altered, whereas AQP4 protein was rather significantly upregulated with a shift in the molecular size of the protein in Cyst−/− vs. WT or Cyst+/− mice. When the animals were treated with a vasopressin V2 receptor specific analog (dDAVP), urine osmolality increased sharply in WT and Cyst+/− but remained unchanged in Cyst−/− mice. Blood Chemistry analysis showed a ~3‐fold increase in BUN levels, hyperkalemia, metabolic acidosis, and lower hematocrit and hemoglobin in Cyst−/− vs. WT or Cyst+/− mice, indicating the onset on renal disease in mutant homozygous mice.In conclusion, Cyst−/− mice exhibited urinary concentrating defect, which is associated with vasopressin resistance of the collecting duct system, and manifested by the reduction in the protein abundance of the apical water‐absorbing channel AQP2. We propose that the impaired water balance and the proximal tubulopathy both contribute to massive volume depletion, which progressively lead to renal injury in cystinosis disease.Support or Funding InformationDialysis Clinic, INC. and NIDDK RO1‐DK‐083582 for H. Amlal and Cystinosis Research Foundation for W. Kao.
Inhibition of Epstein‐Barr Virus by Serotonin‐Dopamine Antagonists
Authors: Gorres Kelly , Hoffman Nate 
Published: April 2020

Epstein‐Barr Virus (EBV), a herpesvirus family member, was the first human cancer‐associated virus discovered. EBV maintains a lifelong latent infection within the host B cells. During periodic reactivation into the lytic phase, the virus replicates and spreads among cells. The virus is reactivated into the lytic phase in response to environmental stress conditions. In cultured EBV‐positive cells, the lytic switch can be initiated by the addition of a variety of small‐molecule inducing agents. Viral reactivation is inhibited by diverse natural products and pharmaceuticals. Viral reactivation can be induced, or blocked, through a number of different mechanisms. Since one viral inhibitor is also a drug used to treat neurological conditions, we investigated the response of the virus to atypical antipsychotic drugs. Previously, we discovered clozapine is an inhibitor of EBV reactivation. Here we screened a panel of atypical antipsychotic drugs for effects on the expression of the viral lytic gene BZLF1. We found antagonists of serotonin and dopamine receptors inhibited the EBV lytic cycle in Burkitt lymphoma cells. The inhibitors may be functioning through their associated receptors or through any of their off target signaling pathways. These results highlight the lesser known roles of serotonin and dopamine signaling outside the central nervous system. Inhibition of EBV through novel mechanisms will be useful in elucidating the natural pathways of viral reactivation and in the treatment of EBV‐associated diseases.Support or Funding InformationThis work was funded by the UWL College of Science and Health and UWL Faculty Research Grants to KLG.
A Comparison of Spherical Harmonic and Sliding Semilandmark Analyses as Methods for Three‐Dimensional Shape Evaluation
Authors: Goldstein Deanna M. , Harper Christine M. , Sylvester Adam D. 
Published: April 2020

The ability to quantify morphological variation is integral to biological research. The use of sliding semilandmarks in geometric morphometric (GM) analyses allows for the evaluation of whole bone morphology in three dimensions. A sliding semilandmark analysis, however, is difficult to complete when structures lack obvious homologous features for landmark placement. Spherical harmonics (SPHARM) offers an alternative method for shape analysis in such cases. Here we evaluate the SPHARM method by comparing results to those from a sliding semilandmark analysis of the calcaneus in African apes and modern humans.Modern human (n=20), chimpanzee (n=20), and gorilla (n=20) calcanei were surface scanned. For the GM analysis, 1007 sliding semilandmarks were evenly distributed across the external surface of each calcaneus. Semilandmarks were slid to minimize the bending energy of the thin plate spline interpolation function relative to an updated Procrustes average. Final landmark configurations were then aligned using Generalized Procrustes Analysis. For the SPHARM analysis, surface models were mapped onto the surface of a sphere (parameterization), and then decomposed into a weighted sum of spherical harmonic functions. Because the spherical harmonics provide a standardized basis for quantifying shape, coefficients of the harmonic functions can be used as shape descriptors. Calcaneal shape variation was summarized using a principal components (PC) analysis of the Procrustes coordinates and SPHARM coefficients. Analyses were compared through calculation of the root mean square (RMS) and maximum distance between surface models of the average specimen from each species, as well as surface models of PC warps created two standard deviations from the average along PCs 1–3.Both sliding semilandmark and SPHARM analyses separate species along the first two PCs. For both analyses, African apes and modern humans separate along PC1. Average RMS between species averages was 0.25 mm. Maximum surface distances between average surface models from the two analyses were all less than 0.23 mm. Average RMS between all respective PC warps was 0.82 mm. Maximum surface distances between PC warps were all less than 1 mm. Although there were minor differences between PC plots produced from the two analyses, the same general morphological distinctions between species were identified. The primary differences between SPHARM and sliding semilandmark‐based surface models of PC warps and species averages occur around sharp edges, which are less defined using the SPHARM analysis, although these differences are small. Overall, SPHARM provides the same shape representation as a sliding semilandmark analysis, making it a comparable method for the evaluation of shape in three dimensions.Support or Funding InformationThis project was supported by NSF grant # BCS ‐ 1824630.
Reducing Turbulent Airflow Lowers Healthy Females Work Of Breathing During Exercise To A Level Similar To Males
Authors: Granger Emily A. , Mann Leah M. , Chan Jason S. , Yu Annie , Dominelli Paolo 
Published: April 2020

ObjectiveImaging studies have shown that healthy females have smaller conducting airways than height matched males. The smaller conducting airways would increase the propensity towards turbulent airflow and this is thought to be the underlying mechanism behind greater resistive and total work of breathing in females. Heliox (21% O2: balance He) is considerably less dense than room air and has a greater propensity towards laminar flow. As such, respiring heliox should produce a physiologically similar effect as increasing airway size and allows for experimental manipulation of work of breathing between the sexes. The objective of the study was to determine if turbulent flow is responsible for the greater work of breathing in healthy females.MethodsSix healthy men (Age=24±4 years) and five healthy women (Age=22±2 years) completed two step‐wise maximal exercise tests on separate days. On one day subjects respired compressed room air and on the other a compressed helium‐oxygen mixture (~21% O2: balance He). Room air and heliox were humidified and delivered in an identical manner. Esophageal pressure was measured with a balloon catheter and allowed for the determination of the work of breathing via integration of transpulmonary pressure‐volume loops. The work of breathing‐ventilation relationship was modelled using a previously described equation consisting of two constants that describe the mechanical work to overcome i) viscous resistance and laminar flow and ii) turbulent resistance.ResultsEnd‐exercise ventilation was significantly greater for females during the heliox trial (82±9 vs. 94±11 L min−1, P=0.02) but there was no difference in work of breathing (122±38 vs. 116±42 J min−1, P=0.45), for room air and heliox respectively. Using the modeled work of breathing data, compared to males, females had a significantly greater work of breathing at ~70 L min−1 during the room air trial and this was due to greater work to overcome turbulent resistance. When comparing the heliox trials between sexes, there was no difference in the work of breathing. Similarly, after ~60 L min−1 compared to the room air trial, the work of breathing among females had significantly lowered during the heliox trial and this was due to a lower contribution of turbulent resistance.ConclusionOur results suggest that increased turbulent flow, as a result of smaller conducting airways, is responsible for the greater work of breathing in healthy young females during exercise.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada RGPIN‐2019‐04615 (PBD). Canada Foundation for Innovation #38432 (PBD).
Cerebrovascular Impairment Due to Maternal Electronic Cigarette Usage During Pregnancy is not Dose Dependent
Authors: Burrage Emily N. , Aboaziza Eiman , Reppert Sarah A. , O’Reilly Juliana , Hare Lane A. , Chantler Paul D. , Olfert I. Mark 
Published: April 2020

Developmental harm to offspring from maternal exposure to electronic cigarettes (E‐cig) during pregnancy is still poorly understood. Similar to cigarettes, we hypothesized the even low‐level E‐cig exposure would produce similar evidence of cerebral vascular dysfunction compared to a higher exposure dose. We examined the effects of maternal E‐cig exposure (Joyetech eGrip OLED using 5‐sec puffs @17.5 W) on cerebrovascular function in offspring (n=2–4 from each dam) from Sprague‐Dawley rat dams exposed to air (Control), E‐cig with 18 mg/ml nicotine (E‐cig18) and without nicotine (E‐cig0). Dams were exposed to low (20 puffs) or high (60 puffs) dose for 1‐hour each day, 5 days/week, starting on gestational day 2 and continued until pups were weaned. Pups themselves we never directly exposed to E‐cig aerosol. Middle cerebral arteries (MCA) were obtained from pups at 3‐months of age, isolated and positioned in a pressurized myobath, and exposed to increasing concentrations of acetylcholine (ACh; 10 ‐9 M to 10 ‐4 M), serotonin (5‐ HT;10 ‐9 M to 10 ‐4 M), and sodium nitroprusside (SNP; 10 ‐9 M to 10 ‐4 M), and in the presence or absence of Tempol (a superoxide dismutase mimetic). At the lower dose, MCA dilation to ACh was impaired by 60±1% and 50±0.1% in E‐cig0 and E‐cig18 groups, respectively, compared to controls (p<0.05). At the higher dose, MCA dilation to ACh was similarly impaired by 63±1% and 60±0.2% in E‐cig0 and E‐cig18 groups, respectively (p<0.05). Incubation with tempol reversed the cerebrovascular dysfunction seen in both E‐cig groups at both doses, suggesting the superoxide pathway is involved in the impairment observed in offspring. At both low and high doses, the vasocontrictory response to 5‐HT, and dilation response to SNP (non‐endothelial‐dependent mechanism), were similar across all groups. These data show that maternal E‐cig usage results in similar cerebrovasculature impairment with either 20 or 60 puffs/day during pregnancy and lactation in adolescent offspring with prior in utero exposure from maternal vaping at suggesting that even low‐levels of maternal E‐cig use confers significant post‐natal vascular health risks for the offspring.Support or Funding InformationWVU Cancer Institute Philip R Dino Innovative Research Grant (IMO); APS STRIDE Fellowship (JO); NIHGMS 5U54GM104942‐03 (PDC)
Inflammation and oxidative damage as risk factors of neurodegeneration after Traumatic Brain Injury in young and aged mice
Authors: Cordaro Marika , Remigante Alessia , Crupi Rosalia , Morabito Rossana , Marino Angela 
Published: April 2020

Traumatic Brain Injury (TBI) causes transitory or permanent structural and functional impairments and may increase the risk of neurodegeneration. As age correlates with development of neurodegenerative disease after TBI, the aim of the present study was to verify the possible onset of neurodegeneration after TBI and the related pathways, namely associated to oxidative stress and neuroinflammation in both young (3 months) and aged mice (12 months). TBI was induced in mice by controlled cortical impact and behavioral tests (open field, elevated plus maze, novel object recognition, Morris water maze) performed after 30 days and 3 months of trauma. Thereafter, the animals were sacrificed and the brains processed for Western blot and immunohistochemical analyses. In particular, IL‐6, IL‐8 and TNF‐a levels accounting for inflammation and malonildialedhyde (MDA), reduced glutathione (GSH) and superoxide dismutase (SOD) levels accounting for oxidative damage have been monitored. With regard to behavioural anaylis at both time intervals after TBI, aged animals exhibited a more pronounced anxiety‐like behavior along with impaired memory than what estimated in young animals. Moreover, a strong increase in neuroinflammation, shown by increased levels of cytokines, and oxidative damage, attested by increased MDA and GSH along with decreased SOD quantity suggested that neuroinflammation and oxidative stress damage were more significantly exhibited by aged than young mice.The present study suggests that aging may exacerbate the damage deriving from TBI and increases the risk of neurodegeneration due to oxidative stress and inflammation response, with specific regard to anxiety‐like behavior. Further studies may be useful to monitor risk factors of neurodegenerative diseases over a longer time lapse after TBI, namely in aged animal, in order to add more knowledge about development of neurodegeneration.Considering the complex multifactorial nature of neurodegenerative diseases, a preventive strategy able to simultaneously target multiple risk factors and disease mechanisms at an early stage is most likely to be effective to slow/halt the progression of neurodegenerative diseases.
The Impact of Chromatin Structure on the Formation of Double Strand Breaks by CRISPR‐CAS9 in the Yeast Genome
Authors: Heitmiller Courtney Anne , Burgess Rebecca C. 
Published: April 2020

DNA is persistently at risk for being damaged by endogenous and exogenous factors. One type of DNA damage is a double‐strand break (DSB), which occurs when both strands of the helix become severed. DSBs are important because upon repair, they could lead to mutations or genomic alteration. The packaging of the DNA into chromatin may affect the susceptibility of an area to DSBs and thus could make the area more prone to mutation. The purpose of this study is to look at how the chromatin environment affects the susceptibility of genes to DSBs in Saccharomyces cerevisiae. A new system, tetracycline‐inducible CRISPR‐Cas9, was used in this experiment to induce DSBs at different sites in the yeast genome Histone modification levels were measured at each site to correlate these chromatin marks with DSB formation frequency. DSB frequency at each genomic locus was measured using quantitative PCR and directly compared to those same sites in unchromatinized DNA to see how chromatin environment and the surrounding histone modifications affects susceptibility to DSBs.To measure the frequency of DSBs at different genomic sites using qPCR, the break site was amplified. DSBs would be indicated through a decrease in product when compared to an uncut control site NFS1 on Chromosome III. While correlation analysis of histone modifications and DSB frequency is still ongoing, preliminary data suggests that repressed genes have higher levels of cutting than active genes after three hours of DSB induction. Of the repressed genes investigated, PPE1 had 82.06% DSB formation and RRT8 had 66.67% after three hours of induction. Active genes RPL22A, TDH2, and TDH3 had on average about 15%, 9.5%, and 56% DSB formation after three hours of DSB induction, respectively. The level of DSB formation in these sites will be directly compared to DSB formation of the same sites on an unchromatinized plasmid, which is currently underway.This research is important because it shows which areas of the genome may be at higher risk for DSBs and whether or that is affected by chromatin structure. Understanding this could help deepen the knowledge of how the genome evolves through spontaneous DSB formation and repair.
Phosphorylation and function of OATP1B1 with tyrosine kinase inhibitors.
Authors: Hayden Elizabeth R. 
Published: April 2020

IntroductionThe organic anion transporting polypeptide OATP1B1 regulates hepatic uptake of many therapeutics. As a result, drug‐drug interactions (DDI’s) are commonly mediated by OATP1B1 activity. Reduced drug elimination via this transporter can lead to increased rates of drug‐associated adverse reactions, such as statin‐induced rhabdomyolysis. Surprisingly, numerous tyrosine kinase inhibitors (TKIs) that are either weak or non‐substrates of OATP1B1 increase systemic concentrations of various known OATP1B1 substrates by an unknown mechanism. We hypothesize that OATP1B1 function is dependent on tyrosine phosphorylation, which can be reduced by specific TKIs.MethodsPrimary human hepatocytes and HEK293 cells transfected with OATP1B1, or tyrosine (Y) to phenylalanine (F) mutant variants of OATP1B1, were used to assess the uptake of 8‐[2‐[Fluoresceinyl]‐aminoethylthio)‐adenosine‐3′,5′‐cyclic‐monophosphate (5–40 μM) or estradiol‐b‐glucuronide (2 μM) in the presence or absence of FDA approved TKIs. A biotinylation labeling assay was utilized to measure surface expression of OATP1B1 treated with and without nilotinib (0–20 μM). Phosphorylation of OATP1B1 was assessed by Western blot and mass spectrometry‐based proteomic analysis. OATP1B1 protein was purified by immunoprecipitation from overexpressing cell lines in the presence or absence of nilotinib. siRNA was utilized to identify kinases that potentially mediate OATP1B1 function. Statistical analysis was performed with Graph‐pad Prism, and each experiment was repeated in triplicate.ResultsNine of 46 FDA approved TKIs inhibit OATP1B1 function over 80%, compared to untreated controls. Nilotinib, the most potent TKI among assessed, does not decrease OATP1B1 expression or membrane localization. Instead, it elicits inhibition of OATP1B1 through a non‐ or mixed‐competitive and reversible mechanism. Nilotinib also reduces OATP1B1‐mediated uptake in primary human hepatocytes (P < 0.001). Mass spectrometry analysis of OATP1B1 revealed a variety of tyrosine phosphorylation sites, among them, phosphorylation at position 625 is greatly reduced in the presence of nilotinib. Moreover, mutation of Y625F results in significantly reduced transport activity compared to the wild‐type OATP1B1 activity (P < 0.001). Finally, we confirmed that SiRNA knocking down of LYN kinase activity, a Src tyrosine kinase, significantly reduces OATP1B1 activity (P < 0.001).ConclusionWe found that OATP1B1 is tyrosine phosphorylated at numerous sites and that position 625 is highly sensitive to nilotinib exposure. The Y625F mutation of OATP1B1 results in decreased transport activity and similarly, loss of LYN expression also leads to lower OATP1B1 transport. These findings indicate that clinically relevant TKIs may promote drug‐drug interactions by interfering with a post‐translational event that is required for OATP1B1‐mediated transport.Support or Funding InformationUniversity at Buffalo School of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, Seed Support Program
STING Activation and its Role in IFN Production
Authors: Link Tina , Rajput Aashita , Kona Abhinav , Lee Chase , Sheth Heli , Vemana Riya , Wright Aryn 
Published: April 2020

The immune system’s ability to respond to foreign DNA is a highly conserved mechanism. One crucial part of this mechanism is Stimulator of Interferon Genes (STING), a central innate immune protein located in the endoplasmic reticulum. It is a pattern recognition receptor that is activated in response to intracellular infection and, ultimately, mediates type 1 interferon (IFN) production. Encoded by the gene TMEM173, STING is expressed in T‐cells, monocytes, natural killer cells, and dermal fibroblasts.. It is composed of four transmembrane helices and a cytoplasmic ligand binding and signaling domain, which form a domain‐swapped dimer assembly. The STING pathway is initiated when double‐stranded DNA (dsDNA) is sensed, causing cyclic‐GMP‐AMP‐synthase (cGAS) to cyclize GTP and ATP and produce 2′3′‐cyclic‐GMP‐AMP (cGAMP), the main ligand that activates STING. Upon STING activation, the ligand‐binding domain closes, inducing a 180 degree rotation of the ligand‐binding domain relative to the transmembrane domain. The C‐terminal tail of STING has been found to autoinhibit the protein by blocking the polymerization interface of inactive STING, preventing auto activation. The conformational change made in response to cGAMP releases the CTT, exposing the polymer interface and allowing disulfide link formation. Alternatively, STING can be activated by bacterial cyclic‐di‐GMP in a cooperative manner, with cyclic‐di‐GMP serving as a partial antagonist of the cGAMP signaling pathway. Structural and biochemical studies have provided elegant insight into the mechanism of differential of STING by cGAMP and cDi‐GMP. STING activates tank binding kinase 1 (TBK1) which phosphorylates transcription factor IRF‐3 and causes it to translocate to the nucleus. Gain‐of‐function mutations of TMEM173 causing elevated transcription of IFNs have been proven to cause autoimmune syndromes such as systemic lupus erythematosus and, most recently, three mutations of TMEM173 have been found to directly cause STING‐associated vasculopathy with onset in infants (SAVI). New research proposes a therapeutic strategy that uses molecules that induces a different STING conformation than cGAMP‐bound STING as a STING inhibitor. This method would prevent constitutive STING activation in autoimmune syndromes while being less immunosuppressive than previously proposed therapies involving STING inhibition. The Walton High School MSOE Center for Biomolecular Modeling SMART Team has designed a 3‐D model of STING using a recently published cryo‐electron microscopy structure that reveals the transmembrane domain structure to further investigate and examine the structure‐function relationship of STING.Support or Funding InformationMSOE Center for Biomolecular Modeling