bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cyclophilin D Sequesters PPARα in Mitochondria to Impair Fatty Acid Oxidation in Cisplatin‐induced Acute Kidney Injury
Authors: Padanilam Babu J. , Noh Mi Ra , Ha LiGyeom , Kim Jinu , Jang Hee-Seong 
Published: April 2020

ObjectiveKidney is a highly metabolic organ and ATP depletion is critical to acute kidney injury (AKI). Mitochondrial fatty acid oxidation (FAO) is the preferred energy source of the kidney and is inhibited during AKI. A pivotal role for the mitochondrial matrix protein cyclophilin D (CypD) in regulating overall energy metabolism is being unraveled. We hypothesize that mitochondrial interaction between CypD and PPARα in renal proximal tubule (PT) where is highly susceptible to acute stimuli modulates FAO in cisplatin‐induced AKI (cisplatin AKI).MethodsUsing genetic and pharmacological intervention and protein‐protein interaction studies, we investigated whether PT‐CypD modulates FAO in cisplatin AKI through mitochondrial CypD‐PPARα binding and its sequestration.ResultsCisplatin injury resulted in histological and functional damage in the kidney, along with downregulated mitochondrial FAO, increased intrarenal lipid accumulation and declined ATP level. However, PT‐specific deletion of CypD protected the malevolent effects. Immunoprecipitation and BioID methods demonstrated mitochondrial translocation of PPARα and its binding to CypD and sequestration. This led to inhibition of nuclear translocation of PPARα and reduced transcription of FAO genes during cisplatin AKI. Genetic or pharmacological inhibition of CypD suppressed mitochondrial CypD‐PPARα binding in cisplatin AKI, preventing the impairment of FAO and intracellular lipid accumulation.ConclusionsThese results uncover a novel mechanism by which mitochondrial interaction between CypD and PPARα impairs FAO in cisplatin AKI. Targeting their interaction may be a potential therapeutic strategy to prevent energy depletion and cell death in AKI.Support or Funding InformationThis work was supported by NIH grants DK‐116987, DK‐120533 and American Heart Association (AHA) Grant in Aid 15GRNT25080031 (BJP), AHA postdoctoral fellowship Grant 15POST25130003 (HSJ), and grants (NRF‐2016R1C1B2012080 and NRF‐2019R1F1A1041410) from the National Research Foundation of Korea (JK).
Probing the unusual functional interaction between mGluR1 and mGluR5.
Authors: Kammermeier Paul J. , McCullock Tyler W. 
Published: April 2020

Metabotropic glutamate receptors (mGluRs) are class C G protein coupled receptors with widespread expression in the central nervous system. Unlike many GPCRs, mGluRs exist and function as obligate, disulfide linked dimers. While all mGluRs can form homodimers, and many can heterodimerize, we have recently published a paper demonstrating that while mGluR1 and 5 may reside in close proximity to each other, and can interact functionally, this interaction does not appear to be due to canonical heterodimerization. Pharmacological evidence supporting this conclusion is largely pharmacological. To ask whether the observed interaction was due to a physical interaction between receptors rather than integration of signaling effects, mutations in each receptor were introduced that uncouple the receptors from G protein signaling, presumably without affecting ligand binding or associated conformational changes. We found that mGluR1 co‐expressed with a double point mutant of mGluR5, Y64A F768D (which right‐shifted the glutamate response and uncouple from G proteins, respectively), the glutamate response of mGluR1 was right shifted. When mGluR1Y74A, a mutant with a 300 fold right‐shifted response, was expressed with mGluR5 F768D, the response was left shifted. Expression of the signaling dead mGluR5 F768D with mGluR1 also altered the pharmacological sensitivity profile, causing mGluR1 to lose sensitivity to 3‐MATIDA and gain sensitivity to MPEP. Together, these findings support the hypothesis that allosteric interactions between the receptors are responsible for the observed functional interactions.Support or Funding InformationSupported by an institutional Drug Targets and Mechanisms pilot award.
Role of Creatine Supplementation in Alleviating Hepatotoxicity Caused by Doxorubicin
Authors: Aljobaily Nouf A. , Viereckl Michael , Aljobaily Hend A. , Albertson Jammie A. , Han Yuyan , Hydock David 
Published: April 2020

Background and aimDoxorubicin is a chemotherapeutic agent that is widely used for several malignancies. It is known to have severe side effects such as hepatotoxicity, cardiotoxicity and increase in nitric oxide concentrations in skeletal muscles. On the other hand, creatine monohydrate is a supplementation used as a therapeutic agent and plays a significant role as regenerative agent. So far, there is not enough information about the effect of doxorubicin on the overall liver functionality and how creatine monohydrate helps in easing the hepatotoxicity; therefore, our aim is to investigate the role of creatine monohydrate supplementation in alleviating the hepatotoxicity caused by doxorubicin.MethodsSprague‐Dawley rats were fed rodent chow, 2% creatine, 4% followed by 2% creatine supplementation for our weeks; and 15 mg/kg doxorubicin was given once the day before they were sacrificed. Peripheral blood and liver were harvested and snap frozen. Serum chemistry was obtained to evaluate the liver function by looking at the levels of Lipemia, T‐Bilirubin, AST, ALP and ALT. Liver damage was evaluated by using Hematoxylin and Eosin staining; whereas liver fibrosis was evaluated by Sirius Red staining. RT‐PCR was performed to assess the gene expression of fibrotic genes, proliferative genes, oxidative stress and apoptosis‐related genes at the transcription level, and western blotting was performed to measure the gene expression at the protein level.ResultsThere was no significant difference found in the serum chemistry in the treatment groups when compared to the control group. However, the histology showed an increase in inflammation and fibrosis in the doxorubicin group when compared to the control, where there was a decrease observed in the group treated with 4% creatine followed by 2% creatine. A significant increase in the gene expression of Bax, CK‐19 and Col1‐a1 has been observed in the 2% creatine + doxorubicin. Further, there was a decrease in the protein expression of CASP3 in the 2% creatine + doxorubicin and 4%/2% creatine + doxorubicin when compared to the doxorubicin group.ConclusionBased on the collected data and observed behavior of groups, we conclude that the gradual dose of creatine monohydrate supplementation does alleviate the hepatotoxicity caused by doxorubicin.Support or Funding InformationGraduate Student Association at the University of Northern Colorado
Acute Pressor Responses in Mice Exposed to Early Life Stress
Authors: McPherson Kasi C. , Becker Bryan K. , Rhoads Megan K. , Jin Chunhua , Pollock David M. , Pollock Jennifer S. 
Published: April 2020

Exposure to adverse childhood experiences, also known as early life stress, is an independent risk factor for the development of cardiovascular disease in adulthood. Previous studies have indicated ELS in rodents may induce impaired blood pressure regulation in response to various pro‐hypertensive stimuli. In the current study, we used maternal separation and early weaning (MSEW), a mouse model of early life stress, to determine the effects of acute behavioral stress on blood pressure regulation (n = 9/group). We utilized exposure to cage switch stress (CSS), a model of acute behavioral stress that subjects mice to a cage previously inhabited by other male mice. MSEW involves maternal separation 4h/day from postnatal days (PD) 2–5 and 8h/day from PD 6–16 then weaned at PD 17. Normally reared litters weaned at PD 21 were used as controls (CON). In normally reared CON and MSEW C57/BL6 male mice, telemetry transmitters were surgically placed with catheters in the carotid artery at 9 weeks of age. After recovery from surgery for 7–10 days, baseline measurements were recorded for 72 hours. Continuous 24 hour systolic and diastolic blood pressures (SBP and DBP) were similar between CON and MSEW mice (SBP: 125 ± 1 vs 121 ± 5 mmHg, p = 0.64; DBP: 89 ± 1 vs 91 ± 1 mmHg, p = 0.24). Additionally, exposure to MSEW did not significantly affect baseline day and night heart rate. We then subjected to CSS for 4 hours. In response to CSS, the peak change in SBP was significantly greater in MSEW mice compared to CON (39 ± 2 vs 27 ± 4 mmHg, respectively, p = 0.02). However, the total pressor response determined by area under the curve for SBP and DBP during the 30 minutes following onset of CSS and 30 minutes following the end of the 4 hour CSS exposure were similar between MSEW and CON mice. Heart rate significantly increased in both MSEW and CON mice compared to baseline (MSEW: 777 ± 10 vs 562 ± 20 bpm, p < 0.0001; CON: 776 ± 4 vs 575 ± 25 bpm, p < 0.0001) and thus the peak change in heart rate was not significantly different between groups (215 ± 18 vs 201 ± 23 bpm, p = 0.64). However, area under the curve analysis indicated MSEW exhibited a blunted heart rate recovery 30 minutes following the end of the 4 hours of exposure to CSS compared to CON (17998 ± 159 vs 18751 ± 287, respectively, p = 0.05). Given the changes in SBP and heart rate following CSS, these data indicate that MSEW mice have an exaggerated pressor response during exposure to acute stress. In conclusion, ELS may predispose exaggerated cardiovascular responses to stress later in life.Support or Funding InformationFunded by NIH grants HL136267 and HL69999 to D.M.P. and J.S.P. and AHA grant 17SFRN33670697 to J.S.P.
Differential expression of renal urate transporters in male and female mice
Authors: Halperin Kuhns Victoria L. , Hoque Kazi Mirajul , Woodward Owen M. 
Published: April 2020

Elevated urate (UA) levels in the serum (hyperuricemia, HUA) can contribute to the development of many diseases, including kidney stones, chronic kidney disease, cardiovascular disease, metabolic syndrome, and gout, the most prevalent form of inflammatory arthritis. Men are 5‐fold more likely to have HUA than women, however, this risk increases in women 4‐fold after menopause. This suggests premenopausal women are protected against developing HUA, but the mechanisms have yet to be elucidated. These differences are echoed in our hyperuricemia mice models. Previously, we found insertion of the orthologous human pathological mutation Q141K (Q140K in mice) in the UA transporter ABCG2 causes significant hyperuricemia in the male mice but not in the female mice. Paradoxically, we also observed female mice have lower fractional UA excretion than males, suggesting complex differences in the renal handling between males and females. We hypothesized that these differences were likely due to either UA transporter expression or regulation. To better understand the gene expression architecture of UA transporters in the mouse kidney, we compared gene expression in male and female wild type (WT) and Q140K ABCG2 mice. RNA‐Seq was performed followed by DESeq2 analysis to determine differentially expressed genes between males and females of each genotype. Targets of interest included the 12 transporter and 3 transcription factor (TF) genes that associate with serum UA levels in humans in recent genome wide association studies. We found small (0.49 – 2.1‐fold) but statistically significant changes in all the UA associated transporter genes, except ABCG2and SLC2A9. Specifically, female mice had lower transcript expression of UA transporters SLC22A12, SLC22A6, SLC17A1, and ABCA1. These genes have variants that associated with lower UA levels in human populations, consistent with the female mice potentially more efficiently excreting surplus UA. Females also had significantly lower levels of the 3 key urate associated transcription factors HNF1A, HNF4A, and HNF4G, providing a potential mechanism of differentiation. Interestingly, the Q140K mice DESeq2 analysis reveals 486 alterations in male Q140K kidney gene expression as compared to WT, including 5 potentially compensatory SLC transporters. None of these changes were observed in female Q140K mice, indicating that changes are the result of the elevation in SUA and not due to mutant ABCG2 protein. These findings strongly suggest female sex may confer protection from developing HUA and associated conditions. Further understanding this mechanism should lead to improved understanding of UA homeostasis and new insights into HUA treatment.Support or Funding InformationNIDDK R01DK114091
Structure Activity Relationship Studies of Psychostimulant Synthetic Cathinones Reveal Nanomolar Antagonist Potency of α‐Pyrrolidinohexiophenone (α‐PHP) at Human Muscarinic M2Receptors
Authors: Chen Yiming , Canal Clinton E. 
Published: April 2020

Synthetic cathinones (SCs), psychostimulant drugs of abuse, are β‐keto analogs of amphetamine that primarily target monoamine transporters as inhibitors or substrates. Certain pyrrolidine‐containing SCs, such as α‐Pyrrolidinohexiophenone (α‐PHP) and methylenedioxypyrovalerone (MDPV), are potent monoamine transporter inhibitors that appear to produce distinct toxic, autonomic—e.g., tachycardia and arrythmia—and neuropsychiatric—e.g., delirium and cognitive impairments—adverse events compared to N‐alkylated SCs and amphetamines, which are monoamine transporter substrates. Although the apparent differences in toxicity could be caused by unique pharmacodynamic effects at monoamine transporters, we hypothesized there is another mechanism. We focused on muscarinic receptors (MRs), as numerous MR antagonists are well‐characterized deliriants that also cause tachycardia. We conducted pharmacological assays assessing human M1, M2, M3, M4, and M5R activity of 10 pyrrolidine‐containing SCs, systematically evaluating the effect of the α‐carbon chain length of methylenedioxy‐containing and unsubstituted α‐propiophenone analogues. [3H]Scopolamine competition binding assays revealed that increasing the α‐carbon chain length of unsubstituted, but not methylenedioxy derivatives led to a stepwise enhancement in affinity (Ki) at all MR subtypes, with highest affinities typically observed at M2Rs. Relative to all other SCs tested, α‐PHP had the highest affinity at M2Rs, with a calculated pKi of 6.28 (Ki= 520 nM). We next assessed the M2R functional effects of α‐PHP using M2R—cAMP and M2R—β‐arrestin recruitment assays; α‐PHP behaved as an M2R competitive antagonist in both assays. α‐PHP antagonized oxotremorine‐elicited cAMP inhibition with an apparent pKb of 7.29 (Kb= 50 nM) and antagonized oxotremorine‐elicited β‐arrestin recruitment with an apparent pKb of 6.22 (Kb= 602 nM). Toxicology studies report that serum concentrations of commonly abused pyrrolidine‐containing SCs, including α‐PHP, can range from 713 to 2,619 nM. Thus, α‐PHP’s potency at M2Rs is physiologically relevant and likely contributes to its toxicity. Given the dominant function of M2Rs to slow heart rate, α‐PHP may cause tachycardia, in part, by inhibition of cardiac M2Rs. This inhibitory effect on the parasympathetic nervous system, in combination with sympathomimetic effects caused by monoamine transporter inhibition could explain the serious adverse cardiovascular events associated with certain pyrrolidine‐containing SCs.Support or Funding InformationFunding: NIDA R21DA040907
Z‐line‐like Structures Are Distinct from α‐actinin‐rich Dense Bodies in Nematode Obliquely Striated Muscle
Authors: Ono Shoichiro , Nickoloff Emily , Ono Kanako 
Published: April 2020

Obliquely striated muscles are present widely in soft‐bodied invertebrates and differ from cross‐striated muscles in that thin and thick filaments are staggered and obliquely aligned. Distinct Z‐lines or Z‐discs are absent from obliquely striated muscles. In general, thin filaments in obliquely striated muscles are considered to be anchored at the dense bodies, although there is no direct evidence of a linkage between the ends of thin filaments and dense bodies. In this study, we used body wall muscle of the nematode Caenorhabditis elegans as a representative obliquely striated muscle and found that the barbed ends of sarcomeric actin filaments are not localized to the dense bodies, but instead aligned to linear Z‐line‐like structures. In C. elegans, α‐actinin (ATN‐1) is one of the major components of the dense bodies. However, sarcomeric actin, as probed by anti‐actin antibody, phalloidin, or GFP‐actin, was only marginally co‐localized with ATN‐1. To specifically localize the barbed ends of sarcomeric actin filaments, we expressed GFP‐tagged CAP‐1, an α subunit of heterodimeric actin capping protein. GFP‐CAP‐1 was localized in a striated pattern filling gaps between dense bodies but was not co‐localized with ATN‐1. These “Z‐line‐like” structures of GFP‐CAP‐1 assembled normally in atn‐1‐knockout worms, indicating that ATN‐1 is not required for linear alignment of the actin barbed ends. In atn‐1‐knockout worms, major cytoplasmic portions of the dense bodies are absent, and only moderate disorganization of sarcomeric actin filaments are observed (Mulder et al., 2010). However, RNA interference of cap‐1 caused larval lethality and strong disorganization of sarcomeric actin filaments in embryonic body wall muscle cells. These results indicate that the barbed ends of sarcomeric actin filaments are aligned to Z‐line‐like structures, which assemble independently of dense bodies, and that capping protein plays an essential role in early sarcomeric assembly.Support or Funding InformationSupported by NIH 5R01AR048615‐18.
The Cryptococcus neoformans capsule polysaccharide glucuronoxylomannan stimulates endothelial barrier permeability and the activation of Src and RhoA
Authors: Khan Mahrukh , Buttan Sagar , Masri Abigail , Martinez Luis , Dores Michael Robert 
Published: April 2020

The human pathogenic yeast Cryptococcus neoformans is capable of crossing the blood brain barrier (BBB), causing over 220,000 cases of fungal meningitis annually. The BBB is comprised of specialized endothelial cells that control the flow of nutrients, signaling molecules and pharmacological agents into the brain. We have identified a novel mechanism for C. neoformans endothelial barrier disruption through the shedding of its capsule polysaccharide glucuronoxylomannan (GXM). GXM stimulates the breakdown of adherens junctions between endothelial cells and stimulates actin stress‐fiber formation, however the molecular mechanisms that control these cellular responses are not known. GXM is proposed to activate toll like receptors expressed on the surface of endothelial cells. Here we demonstrate that exposure of human brain endothelial cells (HBECs) to GXM induces the activation and internalization of TLR‐2 and TLR‐4. In addition, GXM stimulation leads to the activation of Src kinase, a signaling factor that is known to stimulate VE‐cadherin internalization and adherens junction disruption. We show that treatment of HBECs with GXM activates RhoA, a signaling effector that regulates actin stress fiber formation. Our current research seeks to identify new components of these signaling pathways and to determine whether inhibition of these signaling pathways can prevent GXM‐mediated endothelial barrier permeability.Support or Funding InformationNIH NIAID: 1R01AI145559‐01A1
West‐Nile virus replicon particle entry requires the C‐terminal half of the carbohydrate‐recognition domain of the Dendritic cell‐specific ICAM‐3 grabbing non‐integrin related protein
Authors: Ogno Domenic G. , Menting Cody , Martinez Osvaldo 
Published: April 2020

West‐Nile virus (WNV) is an arbovirus usually transmitted to humans via a mosquito vector. Infections commonly result in febrile symptoms while rare severe neuroinvasive cases may result in encephalitis or meningitis. Studies have shown that WNV infection efficiency is enhanced by expression of DC‐SIGNR on target cells, which normally do not express DC‐SIGNR. To investigate WNV tropism, we established 293T kidney epithelial cell lines that stably express vector, DC‐SIGNR and mutants of DC‐SIGNR that lack the entire carbohydrate‐recognition domain (CRD) or lack the C‐terminal half of the CRD. We demonstrate successful surface expression of DC‐SIGNR and its mutants from stably‐transfected 293T cells, but not vector‐transfected 293T cells. Further, we show that monoclonal antibody 120604 which binds specifically to the DC‐SIGNR CRD binds to DC‐SIGNR expressing 293T cells, but not to vector nor any of the DC‐SIGNR mutants expressing cells. Virus replicon particles (VRPs), replication‐incompetent viral particles containing necessary structural proteins for infection and a viral plasmid including a GFP reporter are used to safely and conveniently study viral entry. Entry assays using WNV (NY99) VRPs as well as a variant of WNV (NY99) which contains the beta‐lactamase enzyme show significant entry into DC‐SIGNR expressing cell lines, but not in controls that do not express DC‐SIGNR. Additionally, we show that WNV VRPs do not enter DC‐SIGNR expressing cells that lack the CRD or the C‐terminal half of the CRD suggesting that the C‐terminal half of the CRD is required for successful entry of WNV via DC‐SIGNR. In the future, WNV particle entry into DC‐SIGNR mutation (150(STOP), S139A, S108A) expressing cells will be tested.Support or Funding InformationWinona State University student research and travel grants, Winona State University Foundation Special Projects
Comparison of Giemsa and Wright’s Stain For Differential Analysis
Authors: Gistover Noah , Randolph Tim 
Published: April 2020

Giemsa stain is available in most developing nations for malarial parasite detection. In the absence of Wright stain, Giemsa is also used to perform differential assessments. We are comparing Giemsa and Wright stains using buffers to determine the optimal staining protocol for differential analysis. We compared Giemsa with reverse osmosis drinking water (RO), deionized water (DI), buffer at pH 7.2, to Wright’s stain with RO and DI, using Modified‐Wright’s stain with buffer in an automated slide stainer as the control. Blood samples (n=3) were drawn from the research team and analyzed using these staining protocols. Wright’s stain with DI performed equivalent to control for white blood cell (WBC), platelets (PLT), and red blood cell (RBC). Undiluted Giemsa with DI showed good staining of WBCs and PLTs, and moderate staining of RBCs (grayish discoloration). Diluted Giemsa with buffer stained RBCs poorly (pale ghost cells) but stained WBC, and PLT well. Diluted Giemsa with DI demonstrated good staining of PLTs, poor staining of RBCs, and moderate staining of WBCs. Undiluted Giemsa with RO showed poor staining of RBCs with moderate staining of PLTs and WBCs. Diluted Giemsa with RO has poor staining of RBCs and WBCs, and moderate staining of PLTs. Wright with RO stained RBCs, WBCs, and PLTs poorly. We recommend using Wright stain with DI water for differential analysis but undiluted Giemsa with DI water is an adequate substitute.Support or Funding InformationNG is supported by the DeNardo Education and Research Foundation grant.