bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structural Studies of the RhoGEF Trio as a Target in Uveal Melanoma
Authors: Bandekar Sumit , Barton Brenna L. , Tully Ena S. , Tesmer John J.G. 
Published: November 2023

The C‐terminal Guanine Exchange Factor (GEF) domain of the RhoGEF Trio (TrioC) has recently been implicated in highly metastatic cancers including uveal melanoma and adult T‐cell leukemia. Upon aberrant activation, TrioC transduces growth and motility signals through the small GTPase RhoA. The regulation of TrioC is not understood on a molecular level and this presents a barrier to future inhibitor design efforts. Preliminary biochemical studies have shown that TrioC is regulated via an autoinhibitory constraint that is released upon the binding of the heterotrimeric G‐protein Gαq. I have solved the 2.7Å crystal structure of TrioC in its basal state, revealing high‐resolution detail of the autoinhibition mechanism. Analysis of this structure in comparison to the activated state of a closely related protein, p63RhoGEF reveals a dramatically different orientation of key regulatory regions. Mutational analysis of these regulatory regions are underway using FRET‐based activity assays and differential scanning fluorimetry. Analysis of current data supports that residues in the α6‐αN linker helix and β3‐β4 loop regions are indeed responsible for stabilizing the autoinhibited state of TrioC. A Trio inhibitory peptide (TRIPα) represents the only known inhibitor of TrioC. I have expressed the peptide as an MBP fusion and am attempting to determine a co‐crystal structure of TRIPα in complex with TrioC.Support or Funding Information5T32GM007767‐38, Rackham pre‐doctoral fellowship awarded to S. Bandekar UROP Summer fellowship awarded to B. Barton
Electrostatic Regulation of Phospholipase C β
Authors: Esquina Candi , Rugema Ngango Yvon , Garland‐Kuntz Elisabeth , Lyon Angeline 
Published: November 2023

Phospholipase C (PLC) is an enzyme class that cleaves phosphatindylinositol lipids from cellular membranes to produce second messengers, including inositol‐1,4,5‐triphosphate (IP3) and diacylglycerol (DAG). The PLCβ subfamily is among the best characterized of the PLC super family and is crucial for normal cardiovascular function. Under basal conditions, PLCβ has very low basal activity and is partitioned between the plasma membrane and the cytoplasm. However, it is still unclear how this low activity is maintained from a molecular standpoint. One known autoinhibitory element is the X–Y linker, a loop within the catalytic domain that forms a lid over the active site and participates in interfacial activation. Autoinhibition is thought to require a highly conserved acidic stretch within the linker, as deletions encompassing this region, or the entire linker, result in increased basal activity, decreased efficacy of G protein activation, and decreased thermal stability. These results, together with previous structural studies, suggest that the acidic stretch may regulate activity in part through interactions with conserved basic residues on adjacent surfaces of the catalytic domain. We are using site‐directed mutagenesis, functional assays, and structural studies to establish the contribution of electrostatic interactions to understand how these interactions play a role in the regulation of PLCβ.Support or Funding InformationPurdue University start‐up funds
ERK1/2 Activation Following Renal Injury Is Required For Kidney Injury Molecule‐1 Production
Authors: Collier Justin Brandon , Whitaker Ryan , Schnellmann Rick G 
Published: November 2023

BackgroundBecause substantial damage can occur before detection of acute kidney injury (AKI) new early predictor biomarkers are needed, such as kidney injury molecule‐1 (KIM‐1). KIM‐1 is a transmembrane glycoprotein that is expressed at very low levels in healthy kidney and is highly upregulated during injury. KIM‐1 is shed during injury and can be detected in blood and urine. While KIM‐1 has been studied in various models of AKI, the regulatory mechanisms leading to KIM‐1 upregulation remains limited. Here, we explored the role of ERK1/2 activation on KIM‐1 expression following toxicant exposure to proximal tubule cells and renal ischemia reperfusion injury (IRI) and LPS‐induced sepsis in mice.MethodsMouse kidney proximal tubule cells (TK) were treated with varying concentrations of hydroxyurea (HU), hydrogen peroxide (H2O2), and tert‐butyl‐hydroperoxide (TBHP) with and without pretreatment with the MEK1/2 inhibitor trametinib (10 nM). Trametinib (1mg/kg) was administered 1 hr before renal IRI to C57Bl/6 mice and were euthanized at 3 hr and 24 hr. TLR4 KO and WT mice underwent 3 hr renal IRI or septic AKI for 18 hr using LPS (10 mg/kg) injection. Signaling pathways were explored using RT‐qPCR and immunoblot analysis and kidney function was measured using BUN and serum creatinine.ResultsHU, H2O2, and TBHP exposure to TK cells for 24 hr caused ERK1/2 phosphorylation and upregulated KIM‐1 mRNA. Pretreatment with trametinib prevented KIM‐1 up‐regulation and ERK1/2 phosphorylation at 24 hr. At 3 hr post renal IRI KIM‐1 mRNA increased 4‐fold while pretreatment with trametinib resulted in a 2‐fold increase in KIM‐1 mRNA. Serum creatinine increased 4‐fold 3 hr after IRI and was attenuated by trametinib pretreatment. Cortical KIM‐1 mRNA increased 600‐fold 24 hr after IRI and was reduced 66% using trametinib in IRI. KIM‐1 protein was observed at 24 hr but not at 3 hr post IRI. Trametinib decreased KIM‐1 protein in the kidney 24 hr post IRI. TLR4 KO mice subjected to IRI exhibited increased KIM‐1 mRNA and ERK1/2 phosphorylation after 3 hr. In contrast, TLR4 KO mice exposed to LPS did not exhibit increased KIM‐1 mRNA or protein, nor activated ERK1/2 after 18 hr. WT mice treated with LPS had increased KIM‐1 mRNA and protein and ERK1/2 phosphorylation.ConclusionWe have linked pathophysiological ERK1/2 phosphorylation to the regulation of KIM‐1 within the kidney cortex. ERK1/2 phosphorylation and KIM‐1 mRNA increased following toxicant exposure in TK cells, IRI in mice, and LPS in mice, and trametinib attenuated these increases. LPS‐induced AKI required TLR4 for ERK1/2 activation and subsequent upregulation of KIM‐1. In contrast, IRI mice did not require TLR4 for ERK1/2 activation. These results demonstrate that ERK1/2 is a key initiator of renal injury.Support or Funding InformationGM084147 ‐ RGSF31DK105782 ‐ JBC
Novel Regulation of IGF1R by Ubiquilin1
Authors: Kurlawala Zimple , Gosney Julie , Ceresa Brian P , Beverly Levi 
Published: November 2023

BackgroundInsulin‐like Growth Factor‐1 Receptor (IGF1R) is a receptor tyrosine kinase ubiquitously expressed on cell surfaces of all tissues and is activated upon binding to its ligands (IGF1, IGF2, insulin). Dysregulation of the IGF/IGF1R axis has been implicated in disorders of metabolism, growth and development, progression of cancers like lung and breast and even development of resistance to chemotherapy. Currently, this axis is a major target of research for chemotherapy and other metabolic disorders. However, pharmacological antagonists of IGF1R have failed to show significant benefit in overall patient population. One of the causes is that IGF1R expression and activity does not always correlate with disease progression and response to therapy. This highlights the crucial role of cellular and extracellular factors that modulate activity of IGF1R directly or indirectly. We have identified Ubiquilin1 as a novel interacting partner and regulator of IGF1R. Ubiquilins (UBQLN) are newly discovered proteins and are studied largely in the context of neurodegenerative disorders. Our laboratory recently showed that UBQLN1 is lost in about 50% of non‐small cell lung cancers, indicating a link between UBQLN1 expression and tumorigenesis.HypothesisUBQLN1 regulates expression and activity of IGF1R.MethodsWe characterized regulation of IGF1R by UBQLN1 using the following methods – co‐immunoprecipitation, sub‐cellular fractionation, immunofluorescence, radioligand saturation binding, qRT‐PCR and Western Blot analyses.Results1) UBQLN1 interacts with both phosphorylated and non‐phosphorylated IGF1R. 2) UBQLN1's primary association with IGF1R is through its chaperone‐like region (STI domains) and there exists a secondary association through its UBA (ubiquitin‐associated) domain, which leads to stabilization of IGF1R. 3) Following loss of UBQLN1 in lung adenocarcinoma cells, there is decreased expression of IGF1R at the mRNA and protein levels. 4) Fewer receptors are available for ligand binding on plasma membrane of UBQLN1 deficient cells. 5) The ratio of active: inactive (phosphorylated: non‐phosphorylated) IGF1R is significantly higher in cells that have loss of UBQLN1 expression.ConclusionsWe conclude that UBQLN1 is critical in normal regulation of IGF1R. Loss of UBQLN1 leads to persistent stimulation of IGF1R which may contribute to transforming events in UBQLN1 deficient cells. Understanding the role of UBQLN1 in regulating receptor tyrosine kinase activity can facilitate development of pharmacological drugs targeting novel pathways in cancers as well as metabolic diseases.Support or Funding InformationR01CA193220
Flow cytometry‐based quantification of cyclic AMP in primary human neutrophils.
Authors: Chinn Amy , Michkov Alexander , Insel Paul A , Corriden Ross 
Published: November 2023

Cyclic 3′, 5′adenosine monophosphate (cyclic AMP, cAMP), a ubiquitous second messenger molecule, plays a central role in signal transduction, a large number of cellular functions and drug responses. Although several methods are used to quantify cellular cAMP, such approaches often lack sensitivity and can be difficult to apply to primary cells. Furthermore, currently available assays often require cell lysis and/or large cell numbers to generate readily detectable signals. Here, we describe a fluorescence‐based flow cytometry approach that enables quantification of cAMP even at low cell numbers. In this method, cells are fixed and permeabilized prior to staining using a primary antibody against cAMP and a fluorescent secondary antibody. Use of this method can quantify cAMP levels in freshly isolated human neutrophils, key mediators of the innate immune response and the most abundant phagocytic leukocytes in circulation. The method reproducibly detects and quantifies basal cAMP levels, in addition to cAMP stimulated by forskolin or agonists/antagonist of several Gs/Gi‐linked G protein‐coupled receptors. This approach, in addition to providing a rapid and simple new strategy for the quantification of cAMP, has the potential to facilitate receptor occupancy‐response relationships in terms of second messenger levels and other readouts that can be detected by flow cytometry.Support or Funding InformationSupported by NIH grants R56AI110505, T32HL098062 and T32GM007752.
Derisking Renal Tubular Toxicity Identified During Early Compound Screening
Authors: Mantena Srinivasa Raju , Buck Wayne , Peterson Richard , Liguori Michael 
Published: November 2023

Renal toxicity is common cause of attrition during non‐clinical development. Renal toxicity was identified in multiple series during the early toxicology studies in rodent species. The toxicity was characterized by basophilia of tubular epithelial cells and karyomegaly upon exposure to the parent molecule. Interestingly the tubular injury was restricted primarily to the S1 and S2 segments of the proximal convoluted tubules. Electron microscopic examination of tubular epithelial cells indicated mitochondrial injury as likely mechanism for tubular injury, as evidenced by mitophagy and mitochondrial swelling. We hypothesized that the segment specificity was due to interaction of the compound with transporters (OAT1) that were specifically expressed in these tubules. To test this hypothesis rat were infused with one of the major metabolite which was determined to be the cause of tubular injury alone or in combination with an OAT1 inhibitor, Probenecid. Rats infused with the major metabolite of the drug had moderate to severe tubular injury. In rats infused with major metabolites for seven days along with probenecid partial protection was observed, indicating that transporter mediated uptake was at least partially responsible for the tubular injury. Transporter facilitated tubular epithelial accumulation likely contributed to the mitochondrial injury noted upon electron microscopic examination. A role for transporters should be considered especially when the injury is segment specific and if a transporter role is confirmed there is potential to derisk successfully as evidenced by successful application of this approach with cidofovir and adefovir.Support or Funding InformationAll authors are employees of AbbVie. The design, study conduct, and financial support for this research was provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.
In Vitro Nephrotoxicity Induced by Dibromoanilines in Isolated Renal Cortical Cells from Male Fischer 344 Rats
Authors: Rankin Gary O'Neal , Tyree Connor , Peters Bunni , Norwood Jalen , Anestis Dianne 
Published: November 2023

Dibromoanilines (DBAs) are used as chemical intermediates in the manufacture of dyes, pesticides, drugs and other organic chemicals. Two DBAs, 3,4‐DBA and 3,5‐DBA, also possess direct fungicidal activity. We previously found that among the 3,5‐dihaloanilines, 3,5‐DBA was the most potent nephrotoxicant in vitro using renal cortical slices from male Fischer 344 rats as the animal model. However, the potential nephrotoxicity of the other five DBA isomers is unknown. The purpose of this study was to determine the comparative nephrotoxic potential of the six DBA isomers using freshly isolated renal cortical cells (IRCC) from male Fischer 344 rats. Briefly, IRCC were isolated using a collagenase perfusion technique, and IRCC (~4.1 million cells/ml; 3 ml) were exposed to a DBA isomer (final bath concentrations of 0.25, 0.5 or 1.0 mM) or vehicle (DMSO, 30 μl) for 30 or 60 min. Cytotoxicity was determined by lactate dehydrogenase (LDH) release assays, with LDH release reported as percent of control. At a 30 min exposure, 3,5‐DBA was the most potent nephrotoxicant among the six DBA isomers, inducing an increase in LDH release at 0.5 mM (21 ± 2% treated vs 9 ± 1% control). LDH release was also increased at 30 min by 2,6‐DBA 0.5 mM (14 ± 2% vs 8 ± 1% control). Other DBA isomers required a bath concentration of 1.0 mM or higher to induce cytotoxicity. The largest increase in LDH release at 60 min was also induced by 3,5‐DBA at 0.5 (35 ± 1%) and 1.0 mM (38 ± 1%) vs the control group (14 ± 1%). While 2,6‐DBA 0.25 mM significantly induced LDH release at 60 min, the elevation in LDH release was not different from the LDH release induced by 3,5‐DBA 0.25 mM. Thus, these results demonstrate that 3,5‐DBA is the most potent nephrotoxicant in vitro in IRCC among the six DBA isomers.Support or Funding InformationThis work was supported in part by NIH grant P20GM103434 to the West Virginia IDeA Network of Biomedical Research Excellence.
Effect of Aqueous Extract and Anthocyanins of Calyces of Hibiscus sabdariffa L. (Malvaceae) in Rats with Adenine – Induced Chronic Kidney Disease
Authors: Ali Badreldin H. , Cahliková Lucie , Opletal Lubomir , Karaca Turan , Al Suleimani Yousuf , Al Za'abi Mohammed H , Nemmar Abderrahim , Blunden Gerald 
Published: November 2023

Hibiscus sabdariffa L. calyces (HS) can mitigate several multi‐organ injuries, one of which is chronic kidney disease (CKD). Here, we tested the effect of water extract of HS and a concentrated extract of its isolated anthocyanins on experimental adenine ‐ induced CKD. Rats were orally given, for 28 consecutive days, either adenine, alone or together with either HS aqueous extract or anthocyanins. For comparative purposes, two groups of rats were given lisinopril (10 mg/Kg). One day after the end of the treatment period, blood pressure was measured, followed by collection of urine, plasma and kidney tissues. Adenine ‐ induced CKD was characterized by significant body weight reduction, renomegaly and hypertension, as well as by elevations in plasma creatinine, urea, neutrophil gelatinase‐associated lipocalin activity, urinary albumin, and N‐acetyl‐β‐D‐glucosaminidase activity, and decrease in creatinine clearance. Adenine also significantly increased the concentrations of several inflammatory cytokines and decreased those of oxidative stress indices. Renal tissue sections of adenine–fed rats showed significant increase in renal damage and markers of inflammation and fibrosis. HS aqueous extracts and isolated anthocyanins lessened, mostly in a dose – dependent manner, the severity of CKD to a similar extent to that of lisinopril. HS and its anthocyanins could be considered as possible promising safe dietary agents that could be used to attenuate the progression of human CKD.Support or Funding InformationFinancially supported from a grant from SQU (IG/SQU/MED/01/2014)
The Potential Role of Acetaminophen and Alcohol in Early Stage Renal Disease: A Bayesian Perspective
Authors: Ndetan Harrison T , Einstein George P , Tulp Orien L 
Published: November 2023

In earlier studies, both acetaminophen [AAM] and ethanol [EtOH] have been implicated in renal dysfunction when ingested in variable quantities over a prolonged duration. To determine the potential role of AAM and EtOH on Early Stage Renal Disease [ErSRD], the contribution of threshold dosages of AAM and of light to moderate intakes of EtOH were investigated, and the results analyzed by Bayesian Statistical Methods based on Baye's Theorem. The results of information obtained from the US National Health Examination Survey [2003–3004] were examined, and the data updated with data from a newly designed hospital based case control study conducted in the sub‐Saharan African Region. The results of this study found that renal disease, as determined by measurements of serum creatinine [CTNE], blood urea nitrogen [BUN], Glomerular Filtration Rate [GFR], albumin/creatinine ratios [A/CTNE ratio] values of 0.70 mg/dl, 10.0 mg/dl, 17.0 mg/g, and 135.0 ml/minute/1.73m2, respectively, may occur, marked by statistically significant odds ratios among those who concomitantly ingested therapeutic doses of AAM and ingested light to moderate amounts of EtOH when compared to those who did not ingest the above agents. While light to moderate ingestion of EtOH may not by itself play a significant role, the combination of the two agents appeared to have a synergistic effect on the early development of renal disease in his population. This this study provided both methodological and non‐methodological factors to be considered in the design and implementation of larger scale epidemiologic studies on the interaction of AAM and EtOH on the development of ErSRD.Support or Funding InformationSupported by Institutional Resources of USAT College of Medicine and the Einstein Institute
JTE‐013, an antagonist of sphingosine 1‐phosphate receptor 2, inhibits ovalbumin‐induced allergic asthma in Balb/c mice
Authors: Park Soojin , Kang Saeromi , Im Dongsoon 
Published: November 2023

BackgroundActivation of sphingosine kinases 1 and 2 and secretion of sphingosine 1‐phosphate (S1P) have been implication in allergic responses, asthma and anaphylaxis. Type 1 and 2 receptors for S1P have been suggested as targets of secreted S1P functions in mast cells. However, functions of S1P receptors in vivo has not been investigated in an allergic asthma model.METHODSAntigen‐induced degranulation was measured in RBL‐2H3 mast cells by measuring b‐hexosamidase activity. An ovalbumin‐induced allergic asthma model was used for in vivo efficacy test of JTE‐013, a selective antagonist of type 2 S1P receptor.RESULTSJTE‐013 inhibited the antigen‐induced degranulation of beta‐hexosaminidase in RBL‐2H3 mast cells in a concentration‐dependent manner in vitro. Sensitization and challenge of ovalbumin induced allergic asthma responses, that is, the accumulations of eosinophils and increase of Th2 cytokine levels in bronchoalveolar lavage fluid and lung inflammation. Administration of JTE‐013 before ovalbumin sensitization or before ovalbumin challenge strongly inhibited the accumulation of eosinophils and lymphocytes to bronchoalveolar lavage fluid. JTE‐013 administration also inhibited expression and secretion of Th2 cytokines, that is, IL‐4 and IL‐13. Histologic studies showed that JTE‐013 reduced inflammatory signs in the lung.CONCLUSIONJTE‐013 has anti‐allergic effects and may be utilized as a potent agent for the treatment of asthma.
Anti‐inflammatory effects of serotonin 5‐HT2A receptor activation in ovalbumin‐induced allergic asthma models
Authors: Flanagan Thomas Wayne , Sebastian Melaine Nicole , Nichols Charles David 
Published: November 2023

Only recently has the full therapeutic value of serotonin [5‐hydroxytryptophan (5‐HT)] receptor activation begun to be explored. Currently there are two 5‐HT2A receptor agonists in human clinical trials for the treatment of depression and obesity. An exciting new therapeutic avenue in which 5‐HT2A agonists might be employed is the modulation of inflammation in allergic airways disease. Our lab has previously used an ovalbumin (OVA)‐induced asthma model to demonstrate that administration of (R)‐2,5‐dimethoxy‐4‐iodoamphetamine [(R)‐DOI] prior to allergen challenge prevents many of the symptoms of allergic asthma. Here we have utilized a modified protocol to test the effectiveness of (R)‐DOI in treating persistent allergic asthma. We demonstrate that administration of (R)‐DOI in a chronic model attenuates the elevated airway hyperresponsiveness (AHR) typically observed in an asthmatic response. We also have probed for the expression of inflammatory markers in the lung and BALF. We concurrently are testing for the impact psilocybin and other tryptamines have on AHR in rodents using our OVA model. Overall our strategy is to develop 5‐HT2A receptor agonism as a viable treatment modality against asthma and other inflammatory disorders.Support or Funding InformationAmerican Asthma Foundation Scholar Grant Award
The ‘Prophylactic’ and ‘Therapeutic’ Effects of β2‐Adrenoceptor Blockers in a House Dust Mite (HDM) Driven Murine Asthma Model
Authors: Joshi Radhika , Valdez Daniel , Kim Hosu , Knoll Brian J , Bond Richard A 
Published: November 2023

Asthma is characterized by chronic airway inflammation, excess mucus production and airway hyper responsiveness (AHR). Our previous research suggested that some β2‐adrenoceptor (β2AR) blockers might be beneficial in chronic asthma therapy. Using an ovalbumin (Ova) allergen‐driven murine asthma model, we have previously shown that only certain β2AR blockers such as nadolol and ICI‐118, 551 attenuate the development of an asthma phenotype when administered prior to the antigen challenge (‘prophylactic’ effect); while others including carvedilol and propranolol are ineffective. Recently however, the Ova model has been criticized for its lack of clinical relevance. To address the limitations of this model, we tested the β2AR blockers, nadolol and carvedilol in a clinically more relevant model utilizing house dust mite (HDM) extract. Additionally, we investigated both the ‘prophylactic’ and ‘therapeutic’ (the drugs were administered after the development of asthma phenotype) potential of these β2AR blockers in the HDM model of asthma.Male Balb/c mice were challenged with 25 μg HDM protein by intranasal delivery in two protocols designed to test the ‘prophylactic’ and ‘therapeutic’ effects of nadolol and carvedilol. In both protocols, chronic nadolol treatment significantly lowered the development of airway inflammation and mucus production in response to HDM, while chronic carvedilol treatment was ineffective. In addition, nadolol significantly attenuated the increased AHR response in the ‘prophylactic’ model but only moderately reduced the airway resistance in the ‘therapeutic’ model. Thus, the present studies support our previous findings in the ovalbumin model of asthma and provide further evidence to support nadolol's potential in asthma therapy.Support or Funding InformationSupported by R01AI110007