bluuebunny/crossref_metadata_2025_split huggingface dataset:
Traumatic brain injury in a Drosophila upregulates nitric oxide synthase associated with increased acute behavioral deficits and decreased survival time
Authors: Mackey Tracy , Jones T. B. , Currier Thomas Theresa , Juba Amber N. , Buhlman Lori M. 
Published: May 2022

Traumatic brain injury (TBI) is as an outside force causing a modification in brain function and/or structural brain pathology that upregulates brain nitric oxide (NO), where NO activity is implicated in secondary pathology leading to behavioral deficits. In mammals, TBI‐induced NO production activates immune responses and potentiates metabolic crisis through mitochondrial dysfunction coupled with vascular dysregulation; however, the direct influence on pathology is complicated by the activation of numerous secondary cascades and activation of other reactive oxygen species. TBI in Drosophilahave clearly demonstrated key features of mammalian TBI including temporary incapacitation, disorientation, and activation of innate immunity (inflammation) and autophagy responses observed immediately after injury and related to persisting deficits. We propose to evaluate nitric oxide synthase expression in the absence of vascular disruption and the adaptive immune system to begin creating a model that minimizes extraneous variables to study the longitudinal influence of NO signaling on secondary pathology leading to behavioral deficits where highly targeted interventions can be evaluated.We optimized a modified high impact trauma (HIT) device on NOS‐GAL4>UAS2xEGFP flies (Bloomington Stock Center, Bloomington, IN) where upregulation of Drosophila nitric oxide synthase (dNOS) expression is associated with increased GFP levels. Ten‐day old flies received TBI from the HIT device at multiple angles (30, 45, 60, 75 degrees) followed by assessment of acute neurological behavior, locomotor activity, brain dNOS levels, twenty‐four‐hour mortality, and survival. Data indicate an increase in brain GFP expression, where robust reproducible levels of dNOS could be recorded after a 75‐degree HIT associated with increased incident of temporary incapacitation, disorientation, changes in acute climbing activity, increased 24‐hour mortality, and decreased survival. Results also indicate an effect on dNOS expression as a function of HIT angle and sex. Ongoing studies quantify levels of TBI‐induced NO activity in the brains. Future studies will evaluate different forms of brain pathology and more extensive behavioral deficits where genetic and pharmacological manipulation can be used to tease out direct effects of NO signaling. Due to conserved signaling cascades between flies and mammals, improved outcomes from modulating NO activity can translate to improved targeted therapies to be tested in mammals.
Assessing the Effect of Organic Anion Transporter Inhibition on Circulating Pyridoxic Acid, an Endogenous Transporter Biomarker, using Physiologically‐based Pharmacokinetic Modeling
Authors: Thakur Aarzoo , Prasad Bhagwat 
Published: May 2022

Transporter‐mediated renal secretion plays a critical role in the systemic clearance of ~30% of the Food and Drug Administration (FDA)‐approved drugs and is attributed to clinically significant drug‐drug interactions (DDIs). For example, organic anion transporters (OATs) are inhibited by perpetrator drugs (e.g., probenecid), leading to higher circulating levels of OAT substrates (e.g., furosemide).1 Recently, OAT inhibition has been shown to be associated with increased plasma levels of endogenous substrates, e.g., pyridoxic acid (PDA) and homovanillic acid, which can serve as potential OAT biomarkers.2 The objective of our study was to develop and validate a physiologically‐based pharmacokinetic (PBPK) model of PDA to allow prospective evaluation of the magnitude of potential DDI of an OAT inhibitor‐substrate pair. Probenecid and furosemide were used as selective inhibitor and substrate of OATs, respectively. Physicochemical properties and in vitro data pertaining to the pharmacokinetics of probenecid and PDA were collated from literature. A PBPK model was first constructed to predict the plasma concentration‐time profiles of probenecid after oral administration using Simcyp (v20). Then, utilizing the fraction secreted versus filtered as well as the unbound fraction (fu = 0.092) data of PDA,2 the total elimination and synthesis rates of PDA at steady‐state were used to develop a baseline PBPK model. The effect of OAT inhibition by probenecid on the PDA levels was estimated and the predicted area under the plasma concentration‐time profile (AUC), change in clearance, and the highest plasma concentration (Cmax) values were compared with the observed data.2 The model was able to capture the changes within 2‐fold of the clinical data. Using the predicted AUC profile of PDA and assuming 100% inhibition at Cmax, the magnitude of inhibition was estimated across different time points. The biomarker‐informed magnitude of inhibition showed significant correlation (r2 = 0.9336) with the observed change in furosemide concentrations in the presence of probenecid (Fig. 1). PDA‐informed PBPK model developed here can be used to prospectively predict the magnitude of OAT inhibition, which is particularly applicable to assessing DDI potential of investigational drugs or prescribed drugs in special populations with sparse samples, without the requirement of an exogenous probe substrate.References:

1. Smith, D. E. et al. (1980). Journal of Pharmaceutical Sciences, 69, 571.
2. Shen, H. et al. (2019). Journal of Pharmacology and Experimental Therapeutics, 68, 136.
One Assay, Two Vital Values‐Estimating Intrinsic Permeability and Efflux Using MDCK‐MDR1 Assay
Authors: Zang Grace , Li Chun 
Published: May 2022

BackgroundIntestinal intrinsic permeability and transporter mediated efflux are two major determinants of oral absorption. Typically, wild type Madin‐Darby canine kidney cells (MDCK) and MDCK cells transfected with the human MDR1 gene (MDCK‐MDR1) are used to evaluate intrinsic permeability and efflux, respectively. The MDCK assay provides intrinsic permeability but lacks transporter mediated efflux. Conversely, the MDCK‐MDR1 assay provides efflux ratio and “apparent permeability”. However, the apparent permeability can differ significantly from intrinsic permeability due to efflux. Traditionally, both assays (MDCK, MDCK‐MDR1) are applied separately and sequentially, which can be time‐consuming and costly.In this study, we present a novel approach to estimate intrinsic permeability and efflux simultaneously using the MDCK‐MDR1 assay alone. Intrinsic permeability can be calculated using a scaling method by combining the 3 parameters (bi‐directional apparent permeability values and efflux ratio) determined from the MDCK‐MDR1 assay.MethodsTest compounds of varying permeability and efflux ratios were used to assess the validity of the hypothesis. Results from the MDCK, MDCK‐MDR1 and caco‐2 assays were analyzed. Efflux ratio is estimated by dividing the apparent permeability from the basolateral to apical direction (PappBA) by the apparent permeability from the apical to basolateral direction (PappAB). Different averaging methods were used to calculate intrinsic permeability from MDCK‐MDR1 assay, and the measured intrinsic permeability from the MDCK assay was used as a comparison to find the most accurate averaging method.ResultsOf the different scaling and averaging methods used for estimating intrinsic permeability from MDCK‐MDR1 assay, the best method we found was a “quotient” scheme where calculated intrinsic permeability was adjusted based on the magnitude of efflux ratio. With this adjustment, both the intrinsic permeability and efflux ratio can be reliably obtained from the MDCK‐MDR1 assay for compounds with a wide range of efflux ratios. The calculated intrinsic permeability from the MDCK‐MDR1 assays aligns within 2 fold of the permeability values determined from the MDCK assay. Results from the caco‐2 cell line were also evaluated and applicable to the method mentioned above. Several hypotheses were formulated regarding why the “quotient” method was a better fit than simple apparent permeability averaging.ConclusionThe strategy presented here provides an efficient and effective method to estimate both intrinsic permeability and efflux solely based on the MDCK‐MDR1 assay. Compared to the traditional approach of using MDCK and MDCK‐MDR1 assays separately, the method used here requires less time, cost, and compound for testing. This is particularly attractive for early drug discovery screening needs.
Immune cells play a crucial role in the development of a cardiac dysfunction in cardiac p38 MAPKa KO mice after pressure overload
Authors: Hemmers Anne , Nederlof Rianne , Kalfhues Lisa , Bottermann Katharina , Oenarto Vici , Gödecke Axel 
Published: May 2022

QuestionCardiac metabolic remodeling is one of the early changes driving the progression of heart failure and is present even before overt cardiac dysfunction. Previously we showed that the tamoxifen inducible deletion of cardiomyocyte specific p38 MAPKa (KO) leads to a pronounced left ventricular dilation with a strongly impaired heart function just within 2 days of angiotensin II (ANGII) treatment. Additionally, a strong infiltration of immune cells in the cardiac tissue can be observed.Methods and ResultsUnder baseline conditions no impairment of the heart function in KO mice can be observed. ANGII treatment for 2 days induces a strong left ventricular dilation and poor heart function, indicated by a reduced ejection fraction and an increase in end‐systolic and end‐diastolic volume. KO mice do not get used to constant ANGII treatment even after 7 days of ANGII treatment and cardiac function continues to deteriorate. KO mice transiently loose weight as a consequence of ANGII treatment. On the fourth to fifth day of ANGII treatment, KO animals gain weight again, although the heart function continues to deteriorate. FACS analysis of cardiac tissue revealed a strong infiltration of neutrophils in KO hearts after 48h of ANGII treatment (4.5‐fold increase compared to control hearts). A strong correlation between reduced ejection fraction and increasing amount of neutrophils in the cardiac tissue suggests a particularly important role of neutrophils in the development of cardiac dysfunction. Also an increase of macrophages (1.6‐fold increase), CD4+ T‐helper cells (2.5‐fold increase) and CD8+ cytotoxic T‐cells (2.5‐fold increase) as well as B‐cells (2‐fold increase) could be shown in KO hearts compared to control hearts. After long term ANGII treatment for 7 days, however, the number of neutrophils in the hearts decreased (10‐fold decrease in control hearts and 73‐fold decrease in KO hearts compared to 48h of ANGII treatment) and a strong increase in M2‐like macrophage infiltration, mainly in control hearts (1.6‐fold increase compared to 48h ANGII treatment), could be observed. The infiltrated macrophages were also positive for CCR2, which indicates they originated from monocytes. B‐ and T‐cells were still elevated in KO hearts after 7 days of ANGII treatment. Also in control hearts an increase of B‐ and T‐cells could be observed. In addition, fibrosis developed in the cardiac tissue, which could be shown by histological staining of collagen I and FACS analysis targeting αSMA+ cardiac fibroblasts.ConclusionImmune cells and in particular neutrophils play an important role in the development of cardiac dysfunction after ANGII induced pressure overload in p38 MAPKa KO mice. Over time, however, the number of immune cells in control mice also increases, which underlines the peculiarity of the first neutrophil influx after 48h ANGII treatment for the impairment of cardiac function.
Amphotericin A21 Induces Cytotoxic Effects in Breast Cancer Cell Lines
Authors: Escobar‐Reséndiz Rubi , Ortega‐Blacke Ivan , Galván Arturo , Rodriguez‐Fragoso Lourdes 
Published: May 2022

INTRODUCTIONCancer is a public health problem that causes enormous loss of human life. Worldwide, breast cancer is one of the most diagnosed types of cancer and one that contributes significantly to cancer mortality. One of the situations that contributes to mortality statistics is that the patient may present low efficacy to treatment or the development of resistance. Currently there are various antineoplastic drugs, but these are not enough to face the complications of the disease. Therefore, it is of interest to study new drugs that have antineoplastic potential. Amphotericin B L‐histidine methyl ester, known as Amphotericin A21 (AmB‐A21), is a compound that has been extensively studied by our research group. Toxicological studies have shown that AmB‐A21 is a very safe compound, causing little toxicity in in vivo models. AmB‐A21 has also been found to have a cytotoxic effect, induce apoptosis, and reduce the proliferation and invasion of lung cancer cells (A549 cells). The results of the evaluations suggest that AmB‐A21 has the potential to be an effective and safe antineoplastic drug. The present project aims to evaluate whether AmB‐A21 can also cause antineoplastic effects in breast cancer cell lines, first evaluating the cytotoxic effect of AmB‐A21.AIMTo evaluate the cytotoxic effect of AmB‐A21 on MDA‐MB‐231 and MCF7 cells.HYPOTHESISAmB‐A21 induces cytotoxicity in MDA‐MB‐231 and MCF7 cells.MethodsMDA‐MB‐231 (ATCC HTB‐26) and MCF‐7 (ATCC HTB‐22) lines from breast cancer were used. Both lines were cultured at 37 ° C, with 5% CO2, in DMEM HG medium supplemented with non‐essential amino acids, glutamine, sodium pyruvate, gentamicin and 10% fetal bovine serum.First, growth kinetics were performed for 72 hours (h), to characterize the doubling rate and time of each cell line. Subsequently, dose‐response curves were performed to evaluate the cytotoxic effect of AmB‐A21 and inhibitory concentrations 50 (IC50) were calculated. Finally, the type of cell death that AmB‐A21 induces at IC25 y 50 was identified in MCF‐7 cells.RESULTSThe proliferation rate at 72 h was found to be 2.43 ± 0.38 and 3.10 ± 0.21 for MDA‐MB‐231 and MCF‐7 cells respectively. Regarding the dose‐response curves, it was found that AmB‐A21 induces a concentration‐dependent cytotoxic effect in both breast cancer cell lines, whose IC50 were 178.83 ± 18.8 and 207.24 ± 5.7 µM/mL for the MDA‐MB‐231 and MCF‐7 cells, respectively. The results obtained had a statistically significant difference, with respect to the control. Data are shown as mean ± standard deviation. AmB‐A21 was also found to induce apoptosis cell death in MCF‐7 cells with their IC50 & 25.CONCLUSIONSAmB‐A21 induces cytotoxicity in cell lines originating from breast cancer (MDA‐MB‐231 and MCF‐7) and generates cell death by apoptosis in MCF‐7 cells.
Pharmacological Evaluation of Beta‐Hydroxifosphocarnitine in No‐Alcoholic Esteatohepatitis Induced in Rats
Authors: Sanchez‐Quevedo Janet , Rosado Jorge Luis , Rodriguez‐Fragoso Lourdes 
Published: May 2022

AimEvaluate the pharmacological effect of Β‐Hydroxyphosphocarnitine (β‐HFC) on non‐alcoholic steatohepatitis induced in rats.IntroductionNonalcoholic steatotepatitis (NASH) is a serious global public health problem, it is related to other pathologies with increasing prevalence, manifested in the increased prevalence of NASH. Type 2 diabetes mellitus and obesity are the main pathologies related to NASH. There is no specific drug for NASH, the current drugs used for NASH are related to serious side effects, which is why there is an urgent need in the development of an effective therapy for NASH but with fewer side effects. L‐carnitine has an effect on improving metabolic alterations related to NASH, however, its intestinal absorption is limited. B‐HFC is an analog of L‐carnitine that has been shown to be safe for administration, to maintain pharmacological properties of its precursor and its absorption is greater than that of L‐carnitine. So it can be a good treatment for NASH.MethodThe NASH model was developed in male Wistar rats by ingesting carbonated drink as a source of fructose, coconut oil and carbon tetrachloride. 3 groups were formed with NASH (n = 8); NASH, NASH ‐β‐HFC and NASH‐pioglitazone. Another 3 groups of healthy rats (n = 8); control, control‐β‐HFC and control‐pioglitazone. The effect of β‐HFC in rats with NASH was evaluated by staining of histological sections of liver. The stains used were Masson's trichrome, hematoxylin and osin and Periodic Acid‐Schiff (PAS). Biochemical analysis was performed to evaluate the effect of β‐HFC on the metabolic alterations that characterize NASH.ResultIt was previously reported that β‐HFC reduced the triglyceride levels of rats with NASH, as well as the levels of liver enzymes. Evidencing the effect of β‐HFC in improving lipid metabolism, manifesting itself in less liver damage. For this occasion we will report the effect of β‐HFC on liver glycogen levels. Glycogen is a reserve of the cell, as a source of glucose in prolonged fasting states. Our results demonstrated a higher glycogen reserve in the NASH rats. This result was expected, since the rats had a high fructose intake. It is important to mention that the rats with NASH but treated with β‐HFC presented reduced glycogen levels, this effect can be attributed to a more efficient β‐oxidation process, which could also justify the reduction of triglycerides and fibrosis. Therefore, we could argue that a longer treatment time, the liver alterations in NASH would improve significantly.Conclusionβ‐HFC has an effect on improving liver and metabolic alterations characteristic of the development of NASH.
Continuous Ultrasonic Transit‐Time Blood Flow and Arterial Blood Pressure Monitoring in Freely Moving Sprague‐Dawley Rats via Implantable Telemetry
Authors: Senador Danielle , Azar Toni , Fricke Kyle , Reynolds Christian , Minic Zeljka 
Published: May 2022

BACKGROUNDSimultaneous blood flow and pressure measurements provide a more comprehensive hemodynamic assessment in cardiovascular studies. However, current cardiac output (CO) measurements, by flow probes or imaging, require either anesthesia or a tethered setup, which result in limitations due to anesthesia effects and/or monitoring time.OBJECTIVEThe objective of this study was to use an implantable telemetry device (EndoGear (EG), Transonic Systems Inc., Ithaca, NY) to measure continuous CO (real‐time ascending aortic blood flow), and mean arterial pressure (MAP) in conscious, freely moving rats and to verify the ability of the system to capture well‐known circadian oscillations in major cardiovascular parameters.METHODSAll procedures were approved by the Institutional Animal Care and Use Committee of Wayne State University and complied with the NIH Guide for the Care and Use of Laboratory Animals. Male Sprague‐Dawley rats were implanted with EG transmitters to continuously monitor CO, MAP, temperature, and activity for 5 weeks. An ultrasonic transit‐time flow probe was placed around the ascending aorta via a right lateral thoracotomy (4th intercostal space). The pressure catheter was inserted into the iliac artery and advanced (approximately 2.5 cm) into the abdominal aorta, while the transmitter was implanted intraperitoneally via a midline laparotomy approach. The inductive power receiver associated with the implant was positioned in an abdominal subcutaneous pocket. After surgery, rats received thermal support, prophylactic analgesics, and antibiotics, as well as fluid supplementation. Body weight, food and fluid intake were monitored daily for the first 10 days and then bi‐weekly until the end of the study. Animal checks and necessary handling were conducted at specific times to minimize circadian disturbances.RESULTSChanges in MAP, heart rate (HR, captured from pressure and flow traces), CO, stoke volume (SV, calculated as CO/HR), and temperature revealed, as expected, a clear circadian pattern characterized by lower values during the light phase. MAP decreased by ~5%, from 96.9 ± 0.15 to 92.1 ± 0.19 mmHg (p<0.05). HR decreased by ~10%, from 369 ± 3 to 329 ± 6 bpm (p<0.05). CO decreased by ~14%, from 91.6 ± 1.79 to 78.4 ± 1.89 ml/min (p<0.05). SV decreased by ~7%, from 245 ± 3.5 to 238 ± 1.4 μl (p<0.05). The decreases in temperature and activity during the light phase were subtle yet significant.CONCLUSIONSThe telemetry device was able to effectively measure continuous changes in CO and MAP. Preservation of the circadian rhythm indicates favorable post‐operative outcome after extensive surgery and complex chronic implantation in rats.
Eplerenone‐induced natriuresis and hyperkalemia in mice lacking aldosterone results from aldosterone‐independent mineralocorticoid receptor occupancy
Authors: McCormick James A. , Maeoka Yujiro , Sharma Avika , Su Xiao‐Tong , Wang Wen‐Hui , Ellison David H. 
Published: May 2022

BackgroundMineralocorticoid receptor (MR) antagonists are recommended for patients with resistant hypertension independent of circulating aldosterone levels. Aldosterone, secreted in response to salt depletion or hyperkalemia, activates MR to increase epithelial sodium channel (ENaC) activity. While glucocorticoids can also activate MR, they are metabolized by 11β‐hydroxysteroid dehydrogenase type 2 (11β‐HSD2), protecting MR from illicit glucocorticoid occupation. 11β‐HSD2 is expressed at increasing levels from the DCT through the collecting duct. Here, we hypothesized that glucocorticoid occupies MR to stimulate ENaC in the DCT2 and early connecting tubule.MethodsWe studied aldosterone synthase knockout (AS−/−) mice provided by Dr. JM Luther. Kidney–specific MR knockout (MR−/−) mice were used for validation of Western blot for ENaC and immunofluorescence for MR. We measured plasma and urinary electrolytes at baseline, and following dietary salt restriction with or without administration of the MR antagonist eplerenone.ResultsAS−/− mice displayed higher plasma K+ levels than AS+/+ mice but not salt wasting under baseline. Under low salt (LS) diet, AS−/− mice exhibited higher urine Na+ excretion, but preserved body weight. Cleaved αENaC and γENaC abundances were lower in AS−/− than AS+/+ mice, and were not increased in AS−/− mice after LS diet. Notably, we found nuclear localization of MR was preserved in DCT2 of AS−/− mice. Administration of the MR blocker eplerenone to AS−/− mice fed LS diet led to hyperkalemia and decreased body weight with higher Na+ excretion, mimicking the phenotype of MR−/− mice.ConclusionsOur results provide evidence that MR is occupied in the absence of aldosterone, suggesting glucocorticoid‐binding to MR preserves sodium homeostasis in the DCT2 of AS−/− mice. This likely explains the milder phenotype of AS−/− mice compared with MR−/− mice.