bluuebunny/crossref_metadata_2025_split huggingface dataset:
Anti‐proliferative Effect in HeLa Cells by Cyclodipeptides from Pseudomonas aeruginosa PAO1
Authors: Padilla Laura Hernández , Mixteco Pedro Eduardo Lázaro , Alcantar Lorena Martínez , Pérez Alma Laura Díaz , García Jesús Campos 
Published: April 2020

ObjectiveEvaluation of the anti‐proliferative mechanisms in HeLa cells cultures by cyclodipeptides from Pseudomonas aeruginosa PAO1.MethodsCyclodipeptides from P. aeruginosa PAO1 (PAO1‐CDPs) cultures were obtained by solvent extraction according to Ortiz‐Castro et al. (2011). Cell viability of HeLa cells was determined by the MTT method and apoptosis using Annexin‐V according to Vázquez‐Rivera et al. (2014). Mitochondrial membrane potential determination and real‐time quantification of superoxide was carried out by FACS according to Gómez et al. 2014. Immunodetection was conduced using human antibodies: anti‐Akt, anti‐Akt‐phosphorylated (Ser473), anti‐p70S6 kinase, anti‐phosphorylated‐p70S6 kinase (Thr 389); developed using Goat anti‐Rabbit IgG HRP‐conjugate.AbstractPseudomonas aeruginosa PAO1 produces the cyclodipeptides cyclo(L‐Pro‐L‐Tyr), cyclo(L‐Pro‐L‐Phe), and cyclo(L‐Pro‐L‐Val), whose effects have been implicated in inhibition of proliferation in human tumor cell lines. Our purpose was to elucidate the anti‐proliferative mechanisms caused by the PAO1‐CDPs in HeLa cells cultures. The results indicate that the PAO1‐CDPs both individually as in mixture, inhibited HeLa cell proliferation by arresting the cell cycle at the G0–G1 transition. The PAO1‐CDPs mixture promoted cell death in HeLa line by a dose‐dependent manner, showing half‐lethal dose (LD50) of 60 μM and inducing apoptosis at 0.6 μM. Moreover, PAO1‐CDPs showed a higher pro‐apoptotic activity (~10 fold) than their synthetic analogs. Subsequently, the mitochondrial membrane potential was arrested by the PAO1‐CDPs and induced apoptosis by caspase‐9‐dependent pathway. The anti‐proliferative mechanism in HeLa cells involves inhibition of phosphorylation of both the Akt‐S473 and S6k‐T389 protein kinases, showing a cyclic behavior of their phosphorylation in a time and concentration‐dependent fashion.ConclusionsOur findings indicate that the PI3K/Akt/mTOR/S6k signaling pathway blocking is involved in the anti‐proliferative effect of the PAO1‐CDPs.
Susceptibility Screening of Thymol, Quercetin, Neem, Cinnamaldehyde and Eugenol against Pseudomonas aeruginosa
Authors: Kyeyune Joy , Heuertz Rita M. 
Published: April 2020

Pseudomonas aeruginosa exhibits intrinsic and acquired resistance to antibiotics. An under‐utilized source of antimicrobic activity is phytochemicals. Their use against P. aeruginosa may serve as an alternative to conventional antibiotics in this age of ever‐increasing resistance. The purpose of this study was to investigate susceptibility patterns of P. aeruginosa in the presence of phytochemicals selected from literature citations reporting antibacterial activity. The hypothesis was that phytochemicals thymol, quercetin, neem bark extract, cinnamaldehyde and eugenol inhibit growth of P. aeruginosa. Susceptibility testing of phytochemicals was performed by the Kirby Bauer disk diffusion method and phytochemicals were tested at different concentrations (0–100%) after 2‐fold serial dilutions of the agents into tryptic soy broth. Results showed zones of growth inhibition of P. aeruginosa (ATCC strain 27853) around cinnamaldehyde‐ and eugenol‐impregnated disks when the phytochemicals were presented singly or in combination with each other. Specific results indicated that significant zones of inhibition were evident at 100%, 50% and 25% concentrations of cinnamaldehyde. Zones of inhibition were evident at 100% and 50% eugenol. A combination study of cinnamaldehyde and eugenol, each at 12.5% concentration, produced a notable zone of inhibition (measured at 16 mm diameter): neither cinnamaldehyde nor eugenol displayed zones of inhibition independently at that concentration (of 12.5%). Thymol, quercetin and neem bark extract did not yield visible zones of inhibition at any concentration tested. To conclude, cinnamaldehyde and eugenol each individually possess antimicrobial activity against P. aeruginosa (strain ATCC 27853) as indicated by disk diffusion susceptibility testing. Additionally, the two phytochemicals act synergistically in combination to effectively inhibit bacterial growth. Evidence of synergistic activity at low concentrations not effective individually by either cinnamaldehyde or eugenol offers potential therapeutic value. Other phytochemicals (thymol, quercetin, neem bark extract) failed to inhibit P. aeruginosa growth at any concentration tested. Future studies aim to screen more phytochemicals independently and in combination against other strains of P. aeruginosa to allow for statistical analysis and determination of susceptibility patterns.Support or Funding InformationFinancial support for JK was received from the DeNardo Education and Research Foundation.
In vitro Species Different Metabolism and CYP Phenotyping of Zamicastast
Authors: Loureiro Ana I. , Rebouta Joana , Bonifacio Maria-Joao , Soares-da-Silva Patricio 
Published: April 2020

BackgroundZamicastat
(1) is an inhibitor of dopamine‐β‐hydroxylase, enzyme that catalyzes the hydroxylation of dopamine to norepinephrine in sympathetic nerves and adrenal medulla, and it was shown to increase the survival rate in the monocrotaline rat model of pulmonary arterial hypertension.ObjectivesThe purpose of this study was to evaluate zamicastat clearance and metabolic profile in hepatocytes from different species (human, rat, dog, mouse and monkey) and to identify the CYP450 enzymes isoforms that are involved in zamicastat metabolism. Methods. Metabolite profiling of zamicastat (10 μM) was conducted in human, rat, dog, mouse and monkey cryopreserved hepatocytes. Samples were harvested up to 120 min and analyzed by High Resolution Mass Spectrometry (HR‐MS). The Intrinsic Clearance (CLint) values for zamicastat were assessed in all species. For CYP reaction phenotyping, the study was conducted on human CYP450 recombinant enzymes incubated with zamicastat (5 μM) and NADPH, with samples being harvested up to 45 min and analyzed by LC‐MS/MS.ResultsThe CLint for zamicastat was estimated at 40.3, 32.6, 13.6, 44.2 and 6.29 μl/min/106 cells in mouse, rat, dog, monkey and human hepatocytes, respectively. A total of 21 different metabolites were found where 11 were detected in human, 14 in rat, 7 in dog and 13 in mouse and monkey. In general, the metabolites observed in hepatocytes across species were formed as a result of multiple biotransformation processes, including oxidations, desaturations, acetylations, loss of sulfur, thioureas to ureas and glucuronidations. A potential biotransformation that includes S‐Glutathione conjugation (M21) was only detected in rat species whereas oxidation plus desaturation (M5), loss of sulfur (M7 and M8) and glucuronidation (M20) were detected in all species. Under these experimental conditions, zamicastat was mainly metabolized by CYP2D6. The extent of metabolism, in decreasing order was CYP2D6 > CYP1A2 > CYP2C19 > CYP2C8 > CYP3A4. No zamicastat metabolism was detected when incubated with CYP2B6 or CYP2C9 recombinant enzymes.ConclusionAll the metabolites detected in human hepatocytes were also detected in the pre‐clinical species suggesting that, under the conditions tested no human specific metabolite was observed. The major cytochromes P450 involved in zamicastast metabolism are CYP2D6, CYP1A2 and 2C19.
Amino Acids Influence DMT1 Trafficking in Duodenal Enterocytes and Modulate Iron Absorption
Authors: Woloshun Regina R. , Yu Yang , Xu Xiaodong , Lee Jennifer K. , Zhu Sean , Theophilus Rufus , Ebea Pearl , Vidyasagar Sadasivan , Collins James F. 
Published: April 2020

Iron deficiency (ID), usually due to excessive menstrual bleeding, rapid neonatal and adolescent growth, pregnancy or gastric bypass, is quite common in the U.S. Iron overload (IO) also afflicts thousands of individuals in America, being most frequently associated with Hereditary Hemochromatosis (HH) and β‐thalassemia. Treatment of ID with iron supplements is not always effective, and current treatments for HH, including phlebotomy and iron chelation, are either non‐specific or have negative side effects. Development of improved treatments for these common conditions is thus a priority. We previously demonstrated that select amino acids (AA) can influence trafficking of membrane transport proteins in enterocytes and thus influence electrolyte absorption in the gut. We thus hypothesized that AAs could also influence trafficking of the predominant intestinal iron importer, divalent metal‐ion transporter 1 (DMT1), thus modulating iron absorption. To screen for effects of individual AAs on DMT1 trafficking, ex vivo duodenal loops excised from mice were incubated individually with all 20 AAs. Western blots were performed to quantify DMT1 amounts on the brush‐border membrane (BBM). Two AA formulations were subsequently made that contained mixes of AAs that either increased or decreased DMT1 BBM protein expression. 59Fe flux studies were then performed with mouse duodenal epithelial organ cultures using Ussing chambers after exposure to the AA mixtures. These experiments demonstrated that one AA formulation increased 59Fe flux by ≈4‐fold (p<0.05). In Fe‐deficient mice, short‐term 59Fe gavage studies with these AA demonstrated a significant increase in 59Fe absorption (p<0.05), and 59Fe accumulation in the liver and blood (p<0.05). To test the other AA formulation (which decreased DMT1 BBM protein expression), we utilized a mouse model of HH, hepcidin (Hamp) KO mice, in which intestinal iron absorption is inappropriately elevated. Importantly, 59Fe flux studies with Hamp−/− mice showed ≈10‐fold reduction in iron transport (p<0.05) when exposed to the AA mixture. A 3‐week daily gavage of these AA in weanling Hamp−/− mice demonstrated a significant decrease in serum ferritin (p<0.05). We thus conclude that select AAs can influence DMT1 trafficking to and from the BBM and thus alter iron flux. Although additional in vivo testing is ongoing, these findings could lead to the development of AA formulations that could be used to mitigate pathological changes in intestinal iron transport associated with both ID and IO.Support or Funding InformationThis work was supported by NIH grants R01 DK074867 and R01 DK109717 (to JFC).
Vagal Feedback Obscures the Effects of Systemic Opioids on Respiratory Rate in the preBötzinger Complex in Young Rabbits in vivo
Authors: Stucke Astrid G. , Palkovic Barbara , Callison Jennifer J. , Marchenko Vitaliy , Stuth Eckehard A.E. , Zuperku Edward J. 
Published: April 2020

IntroductionWe have previously shown in vagus‐intact young rabbits that respiratory rate depression from clinical dose‐rates of remifentanil was partially reversed by naloxone injection into the Parabrachial Nucleus (PBN)
(1). On the contrary, naloxone injection into the preBötzinger Complex (preBötC) changed the ratio between inspiratory (TI) and expiratory (TE) duration but did not reverse respiratory rate depression
(2). To determine whether the entrainment of the respiratory rhythm to the ventilator contributed to the observed lack of respiratory rate reversal, we repeated the study in vagotomized animals.MethodsThe study was approved by the local Animal Care Committee and conformed to NIH standards. Young New Zealand White rabbits (16–39d, 155–547g) were anesthetized, tracheotomized, ventilated, decerebrated and vagotomized. Phrenic nerve activity was recorded from the c5 rootlet, time averaged and used to calculate TI, TE and peak phrenic activity (PPA). Gridwise localized pressure microinjections of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA, 50 μM, 35 nl) utilizing a multibarrel glass pipette with a tip diameter of 20μm into the ventral respiratory column rostral to obex identified the preBötC where AMPA injection caused significant tachypnea. We infused remifentanil until respiratory rate was depressed to 50% of control. After reaching steady‐state, we microinjected naloxone (1mM, 350nl) into the bilateral preBötC and, once the effect had reached steady‐state, we injected naloxone (100mcM) intravenously. Data are provided as mean±SD. Comparison of drug effects within one group used 1‐way repeated‐measures ANOVA. Comparison of drug effects between the current study and an earlier study in non‐vagotomized animals used 2‐way ANOVA.ResultsIn seven rabbits, remifentanil infusion reduced respiratory rate from 29±14 breaths per minute (bpm) to 15±8 bpm, which was due to an increase in TI from 0.9±0.2s to 1.4±0.4s and an increase in TE from 1.6±1.1s to 3.4±1.8s (all p<0.001). Peak phrenic activity (PPA) was not significantly decreased (78±23%). Naloxone injection into the preBötC increased respiratory rate to 21±11bpm (p=0.013) due to a decrease in TI to 1.1±0.4s (p=0.028) and TE to 2.3±1.4s (p=0.009). PPA did not change. The difference from our previous study where we did not observe a reversal of respiratory rate depression (11±6 to 15±9bpm, n=12, p=0.198) was not significant. However, there was a difference between the change in TI:TE ratio, which increased more in vagus‐intact animals (0.5 to 1.1 vs. 0.5 to 0.5, p=0.04).ConclusionSystemic opioids affect respiratory phase duration through action in the preBötC in young rabbits, however, the effect is obscured when respiratory rhythm is entrained to the ventilator in vagus‐intact animals.Support or Funding InformationR01GM112960‐01A1 (Dr. Stucke) and VA merit grant I01BX000721 (Dr. Zuperku)
Neuronal Nitric Oxide Synthase Associated Protein: Nos1ap mediates Sympathoexcitation through Paraventricular Nucleus of the Hypothalamus
Authors: Sharma Neeru M. , Katsurada Kenichi , Nandi Shyam S. , Gao Lie , Patel Kaushik P. 
Published: April 2020

Exaggerated sympathoexcitation is associated with multiple disorders, including hypertension, heart failure, diabetes leading to various cardiovascular complications. Within the paraventricular nucleus of the hypothalamus (PVN), nitric oxide (NO•) is synthesized by neuronal nitric oxide synthase (NOS1) and acts as a non‐conventional neurotransmitter that inhibits neuronal firing and consequent sympathetic outflow. NOS1 is tethered to N‐methyl D‐aspartate receptor (NMDAR) channels harboring NMDAR subunit 2B by the scaffolding protein postsynaptic density 95 (PSD95). Activation of the NMDAR at the synaptic membrane initiates a Ca2+ influx that stimulates the production of NO•. NOS1 adaptor protein (Nos1ap) competes with PSD95 for NOS1 binding and thus influences the production of NO•. We investigated the effect of over‐expressing Nos1ap in the PVN on sympathetic outflow using Cre recombinase‐conditional Nos1ap‐transgenic (Tg) mice. The Nos1ap Tg mice were microinjected with lentiviral expressing Cre‐GFP or GFP bilaterally into the PVN following our standardized method, referred to as CAPON‐OE and Control, respectively. Two weeks of post‐viral administration, the sympathetic outflow, was evaluated. CAPON‐OE mice displayed a significantly elevated urinary excretion of norepinephrine, an index of overall sympathetic outflow compared to Control (0.64±0.11* vs. 0.17±0.03 ug/day). Basal renal sympathetic nerve activity, measured as a percent of maximal activity was elevated 1.5‐fold in CAPON‐OE (46.09 ± 3.56* vs.31.04 ± 3.24) mice. Furthermore, FosB (a member of the AP‐1 family of transcription regulatory proteins) immunocytochemistry revealed increased expression in the PVN of CAPON‐OE mice when compared to control mice suggesting a chronic activation of PVN neurons after Nos1ap over‐expression. Interestingly, a robust over‐expression of Nos1ap in the PVN of CAPON‐OE (0.49±0.04* vs. 0.21±0.03) compared to control mice does not lead to any significant change in NOS1 expression. However, DAF‐FM staining of PVN slices showed decreased NO• levels in CAPON‐OE mice when compared to control mice suggesting that the decrease in NMDAR‐mediated NOS1 activity due to sequestering of NOS1 by Nos1ap leads to less NO• production. Taken together, our data indicate that specific over‐expression of Nos1ap within the PVN reduces the levels of NO• generated by NOS1, leading to increased sympathetic outflow.Support or Funding InformationSupported by AHA ‐ 14SDG19980007 and NIH grant HL62222.
Role of Rhodopsin in Retinitis Pigmentosa
Authors: Link Tina , Stanovski Lisa , Handa Samir , Malladi Pranav , Nathan Ajay , Maraghi Sophie , Thomas-Wilkinson Shiloh 
Published: April 2020

The ability of vertebrates, including humans, to see light relies on a visual transduction cascade in which rhodopsin’s ability to stimulate the nervous system plays a central role. Rhodopsin is a 348 amino acid protein composed of seven transmembrane alpha helices located within the rod photoreceptor cells of the retina. Rhodopsin is a member of the much broader protein superfamily, the G‐protein coupled receptors (GPCRs). GPCRs are cell surface signaling receptors that convert extracellular signals into intracellular signaling pathways through the activation of G proteins. There are about 800 different human GPCRs and members of this superfamily are responsible for our sense of smell, taste, and vision. The “wild type” rhodopsin uses its retinal pigment, a derivative of vitamin A, in conjunction with its G protein partner, transducin, to detect light, particularly in night vision. Retinal serves as the “cofactor” of rhodopsin that directly absorbs a photon, causing its isomerization and a change in the structure of rhodopsin that is sensed by transducin, leading to an intracellular signaling cascade that results in a signal being sent through the optic nerve to the brain that light has been detected. Any one of over 150 rhodopsin mutations already documented in humans are known to result in loss of rhodopsin’s light‐sensing function, leading to Retinitis Pigmentosa (RP). RP is a rare, slow, progressive, genetic disease which damages the retina leading to loss of vision, particularly among young adults. The most common cause of RP is a mutation‐induced misfolding of rhodopsin. The many forms of proposed treatment of RP are largely focused on improving folding of rhodopsin and inducing proteostasis, the process in the cell that promotes correct folding and quantities of proteins. Pharmacological chaperones, such as retinoids or non‐isomerrizable retinoid analogues directly target protein structure and have produced lab results that show enhanced folding and protection of other cells from the effects of mutated rhodopsin. However, replicating this in living organisms has not been possible yet. Nutritional supplements, histone deacetylase inhibitors, such as Valproate, and molecular chaperone inducers have also been tested, but only produce laboratory results that positively affect only a few aspects of mutated rhodopsin and have not yet yielded promising results in humans. Seemingly, the most promising forms of treatment for RP and rhodopsin mutations are gene therapy and CRISPR Cas‐9 genome editing to correct the genetic defects that underlie RP. However, whether any of these approaches will eventually form the basis for effective treatment or cure of RP is not yet known. The Walton High School MSOE Center for Biomolecular Modeling SMART Team has designed a 3‐D model of rhodopsin to further investigate and examine the structure‐function relationship of the protein.Support or Funding InformationMSOE Center for Biomolecular Modeling
Teaching and Assessing Pharmacology Learning Using Different Approaches in UCSF’s Integrated Bridges Curriculum – Omnes viae Romam ducunt?
Authors: Tuan Rupa Lalchandani , Kruidering-Hall Marieke 
Published: April 2020

OBJECTIVETo evaluate the association between different teaching strategies of pharmacology content and performance on open‐ended question (OEQ) exams.BACKGROUNDThe UCSF Bridges curriculum is an integrated, organ systems‐based curriculum. Students learn via multiple modalities including A) live lectures, B) small groups, C) online videos, D) click‐through online PowerPoints and E) interactive case‐based wrap‐up sessions co‐taught with clinicians. Summative assessments for each block are OEQ and require application of knowledge. Pharmacology‐specific OEQ scores were gathered from all blocks that taught and assessed pharmacology: Ground School, “GS” (modalities A&B&C&E); Airways, Blood, Circulation, “ABC” (A&B&D); Renal, Endocrine, GI, and Nutrition, “REGN” (A&B&C&E+flashcards on exam); Pathogens & Host Defense, “PHD” (C&E+flashcards on exam); and Brain, Movement, & Behavior, “BMB” (C&E).METHODSWe collected pharmacology OEQ scores from medical students in the 2017–2018 academic year. OEQs were scored by trained faculty on a scale of 1–6 using a holistic rubric: “meets expectations” (5,6), “borderline” (3,4) or “does not meet expectations” (1,2). OEQ scores were averaged and results were analyzed using repeated measures analysis of variance in SPSS software. Three students who did not complete all blocks were removed from analysis.RESULTSData was considered continuous and scores were not normally distributed. Mean performance on pharmacology OEQs was significantly different across blocks (GS: 5.56±0.03; ABC: 5.21±0.03; REGN: 5.07±0.03; PHD: 4.99±0.03; BMB: 5.45±0.03; ± SEM, n=149).CONCLUSIONThe class average in all blocks was >4.99, suggesting that a variety of teaching methods are effective. Small differences across blocks may be related to the specific methods used in each block. We are currently finalizing data collection from the 2018–2019 academic year, which will be analyzed and added to the current study.