bluuebunny/crossref_metadata_2025 huggingface dataset:
Learning Focus: Scaffolded Primary Literature Curriculum for Improved Student Skills
Authors: Miller Keith R. , Boyd-Kimball Debra
Published: April 2020

Essential student skills in the molecular life sciences include the ability to locate relevant chemical literature, to critically analyze and evaluate primary literature, and to communicate scientific understanding in written and oral forms. Often, students have difficulty using primary literature and there is a lack of resources for purposeful development of these skills. To improve undergraduate skills for reading, writing, and critiquing primary literature, a curriculum was developed for an introductory biochemistry lecture to allow students to engage with primary literature in a controlled environment and gradually increase their usage. The curriculum consisted of case studies relating to lecture topics and figure summaries, where students would locate a primary, peer‐reviewed research article and summarize one of the figures from the article. After gaining experience with primary literature in these assignments, students wrote a literature review on a protein’s structure‐function relationship. This scaffolded primary literature curriculum was intended to shift student learning from passive to active engagement by applying their knowledge to reading and critiquing science to improve their confidence. From 2016–2019, the curriculum was assessed using student evaluations, pre‐ and post‐surveys, and student writing samples. Students reported that they felt more comfortable understanding the background necessary to use and critique primary literature, and communicate about that literature in written form. Student performance on these assignments showed that statistical improvement in assignment scores was not achieved until the third case study or figure summary.
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

Objective

Evaluation of the anti‐proliferative mechanisms in HeLa cells cultures by cyclodipeptides from Pseudomonas aeruginosa PAO1.

Methods

Cyclodipeptides 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.

Pseudomonas 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.

Conclusions

Our 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 Information

Financial 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

Background

Zamicastat
(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.

Objectives

The 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.

Results

The 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.

Conclusion

All 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 Information

This work was supported by NIH grants R01 DK074867 and R01 DK109717 (to JFC).
Expression Regulation of Hox and paraHox Genes by Perfluorochemicals in Mouse Liver
Authors: Zhang Yue , Cheng Xingguo
Published: April 2020

Homeodomain proteins, encoded by Hox genes, play important roles in the development and morphological diversification of organisms. Perfluorochemicals (PFCs), which are persistent environmental pollutants and frequently detected in human blood, interfere with animal development. PFCs produce numerous adverse effects in the liver of research animals, such as hepatomegaly and hepatic dyslipidemia, primarily via activation of peroxisome proliferator‐activated receptors (PPARs) and/or constitutive androstane receptor (CAR). No report shows that PFCs alter the expression of Hox genes. This study was designed to determine the regulation of Hox (including Hox‐a to ‐d subfamily members) and paraHox (including Gsx, Pdx, and Cdx gene family members) gene expression by perfluorochemicals including perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and perfluorodecanoic acid (PFDA) in mouse liver. A single dose of 0.1 mmole/kg PFOA, PFNA or PFDA was intraperitoneally administered to adult male C57BL/6 wild‐type mice, as well as age‐matched male PPARα‐null and male CAR‐null mice (n=6/genotype/treatment). Four days later, mouse livers were collected and processed. PFNA induced mRNA expression of Hoxa5, b7, c5, c8, and d10 in the livers of wild‐type and CAR‐null mice, but not in PPARα‐null mouse liver, indicating a PPARα‐dependent mechanism. In addition, PFOA, PFNA, and PFDA all induced mRNA expression of Hoxb7, c5, and d10 in wild‐type but not PPARα‐null mouse liver. Furthermore, Wy‐14643, a classical PPARα agonist, induced mRNA expression of Hoxc5 in wild‐type but not PPARα‐null mice. However, Wy‐14643 did not alter mRNA expression of Hoxa5, b7 or d10 in either wild‐type or PPARα‐null mouse liver. TCPOBOP, a classical CAR agonist, did not alter mRNA expression of Hoxa5, b7, c5 or d10 in mouse liver. Moreover, PFNA increased cytoplasmic levels of Hoxc5 protein in mouse liver. In conclusion, perfluorochemicals induced the expression of certain Hox genes such as Hoxc5, mainly via PPARα activation.
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 Information

Supported 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 Information

MSOE 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

OBJECTIVE

To evaluate the association between different teaching strategies of pharmacology content and performance on open‐ended question (OEQ) exams.

BACKGROUND

The 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).

METHODS

We 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.

RESULTS

Data 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).

CONCLUSION

The 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.
Stimulation of Auricular Vagal Nerves Attenuates Pressor Cardiovascular Responses through Influence on Medullary nuclei
Authors: Guo Zhiling , Samaniego Tracy , Malik Shaista
Published: April 2020

Stimulation of the auricular branch of the vagus nerve (ABVN) is used to manage cardiovascular disorders, including elevated blood pressure. However, precise mechanisms underlying its effects remain unclear. One previous study showed that ABVN stimulation with acupuncture at the center of the inferior concha (the “heart acupoint,” CO15) activates neurons in the nucleus tractus solitarii (NTS) through vagal inputs. Still, its actions on NTS processing and downstream influence on the nucleus regulating cardiovascular function have not been studied. Thus, the present study evaluated the effect of ABVN stimulation on pressor cardiovascular responses and the underlying neural mechanisms associated with medullary nuclei. We hypothesized that ABVN stimulation with acupuncture activates the NTS and ultimately inhibits rostral ventrolateral medulla (rVLM), a critical region controlling cardiovascular function, through opioid and GABA mechanisms. In Sprague‐Dawley rats, repeated pressor cardiovascular reflexes were induced by repetitive gastric distension (GD) under anesthesia. ABVN stimulation with manual acupuncture was performed at the CO15 acupoint with gentle rotation of the acupuncture needle at a frequency of □2 Hz for four min every 10 min during a 30 min period, 2 min in each side of the ear. During sham‐ABVN stimulation, the acupuncture needle was inserted into the same site without subsequent rotation of the needle. Unilateral microinjection of the chemical (50 nl) was conducted in the NTS and rVLM. We found that GD‐induced pressor cardiovascular reflexes significantly attenuated by ABVN stimulation (n=8; P<0.05), but neither by sham‐ABVN stimulation nor mechanical stimulation with the acupuncture needle applied at the HX9 acupoint, which is located at the ear tip overlying lesser occipital nerve, the auricular somatic nerve. The modulation of pressor reflexes by ABVN stimulation was significantly (P<0.05) reversed by microinjection of kynurenic acid (25 mM; n=7) into the NTS as well as naloxone (1 mM; a non‐selective opioid receptor antagonist; n=8) or gabazine (27 mM; a GABAA receptor antagonist; n=8) into the rVLM, but not by their vehicles, 0.9% normal saline. Furthermore, in rats subjected to no GD intervention, we noted that ABVN stimulation induced markedly increase in c‐Fos expression in both NTS and rVLM, compared to sham‐ABVN stimulation. Our data suggest that ABVN stimulation with manual acupuncture excites the NTS through a glutamatergic mechanism, which, in turn, attenuate excitatory cardiovascular reflexes evoked by visceral stimulation through GABA and opioids in the rVLM.

Support or Funding Information

The National Center for Complementary & Integrative Health, R01 grant AT009347 and diversity supplement award to this grant.
Berberine (BBR) Induced Bile Salt Export Pump (Bsep) Expression in Mouse Liver and Cultured Hepatoma Cells
Authors: Le Yuan , Cheng Xingguo
Published: April 2020

Bile salt export pump (Bsep) is responsible for the biliary excretion of majority of bile salts in the liver. Dysfunction of Bsep often leads to disruption of the enterohepatic circulation of bile salts and thus cholestasis. Berberine (BBR), an active component of certain traditional herbal medicine such as Goldenseal, shows anti‐oxidative and anti‐inflammatory properties. Nuclear factor erythroid 2‐related factor (Nrf) 2 plays important roles in fighting against oxidative stress and inflammation. We recently showed that BBR increased Bsep mRNA expression in mouse liver. However, the underlying mechanism by which BBR induces Bsep expression remains largely unknown. The present study was designed to determine whether BBR induces Bsep expression via activation of Nrf2 signaling. BBR dose‐dependently increased mRNA and protein expression of Bsep in mouse liver. The studies, conducted in cultured mouse and human hepatoma cells, showed that BBR (1) increased Bsep/BSEP mRNA and protein expression, (2) enhanced the cellular efflux of Dichlorofluorescin diacetate, a known Bsep/BSEP substrate, and (3) stimulated the Bsep/BSEP gene promoter‐driven luciferase reporter activity. To determine whether Nrf2 activity is responsible for BBR‐induced Bsep expression, we knocked down Nrf2 expression in cultured hepatoma cells by using silence RNA (siRNA) that specifically targets Nrf2. Our results showed that after Nrf2 knock‐down, BBR no longer induced BSEP mRNA expression in human hepatoma cells. In contrast, in mouse hepatoma cells, Nrf2 silence partially attenuated BBR‐induced Bsep expression. In addition, promoter partial‐deletion followed by promoter‐driven luciferase reporter assay showed that deletion of putative Nrf2 binding site(s) in mouse and human Bsep/BSEP gene promoter abolished BBR‐induced luciferase activity in human but not mouse hepatoma cells. In conclusion, BBR induces human BSEP expression via Nrf2 signaling. In contrast, BBR induced Bsep expression in mouse liver or hepatoma cells independent of Nrf2 signaling.
Signalosome nucleation enables digital innate immune proinflammatory responses
Authors: Gama Alejandro Rodriguez , Miller Tayla , Lange Jeffrey , Halfmann Randal
Published: April 2020

Pathogen sensing in innate immune cells begins with the activation of Pathogen Recognition Receptors (PRR) that result in the formation of polymeric protein self‐assemblies known as signalosomes. The proinflammatory response resulting from signalosome formation is thought to produce a binary all‐or‐none response. How this is controlled remains unclear. Because the activation of ordered self‐assemblies like signalosomes implies a rate‐limiting nucleation step, we hypothesized that a nucleation barrier imposed by the signalosome assembly enables the digital all‐or‐none response. Here we focused on the signalosome CARD9‐Bcl10‐MALT1 (CBM), which functions downstream of fungal PRR, Dectin‐1, which activates NF‐κB. We employed DAmFRET, a recently developed method that allows to determine the frequency of protein nucleation in cells using flow cytometry. We determined that CARD9 and Bcl10 form nucleation‐limited polymers in cells with critical concentrations similar to those from in vitro studies. We then dissected the nucleation steps required for signalosome formation. Co‐expression of artificial CARD9 polymer seeds with Bcl10 resulted in the complete nucleation of Bcl10 monomeric protein. Remarkably, CARD9 seeds containing mutations that impair antifungal signaling in humans were unable to nucleate Bcl10, suggesting that a direct seeding effect of CARD9 over Bcl10 is critical for its function. We next asked whether the formation of the signalosome drives the digital activation of NF‐κB. To do so, we coupled a transcriptional fluorescent NF‐κB reporter to DAmFRET. We determined that cells containing Bcl10 assemblies strongly induced NF‐κB activation, while cells expressing a similar protein concentration in the monomeric state did not. We asked next whether mutations that alter the nucleation barriers likewise modulate NF‐κB activation. Disruption of polymer formation with mutation E53R as well as increasing the critical concentration of polymerization with mutant R36E, both impaired NF‐κB activation. In contrast, mutation R58G decreased the nucleation barrier while also exacerbating the activation of NF‐κB. Our results indicate a critical role of nucleation barriers in governing the functional outcome of signalosomes. We anticipate the impact of our work could uncover the molecular mechanism of innate immune digital proinflammatory activation, which may serve as a basis to understand the cause of autoinflammatory diseases.

Support or Funding Information

Stowers Institute for Medical Research
Apolipoprotein‐M bound sphingosine‐1 phosphate enhances heparan sulfate expression and endothelial nitric oxide synthase phosphorylation in cultured endothelial cells
Authors: Collignon Taylor E. , Norton Tabitha E. , Motawe Zeinab Y. , Breslin Jerome W.
Published: April 2020

Sphingosine‐1 phosphate (S1P) is blood borne bioactive sphingolipid metabolite that has protective properties against microvascular leakage under inflammatory conditions and also can promote endothelial NO production. Previous investigations suggest that functions are mediated by S1P receptor‐1 (S1pr1) but are also limited by action of S1P on other S1P receptors. Apolipoprotein‐M (ApoM), which has been shown to act as a carrier of S1P, may help target S1P to S1pr1, producing biased S1pr1 signaling. In the current study, we hypothesized that combination of ApoM will improve the ability of S1P to enhance 1) endothelial barrier function, and 2) phosphorylation of eNOS on its activation site. We used confluent monolayers of human umbilical vein endothelial cells (HUVEC) to model the endothelial barrier. Transendothelial electrical resistance (TER), determined by electric cell‐substrate impedance sensing, served as an indicator of barrier function. Because endothelial glycocalyx health is related to barrier function, we also determined the degree of heparan sulfate expression on the cell surface, visualized by immunofluorescence microscopy of fixed, non‐permeabilized HUVEC. Western blotting was used to measure eNOS phosphorylation on its activation site (S1177). These endpoints were evaluated in control cells and cells that were treated with ApoM (0.5 μM), S1P (1 μM), or the combination of both. The results show that the addition of ApoM‐bound S1P to HUVEC significantly increased and sustained the increase in TER of the HUVEC monolayers compared to the vehicle and S1P alone. ApoM‐bound S1P also increased heparan sulfate expression on the plasma membrane and elicited enhanced phosphorylation of eNOS on S1177 compared to either ApoM or S1P alone. Collectively, the findings support that ApoM enhances the ability of S1P to promote endothelial barrier function, surface heparan sulfate expression, and activation of eNOS. These findings suggest high potential for using ApoM‐bound S1P as a therapeutic target for ameliorating endothelial dysfunction.

Support or Funding Information

This work was supported by NIH R01GM120774 and American Heart Association 18UFEL33960365. Tabitha E. Norton and Taylor E. Collignon contributed equally to this work.
Regulation of food intake, insulin sensitivity and sympathetic nerve traffic by hypothalamic steroidogenic factor 1 neurons
Authors: Jiang Jingwei , Morgan Donald A. , Cui Huxing , Rahmouni Kamal
Published: April 2020

The ventromedial nucleus of the hypothalamus (VMH) was the first site to be recognized as a key player in body weight regulation. The VMH has also been implicated in brown adipose tissue (BAT) thermoregulation, sympathetic nerve activity and glucose homeostasis. Within the VMH, neurons expressing steroidogenic factor 1 (SF1) which are equipped with receptors that binds various hormonal signals have emerged as key mediators of metabolic control. In this study, we assessed how Designer Receptors Exclusively Activated by Designer Drugs (DREADD)‐mediated activation of SF1 neurons affect food intake, glucose homeostasis, insulin sensitivity and sympathetic nerve activity. Injection of an adeno‐associated virus expressing a Cre‐dependent mCherry tagged‐hM3Dq (AAV‐hM3Dq) into the VMH of SF1Cre+ mice led to VMH‐restricted expression of mCherry validating our strategy. In SFCre+ male mice, DREADD‐mediated activation of SF1 neurons caused a dramatic feeding stimulatory response within 4h of CNO injection (0.75±0.16g relative to 0.13±0.07g in control SF1Cre− mice, P<0.05). Interestingly, in male mice SF1 neuron‐specific activation induces BAT sympathetic nerve activation without affecting renal sympathetic tone. In female mice, food intake and BAT sympathetic traffic were not altered after activation of SF1 neurons. On the other hand, insulin sensitivity was significantly attenuated only in females following SF1 neuron activation (100.20±13.07mmol/L vs 25.25±12.15mmol/L in control at 2h, P<0.05). Glucose homeostasis was not affected upon SF1 neuron activation in both male and female mice. These results show that VMH SF1 neurons regulate food intake, sympathetic tone and insulin sensitivity in a sex‐specific manner. Our findings also provide new insight into a previously unappreciated role of VMH SF1 neuron in the differential control of regional sympathetic nerve traffic.
Interactions of Adolescent Over‐Nutrition and Adult Alcohol Exposure on Metabolic Function in Mice
Authors: Coker Caitlin R. , Aguilar Elizabeth A. , Arnold Amy C. , Silberman Yuval
Published: April 2020

Over‐nutrition with calorically dense diets high in fat (high fat diets, HFD) during adolescence may result in long‐lasting changes in mechanisms regulating adult insulin and glucose metabolism. These changes may be further influenced by adult alcohol (EtOH) intake leading to increased risk of metabolic disorders, such as insulin and glucose dysregulation typically seen in Type II Diabetes. We hypothesized that over‐nutrition during adolescence promotes long‐lasting perturbations in insulin signaling leading to enhancement of EtOH‐induced metabolic disruptions during adulthood. Male and female C57Bl/6J mice had ad libitum HFD or control diet from post‐natal day (PND) 28–43, after which they were all given control diet. Mice were then exposed to EtOH either one time (acute) or three times (chronic) beginning on PND70 via vapor chamber exposure to ensure equal blood ethanol concentration (BEC) levels across groups; control animals were not exposed to EtOH. Glucose and insulin tolerance tests (GTT, ITT) were performed following the last EtOH exposure. In control diet male mice, repeated EtOH exposure worsened 4 hour fasting glucose levels without altering insulin or glucose tolerance or altering 12 hour fasting glucose levels. Mice receiving adolescent HFD showed significantly increased 4 hour and 12 hour fasting glucose levels but EtOH exposure reduced these heightened levels without altering ITT or GTT results. These findings suggest that EtOH causes divergent changes in glucose control in adult mice based on history of adolescent HFD exposure which may be due to alterations in insulin and glucose counter‐regulatory mechanisms unrelated to insulin and glucose tolerance. Overall, female mice appear to be protected from adolescent HFD effects on adult metabolic dysregulation with EtOH producing divergent metabolic effects depending on length of EtOH exposure. Together, these results suggest there are sex differences in the effects of adolescent over‐nutrition and adult EtOH exposure on insulin and glucose function. Experiments are currently underway to determine if hypothalamic neuroimmune activity regulates the interaction of adolescent over‐nutrition and adult EtOH exposure on whole‐body insulin and glucose homeostasis.

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F31 AA027697

R01 AA026865
L‐Menthol Inhibits Cardiac Sympathoexcitation Caused by Inhaled Irritants in the Spontaneously Hypertensive Rat
Authors: Hooper Justin Shane , Taylor-Clark Thomas E.
Published: April 2020

Inhalation of irritants (e.g. particulate matter and ozone) triggers various efferent responses through activation of transient receptor potential ankyrin 1 (TRPA1) and TRP vanilloid 1 (TRPV1) channels expressed on airway vagal afferent nerves. In the airways they cause cough and mucus secretion and in conscious, healthy animals parasympathetic‐mediated reflexes include bradycardia and atrioventricular block. However, inhalation of air pollutants including particulate matter and ozone evokes sympathoexcitation (tachycardia, hypertension) in patients with cardiovascular disease. Interestingly, selective activation of airway vagal afferent nerves in spontaneously hypertensive (SH) rats via inhalation of the TRPA1‐specific agonist allyl isothiocyanate (AITC) evoked sympathetic‐mediated tachycardia and premature ventricular contractions (PVCs) consistent with reflexes evoked by inhaled pollution in patients with cardiovascular disease.

Airway reflexes such as cough have been shown to be inhibited by nasal stimulation with L‐menthol via activation of cold‐sensitive trigeminal pathways which regulate brainstem respiratory networks. Here, we investigated the cardiac reflexes evoked by inhalation of L‐menthol (150mg/ml) alone and in combination with AITC (4.7mg/ml) in conscious SH rats. L‐menthol inhalation alone had no effect on heart rate, but, when inhaled during AITC inhalation, L‐menthol selectively inhibited the AITC‐evoked tachyarrhythmia. Inhibition of the AITC‐evoked sympathoexcitatory reflexes was not seen in identical studies performed with inactive D‐menthol (150mg/ml). Therefore, we conclude that pulmonary‐cardiac reflex tachyarrhythmia are reduced by activation of nasal menthol‐sensitive pathways. This data supports the potential for prevention of aberrant irritant‐evoked pulmonary‐cardiac reflexes by nasal menthol treatment.

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NIH r56
Defining Opioidergic, Glutamatergic Pontine Respiratory Patterned Neurons
Authors: Saunders Sandy , Levitt Erica
Published: April 2020

Opioid overdose deaths have rapidly increased largely due to illicitly manufactured fentanyl. The main cause of death from opioid overdose is respiratory depression. Opioid‐induced respiratory depression is characterized by decreases in respiratory rate, chemoreceptor reflexes and impairment of the upper airway. Pharmacological inhibition of the Kölliker‐Fuse (KF) nucleus produces strikingly similar impairments to breathing as opioids, thus warranted investigation. Using the arterially perfused preparation, we have previously found that application of CTAP, a selective mu opioid receptor antagonist, directly into the KF prevents fentanyl induced apnea. Although the KF is sufficient to restore phasic motor output, it did not restore eupneic 3‐phase respiratory patterning. To determine the identity of KF neurons modulated by opioids, we performed extracellular recordings before, during and after fentanyl administration. Respiratory phase was determined by phrenic and vagal motor output. A subpopulation of inspiratory and post‐inspiratory pattered neurons stopped firing upon fentanyl administration, whereas, a variety of respiratory patterned neurons continued to fire in the presence of fentanyl, albeit some with changes in frequency. Although many medullary glutamatergic respiratory populations have been identified, the firing pattern of pontine respiratory modulated glutamatergic neurons remain unknown. In separate experiments, a promoter specific optogenetic approach was used with extracellular recording to identify Ca2+/Calmodulin‐dependent protein kinase II (CaMKIIa) expressing, presumably glutamatergic, neurons. These results suggest that KF neurons are differentially impacted by opioids and during overdose may promote aberrant glutamatergic transmission to downstream respiratory nuclei.

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This work was supported by NIH R01 DA047976.
Targeting cerebrovascular sphingosine‐1‐phosphate signaling to treat heart failure‐induced brain dysfunction
Authors: Vanherle Lotte , Uhl Franziska Elisabeth , Matthes Frank , Meissner Anja
Published: April 2020

Chronic cardiovascular diseases increasingly emerge as risk factors for neurodegeneration. Heart failure for instance, induces a chronic reduction in cerebral perfusion. Such chronic brain perfusion deficits are frequently accompanied by cognitive decline, which not only seriously impacts the patients’ quality of life but also reduces treatment compliance. Statistically, cognitive impairment is 5 times more prevalent in heart failure patients relative to the age‐matched general population. We previously demonstrated that heart failure associates to augmented signaling of a potent bioactive phospholipid called sphingosine‐1‐phosphate (S1P). During heart failure, such enhanced S1P signaling roots from dysfunctional cystic fibrosis transmembrane regulator (CFTR) expression and activity. CFTR controls the bioavailability of cerebrovascular S1P and hence, its pro‐constrictive effects. In heart failure, reduced microvascular CFTR expression correlates with enhanced myogenic tone and reduced cerebral blood flow.

In this study, we investigated the therapeutic potential of CFTR corrector compounds to normalize cerebrovascular S1P signaling and thereby, improve neurological function during experimental heart failure in mice.

Systemic CFTR corrector treatment stabilized cerebrovascular CFTR expression, improved myogenic tone and rectified deficient cerebral perfusion in heart failure, without altering the systemic parameters of cardiac output or total peripheral resistance. This normalization associated to recovery of neuronal integrity (i.e., dendrite morphology and dendritic spine density) and cognitive function. The heart failure‐associated dendrite atrophy (i.e., a 22% reduction in dendrite length) was not altered in mice treated with CFTR corrector compound. Longitudinal pre‐treatment (i.e., at 10 weeks post‐infarction) and post‐treatment analyses of the same vehicle‐ and CFTR corrector‐treated HF mice confirmed that CFTR corrector treatment reversed the impaired rhino‐cortical‐dependent, short‐term retention of object familiarity caused by heart failure.

Our results put forward a new mechanistic understanding of heart failure‐associated brain complications, and CFTR correctors as potential treatment option for heart failure‐associated cognitive decline.

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This work is supported by the Knut & Alice Wallenberg Foundation and the Swedish Research Council.
Transcriptional regulations induced by estradiol and intermittent hypoxia in the lungs of ovariectomized female rats.
Authors: Jochmans-Lemoine Alexandra , Ribon-Demars Alexandra , Ganouna Gauthier , Marcouiller François , Pialoux Vincent , Bairam Aida , Joseph Vincent
Published: April 2020

Intermittent hypoxia (IH) leads to oxidative stress and inflammation contributing to tissue and cellular dysfunctions in sleep apnea patients. We showed that estradiol (E2) reduces oxidative stress markers in the brain and aorta of female rats exposed to IH (model of sleep apnea), but it remains unknown if the effects of E2 in IH occur in other tissues such as the lungs, in which E2 receptors are present. Interestingly, both IH and E2 receptors act through specific transcription factors, changing the expression of target genes, and potential interactions between these transcription networks are not known. We therefore asked if IH exposure induces oxidative stress and alters the gene expression pattern in the lungs of ovariectomized female rats, and if E2 supplementation prevents these effects. Female Sprague‐Dawley rats were ovariectomized (OVX) and implanted with osmotic minipumps delivering vehicle (OVX‐VEH), or E2 (0.5 mg/kg/day). Two weeks following surgery, the rats were exposed to room air (RA), or IH for 7 days (nadir 10% O2, 10 cycles/hour, 8 hours/day). Lung samples were used to measure the oxidative stress marker AOPP (Advanced Oxidation of Protein Products) and determine the gene expression profiles (RNA microarray, ThermoFisher Clariom S rat assay). In OVX‐VEH female rats, E2 treatment, but not the IH exposure, increased AOPP level. In OVX‐Veh rats, IH upregulated the expression of 11 genes and down regulated 5 genes, none of these genes are involved in oxidative stress. E2 in RA rats upregulated 130 genes and downregulated 112 genes including CYP1a1, a cytochrome involved in production of reactive oxygen species. In rats treated with E2, the IH exposure increased the expression levels of 270 genes and decreased the expression of 828 genes (including anti‐oxidant genes). We conclude that in the lungs of OVX female rats, IH has only limited effects on oxidative stress and on genes transcriptional pattern. E2 treatment under RA conditions apparently increases oxidative stress and has more important transcriptional effects than IH exposure. Finally, E2 treatment has profound effects on the transcriptional response to IH exposure, and helps controlling the oxidative stress response. This important interaction between E2 and IH might be relevant to better understand the cellular responses to IH and sleep apnea in women.

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Supported by CIHR.