National Center For Research Resources Research Articles

Neurotoxic Dopamine Transporter Substrates Differentially Affect DAT Regulation, Uptake, and Reverse Transport

Synaptic action of the neurotransmitter dopamine (DA) is primarily terminated by reuptake into presynaptic dopaminergic neurons via dopamine transporters (DATs). DATs are integral plasmalemmal proteins whose reuptake function is tightly coordinated by interplay between the post translational modifications phosphorylation and palmitoylation. The psychostimulant amphetamine (AMPH) is a DAT substrate known to empty sequestered DA from vesicles and induce protein kinase C (PKC)‐dependent DAT phosphorylation, resulting in reverse transport of DA (efflux) greatly increased DA signaling. Elevated DA concentrations similar to those produced by AMPH create neurotoxic conditions as DA oxidation produces free radicals and hinders mitochondrial respiration, thus strict spatial and temporal control of cytosolic and synaptic DA is essential for neuronal health. DA induced oxidative stress is an important pathogenic mediator in Parkinson's disease (PD), and animal models of PD are generated by the neurotoxins 1‐methyl‐4‐phenylpyridinium ion (MPP+) and 6‐hydroxydopamine (6OHDA) which selectively target DAT‐expressing neurons, are translocated to the cytosol by DAT, and perturb the oxidative balance within the cell, producing nigrostriatal DA neuron death observed in PD. It is therefore intriguing to investigate DAT‐involved mechanisms in terms of PD etiology as atypical DAT function could contribute to DA oxidative stress and is hypothesized to play a role in other neurological disorders including attention deficit/hyperactivity disorder (ADHD) and addiction. DAT expression occurs around synaptic terminals, along axons, and on dendritic spines, and efflux via DATs located on dendrites in the substantia nigra has been shown to diminish dopaminergic signaling. It is possible, therefore, for aberrant regulation and localization of DA clearance and DAT‐mediated DA efflux to profoundly influence the excitability of neurons and play a significant role in neurodegeneration in the progression of PD. This is even more interesting in the light of recent studies revealing DAT polymorphisms in patients diagnosed with a psychiatric disorder or early onset PD that display an elevated basal DA efflux phenotype. In this study, we are investigating the effect of DAT substrates and PD‐inducing neurotoxins on DAT phosphorylation, palmitoylation, down‐regulation, and reverse transport to better understand aberrant DAT mechanisms which may contribute the onset of PD. Our findings thus far indicate MPP+ and 6OHDA differentially alter DAT function. 6OHDA inhibits basal and attenuates PMA‐induced DAT phosphorylation, does not significantly reduce DAT function, and decreases DA efflux. MPP+ does not change basal or PMA stimulated DAT phosphorylation yet it induces significant DA efflux which may be an additional means by which MPP+ produces neurodegeneration. This MPP+ induced efflux was not blunted by pretreatment with the PKC inhibitor bisindolylmaleimide (BIM). Although AMPH‐induced efflux was blunted after BIM treatment, significant efflux remained suggesting mechanisms independent of PKC mediated DAT phosphorylation for the stimulation of DA efflux.Support or Funding InformationSupported by the National Institute on Drug Abuse Grant DA 031991 JDF, P20 RR017699 (to U.N.D.) from the COBRE program of the National Center for Research Resources, and P20 RR016741 (to U.N.D.) from the INBRE program of the National Center for Research Resources.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Development of Phage Display of Nisin

Since its development in 1985, phage display has proven to be a powerful platform for the selection of peptide ligands. Recently, strategies for the in vitro or in vivo modification of phage-displayed peptides have been developed to allow display and maturation of monocyclic or bicyclic peptides, thereby taking advantage of the increased rigidity and improved resistance to proteolysis demonstrated by such compounds. Ribosomally synthesized and post-translationally modified peptide (RiPP) natural products are an ideal platform for the generation of large peptide libraries containing one or more macrocycles because the primary sequence is genetically encoded and the modification enzymes responsible for macrocycle installation tolerate varied substrates. We have developed and validated a system that allows for assembly on phage of the lanthipeptide nisin, a potent antimicrobial peptide containing 5 post-translationally installed macrocycles. We first demonstrated N-terminal display of nisin. We then generated a library of more than 1 million nisin variants and enriched binders to the small molecule lipid II. We finally characterized the top hit from the selection, revealing both that the modification enzymes accept it as a substrate and that it binds lipid II. Having thus demonstrated the feasibility of N-terminal display of nisin, we expect future work to leverage this platform for display and optimization of a wide array of RiPP natural products. Support or Funding Information This work was supported by the National Institutes of Health (R37 GM 058822 to W.A.v.d.D and F31 GM113486 to K.J.H). The Bruker UltrafleXtreme MALDI TOFTOF mass spectrometer was purchased in part with a grant from the National Center for Research Resources, National Institutes of Health (S10 RR027109 A). Development of phage display of the ribosomally synthesized and post-translationally modified peptide (RiPP) nisin. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Proteomic analysis of germinal vesicles in the domestic cat model reveals candidate nuclear proteins involved in oocyte competence acquisition.

Do nuclear proteins in the germinal vesicle (GV) contribute to oocyte competence acquisition during folliculogenesis? Proteomic analysis of GVs identified candidate proteins for oocyte competence acquisition, including a key RNA processing protein-heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1). The domestic cat GV, which is physiologically similar to the human GV, gains the intrinsic ability to resume meiosis and support early embryo development during the pre-antral-to-antral follicle transition. However, little is known about nuclear proteins that contribute to this developmental process. GVs were enriched from pre-antral (incompetent) and antral (competent) follicles from 802 cat ovaries. Protein lysates were subjected to quantitative proteomic analysis to identify differentially expressed proteins in GVs from the two follicular categories. Two biological replicates (from independent pools of ovaries) of pre-antral versus antral samples were labeled by tandem mass tags and then assessed by liquid chromatography-tandem mass spectrometry. Proteomic data were analyzed according to gene ontology and a protein-protein interaction network. Immunofluorescent staining and protein inhibition assays were used for validation. A total of 174 nuclear proteins was identified, with 54 being up-regulated and 22 down-regulated (≥1.5-fold) after antrum formation. Functional protein analysis through gene ontology over-representation tests revealed that changes in molecular network within the GVs during this transitional phase were related to chromatin reorganization, gene transcription, and maternal RNA processing and storage. Protein inhibition assays verified that hnRNPA2B1, a key nuclear protein identified, was required for oocyte meiotic maturation and subsequent blastocyst formation. Data are available via ProteomeXchange with identifier PXD007211. Proteins identified by proteomic comparison may (i) be involved in processes other than competence acquisition during the pre-antral-to-antral transition or (ii) be co-expressed in other macrostructures besides the GV. Expressional and functional validations should be performed for candidate proteins before downstream application. Collective results generated a blueprint to better understand the molecular mechanisms involved in GV competence acquisition and identified potential nuclear competence markers for human fertility preservation. Funded by the National Center for Research Resources (R01 RR026064), a component of the National Institutes of Health (NIH) and currently by the Office of Research Infrastructure Programs/Office of the Director (R01 OD010948). The authors declare that there is no conflict of interest.

Abstract 3252: Synthesis of N-substituted [Benzoylamino]-3-Ethyl-1,2,3,6-tetrahydropyridines as potential anti-cancer agents

Abstract Chronic inflammation is believed to promote cancer through cellular and genomic damage. Inflammation creates a microenvironment within the tissue that enhances cell proliferation. In 2016, American Cancer Society estimates that 1,685,210 new cancer cases are expected to be diagnosed in the United States. Cyclooxygenase (COX) is an enzyme found to play an essential role in inflammation and converts arachidonic acid to prostaglandin-like molecules. The three isomers of COX are COX-1, COX-2, and COX-3. COX-2 has been found to be up-regulated in inflammation and some cancers, such as breast cancer and prostate cancer. Tetrahydropyridines (THPs) developed by our research group were effective in inhibiting inflammation, COX, and the growth of some breast cancer cells. Previous research has shown that pharmacological activities of the THP derivatives depend greatly on the nature and position of the substituents on the THP ring structure. Here, we present the synthesis of a series of novel THP derivatives. 3-Ethylpyridine is reacted with O-mesitylenesulfonylhydroxylamine (O-MSH) to furnish N-amino-3-ethylpyridinium mesitylenesulfonate. The reaction of N-amino-3-ethylpyridinium mesitylenesulfonate with substituted acid chlorides gives the stable crystalline pyridinium ylides. A sodium borohydride reduction of ylides furnishes the target compounds, N-substituted [benzoylamino]-3-ethyl-1,2,3,6-tetrahydropyridines. Five novel N-substituted [benzoylamino]-3-ethyl-1,2,3,6-tetrahydropyridines were synthesized, purified, and characterized. The evaluation of these compounds cytotoxicity studies on MCF-7 estrogen receptor positive breast cancer cells, MDA-MB-231 estrogen receptor negative breast cancer cell lines, and Ishikawa cells, using the CellTiter-Glo (CTG) luminescent cell viability assay is underway. This research was supported by the National Center for Research Resources and the National Institute of Minority Health and Health Disparities of the National Institutes of Health through Grant Number 8 G12MD007582-28. Citation Format: Elizabeth Henderson, Madhavi Gangapuram, Suresh Eyunni, Kinfe Redda, Tiffany Ardley. Synthesis of N-substituted [Benzoylamino]-3-Ethyl-1,2,3,6-tetrahydropyridines as potential anti-cancer agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3252. doi:10.1158/1538-7445.AM2017-3252

Abstract 173: Synthesis of substituted tetrahydroisoquinoline derivatives as anticancer agents

Abstract Breast cancer is the second leading cause of cancer-related deaths in women today and is the most common cancer among women, excluding non-melanoma skin cancers. Over 246,000 women will be diagnosed with breast cancer and 40,450 are expected to die of it in 2016. Estrogen receptors ERα, ERβ and their associated steroid hormones play vital role in breast cancer development and progression. Estrogen binds and activates the estrogen receptors in certain breast cancer cells. Endocrine therapy which aims to block ER action (antagonism) on breast tumor cells and thereby stopping the proliferation of cancer cells is a very useful strategy in treating breast cancer. Tamoxifen is the first selective estrogen receptor modulator (SERM) and is the leading drug used in treating breast cancer. Although Tamoxifen has shown great benefit in treating breast cancer, its agonistic effect on the uterus is said to be associated with an increased risk of developing endometrial cancer. We are interested in designing a single chemical entity which acts at specific multiple biomolecular targets. This strategy may exert favorable advantages in improving efficacy at desired multiple targets and lower incidence of side effects. Thus, alternative chemical entities, preferably non-steroidal molecules which act as estrogen receptor modulators and microtubule disruptors are sought. The tetrahydroisoquinoline (THIQ) core structure is an important pharmacophore in natural products and small molecules which act as drug molecules. The steroidomimetic tetrahydroisoquinoline moieties were reported to be selective SERMs and microtubule disruptors. Based on these considerations and in continuation with our previous research, herein we report in vitro cytotoxic and in silico docking studies of substituted THIQs. Novel substituted THIQ derivatives were designed and synthesized. N-amination of substituted isoquinolines by the aminating agent, O-mesytelene sulfnylhydroxylamine led to the formation of ylides. The ylides were reduced using sodium borohydride to yield the desired substituted tetrahydroisoquinolines in moderate to good yields. These compounds were evaluated for their cytotoxic effects using MCF-7, MDA-MB-231 and Ishikawa cells using the CellTiter-Glo luminescent cell viability assay. Among all the compounds screened, 4-ethyl-N-(8-hydroxy-3,4-dihydroisoquinolin-2(1H)-yl)benzamide showed IC50 values of 0.61, 1.36 and 0.09 µg/ml on MCF-7, MDA-MB-231 and Ishikawa cells respectively. This research was supported by the National Center for Research Resources and the National Institute of Minority Health and Health Disparities of the National Institutes of Health through Grant Number 8 G12MD007582-28. Citation Format: Kinfe Ken Redda, Madhavi Gangapuram, Suresh Eyunni. Synthesis of substituted tetrahydroisoquinoline derivatives as anticancer agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 173. doi:10.1158/1538-7445.AM2017-173

0300 STRESS SYSTEM DYSREGULATION IN INSOMNIA DISORDER

Insomnia disorder (ID) is highly co-morbid with chronic pain conditions. Dysregulation of the hypothalamus-pituitary adrenal (HPA) stress system suggests a contribution to both ID and chronic pain. We investigated HPA system integrity in individuals with ID, indicated by measures of resting cortisol levels and IL-6 expression in monocytes, glucocorticoid (GC) sensitivity (i.e., sensitivity of cells to the counter-inflammatory signal cortisol), and reactivity of cortisol to a repeated challenge. We hypothesized deterioration of HPA system integrity may represent a mechanistic pathway by which ID increases chronic pain vulnerability. This is an ongoing study with N=19 participants (ages 18–49). Seven participants (1 male) diagnosed of insomnia disorder (ID) based on DSM-V criteria and 12 healthy controls (HC;3 male) completed an overnight polysomnographic (PSG) sleep recording in the Clinical Research Center. The following morning, resting blood samples were collected for measurements of basal serum cortisol levels, IL-6 positive monocytes, and GC sensitivity of monocytes as assessed by flow cytometry. To determine serum cortisol reactivity, participants completed a cold pressor test (CPT) challenge three times in succession (1300,1430,1600) by placing their hand in a temperature-controlled cold water bath for at least one minute. Blood was drawn 20 and 50 minutes after each CPT to measure post-challenge cortisol levels. ID participants had significantly shorter PSG sleep duration (ID:341 ± 110 min; HC:422 ± 31 min). Basal cortisol levels, IL-6 positive monocytes and GC sensitivity did not differ between groups, but cortisol levels in the ID group were significantly higher 20 min (ID:14.24 ± 3.34μg/dL; HC:10.86 ± 2.38μg/dL) and 50 min (ID:11.46 ± 2.40μg/dL; HC:8.99 ± 2.04μg/dL) after the first CPT challenge, and 20 min after the second CPT challenge (ID:11.24 ± 3.59; HC:7.92 ± 1.59μg/dL). While basal levels of cortisol, IL-6 expression by monocytes, and GC sensitivity did not significantly differ between ID and HC cortisol reactivity in the first and second CPT challenge was increased. These preliminary results suggest a stronger stress system responsiveness to novel challenges, which may mechanistically contribute to the relationship between ID and vulnerability to chronic pain. Investigator-initiated grant from Merck Inc., NIH/UL1 RR02758 and M01-RR-01032 from National Center for Research Resources to Harvard Clinical and Translational Science Center.

0208 CIRCADIAN MISALIGNMENT IMPACTS ON HUMAN COGNITIVE PERFORMANCE

Shift work increases the risk for disorders of sleep and human error. Overnight operations pose a challenge because our circadian biology promotes nocturnal sleep and daytime vigilance and performance, which may underlie cognitive vulnerability at night. Here we investigated if daily circadian misalignment, typical for night shift work, adversely impacts cognition across diverse cognitive domains. Thirteen healthy young individuals (27.8 ± 9.5 y; 7 men) underwent two 8-day protocols including either 4 days of circadian alignment (day shifts) or misalignment (night shifts; 12-h inverted behavioral/environmental cycles). Cognitive testing included tasks of sustained attention (Psychomotor Vigilance Task; PVT), cognitive throughput (Addition Task; ADD), information processing (Digit Symbol Substitution Task; DSST) visual-spatial performance (Unstable Tracking-Task; TKT) and declarative memory (Probed Recall Memory; PRM), all of which are sensitive to increased sleep pressure and circadian phase. Circadian misalignment over successive days increased cognitive vulnerability by ~10–20% on sustained attention, cognitive throughput, information processing and visual-motor performance, as compared circadian alignment. Attention, as indexed by PVT performance, was acutely impaired during the first 2 days of misalignment with subsequent improvement after 3 days (interaction of “circadian alignment/misalignment”, “day” and “time since awakening”, p=0.006). Conversely, learning, as indexed by changes in ADD, DSST and TKT performance, significantly improved across multiple days of circadian alignment, while no improvement occurred under misalignment (interaction of “circadian alignment/misalignment” and “day”, p<0.05). Lastly, we investigated if the effects of circadian misalignment on performance were also mediated by prior sleep-wake history. Accordingly, lower sleep efficiency in the sleep episode before cognitive testing significantly impaired performance (interaction of “sleep efficiency”, “circadian alignment/misalignment” and “night”; p<0.05). Our data indicate that daily circadian misalignment may explain cognitive vulnerabilities experienced by night workers, and provide a biological framework for the development of countermeasures against adverse cognitive effects in this vulnerable population. National Heart, Lung, and Blood Institute (NHLBI) Grant R01 HL094806, Clinical Translational Science Award UL1RR025758, and Brigham and Women’s Hospital from the National Center for Research Resources.

Evaluation of the High Mobility Group A1 proteins (HMGA1) as a key mediate in the anticancer activity of ‐EF24.

The architectural chromatin binding proteins High Mobility Group A1 (HMGA1) are some of the most overexpressed proteins in malignant cancers and induce neoplastic transformation. The protein is increased as the last step of a prominent colon cancer progression pathway and mediates drug resistance, therefore correlating with a poor patient prognosis. HMGA1‐‐mediated chemoresistance results from a self‐protective process called cellular senescence. Analogs of the antioxidant, curcumin, when used in combination with traditional chemotherapeutic agents, are useful treatment options for drug resistant tumors. This study had two specific aims. The first being to investigate how colon cancer cells HCT116 respond to treatment with EF24. The second aim was to evaluate how hmga1 expression changed as a result of treatment with EF24. Our preliminary findings showed that cell viability decreased after 24‐hour treatment with low‐dose EF24, as indicated by an MTS assay, with notable discrepancies between cells that underwent a pulsed treatment regimen versus those that underwent continuous treatment. Furthermore, we demonstrated that cells exhibited fragmented DNA when treated with low‐dose EF24, which is characteristic of apoptotic cells. At higher, pulsed doses, senescence activity increased indicating the induction of a senescence pathway. Lastly, gene expression studies indicated that hmga1 was significantly down regulated in cells treated with continuous, low‐‐dose EF24. Further investigation of this pathway could lead to decreased toxicity and increased viability of combination cancer therapies.Support or Funding InformationThe research support is provided by the National Center for Research Resources (5 P20 RR16461), the National Institute of General Medical Sciences (8 P20 GM103499) from the National Institutes of Health, and the Winthrop McNair Scholars program.

Using Transcriptome Analysis to Solve the Puzzle of DNA Repair in Bdelloid Rotifers

Bdelloid rotifers are microscopic aquatic animals that have apparently survived for more than 40 million years without sex, males, or meiosis. Bdelloids have an efficient DNA repair system that gives them the ability to recover from levels of ionizing radiation that are lethal to other eukaryotes. In bdelloids, DNA double strand breaks induced by high doses (&gt;1000 Gray) of ionizing radiation are repaired without significant loss of viability. The objective of this project is to gain insight into the mechanisms of DNA repair in bdelloids by identifying genes involved in this process. This was done using transcriptome sequencing (RNA‐seq) to characterize genes that are differentially expressed during DNA repair in the bdelloid Adineta vaga. To induce DNA double‐strand breaks, bdelloid cultures were irradiated with a total dose of 280 Gray of ionizing radiation using a cesium‐137 source. RNA was then isolated from these cultures at three time points (0, 30 and 60 minutes) post‐irradiation and also from non‐irradiated controls. RNA‐seq was done using the TruSeq RNA Library Preparation Kit and the Illumina HiSeq 2500 platform. From this data, over 500 genes were differentially expressed during DNA repair. Real‐time PCR was done to validate some differentially expressed genes from RNA‐seq. Gene ontology enrichment analysis was carried out to identify biological processes and pathways that were differentially expressed in irradiated bdelloids. Many upregulated genes were related to DNA repair functions (e.g. ligation, non‐homologous end‐joining, DNA polymerases) while the majority of downregulated genes were associated with transcription, translation and cell cycle regulation. Interestingly, over 20% of differentially expressed genes are apparent “bdelloid‐specific genes,” meaning that they lack homologs in other eukaryotes. However, in some cases these bdelloid‐specific genes represent inadequately annotated gene models; for example, the Random Amplification of cDNA Ends (RACE) method was used to identify a previously unannotated map kinase‐activated protein kinase 2 homolog among these genes. Conversely, other differentially expressed genes appear to be genuine bdelloid‐specific genes since further investigation using computational and molecular methods failed to identify their homolog. Overall, this ongoing gene expression analysis has identified several candidate genes for having functions in DNA repair, which will provide further insight into the mechanism of DNA repair in bdelloid rotifers.Support or Funding InformationThis work was supported by grants from the National Center for Research Resources (5P20RR016460) &amp; National Institute of General Medical Sciences (8P20GM103429) from the NIH.

Lindane (Gamma‐Hexachlorocyclohexane) Exposure Impairs [Ca2+]i‐Mediated Vascular Reactivity

Gama hexacholorocyclohexane (γHCH; lindane), Gammallin is an organochlorine chemical that has been used as an agricultural insecticide as well as a pharmaceutical treatment for lice and scabies. It is reported to be neurotoxic, interfering with GABA neurotransmitter functions and other biological functions from brain to endocrine and cardiovascular systems. Despite being a banned persistent organic pollutant, it's still in use and can leach and contaminate our foods and water. We have investigated the effects and the mechanism(s) of lindane‐induced vascular dysfunctions in rat aorta. Rats treated with low dose of single pesticide Lindane (100 or 300 μmol/kg;1/5LD50; SP) or multiple pesticides (MP) (aldrin, endosulfan, lindane, 4,4‐DDT, and endrin: 1/100, 1/50, and 1/25 of the LD50, MP) orally or dermal application for 2 or 4 weeks showed significantly attenuated vascular relaxation (57 ± 5% to 36 ± 0.8%; ACh 10−3 M), MP attenuated relaxation (57 ± 5% to 19.8 ±5.8% and 19.0 ±4.0% respectively for 1/100 and 1/25 LD50). Paradoxically, 4 weeks SP enhanced relaxation to ACh (60.5 ± 4.6% to 83.5 ± 8.8%). In aorta from dermal group (SP), relaxation to ACh was significantly attenuated (59.4 ± 5.0 to 23.7 ± 6.0%). Vasoconstriction to PE (10−8 ‐10−3 M) were significantly attenuated by 4 weeks' treatment with oral SP or dermal MP reducing the contraction (0.64±0.06 to 0.15 ± 0.02gm or 0.49±0.01 gm; 10−3 M), but not for 2 weeks oral MP for PE‐induced contraction. Further investigation into the mechanism behind the observed vascular dysfunction reveals that in CHO cell line stably expressing M1‐ACh receptors, Lindane diminishes carbachol (10−8 to 10−3 M)‐induced increases in [Ca2+]i mediated by M1‐ACh receptor. Lindane diminishes Carbachol effects only at Carbachol's maximum activation and these results indicate that the vascular dysfunction by the pesticides could be due to disruption of intracellular Ca2+ regulation. Thus, 24‐hour Lindane exposure facilitates IP3‐mediated [Ca2+]i signaling. Month‐long Lindane exposure induces vascular dysfunction possibly via disruption of IP3‐mediated in [Ca2+]i signaling leading to dysregulated vasoresponsiveness to hormones/neurotransmitters contributing to cardiovascular dysfunctions.Support or Funding InformationThis research was supported by the National Heart, Lung, and Blood Institute (Grants HL03674 and HL070669) and by the use of TSU Research infrastructure support provided by grants G12RR003045 and CO6RR012537 awarded by the National Center for Research Resources, National Institutes of Health (NIH). The G12 program is now a part of the National Institute on Minority Health and Health Disparities (NIMHD) and the C06 program is in the Office of Research Infrastructure Programs in the Office of the Director, NIH.

Upregulation of RGS2: A New Mechanism for Pirfenidone Amelioration of Pulmonary Fibrosis

BackgroundPirfenidone was recently approved for treatment of idiopathic pulmonary fibrosis. However, the therapeutic dose of pirfenidone is very high, causing side effects that limit its doses and therapeutic effectiveness. Understanding the molecular mechanisms of action of pirfenidone could improve its safety and efficacy. Because activated fibroblasts are critical effector cells associated with the progression of fibrosis, this study investigated the genes that change expression rapidly in response to pirfenidone treatment of pulmonary fibroblasts and explored their contributions to the anti‐fibrotic effects of pirfenidone.MethodsWe used the GeneChip microarray to screen for genes that were rapidly up‐regulated upon exposure of human lung fibroblast cells to pirfenidone, with confirmation for specific genes by real‐time PCR and western blots. Biochemical and functional analyses were used to establish their anti‐fibrotic effects in cellular and animal models of pulmonary fibrosis.ResultsWe identified Regulator of G‐protein Signaling 2 (RGS2) as an early pirfenidone‐induced gene. Treatment with pirfenidone significantly increased RGS2 mRNA and protein expression in both a human fetal lung fibroblast cell line and primary pulmonary fibroblasts isolated from patients without or with idiopathic pulmonary fibrosis. Pirfenidone treatment or direct overexpression of recombinant RGS2 in human lung fibroblasts inhibited the profibrotic effects of thrombin, whereas loss of RGS2 exacerbated bleomycin‐induced pulmonary fibrosis and mortality in mice. Pirfenidone treatment reduced bleomycin‐induced pulmonary fibrosis in wild‐type but not RGS2 knockout mice.ConclusionsEndogenous RGS2 exhibits anti‐fibrotic functions. Upregulated RGS2 contributes significantly to the anti‐fibrotic effects of pirfenidone.Support or Funding InformationThis work was supported by NIH grant R01HL116849 to Y. Tu and T.B. Casale; R01HL097796, P01 HL114471 and P30 ES013508 to R.A. Panettieri; Nebraska State LB595 grant to Y. Tu and P.W. Abel; and the NIH National Center for Research Resources G20‐RR024001.

Application of the Third International Consensus Definitions for Sepsis (Sepsis-3) Classification: a retrospective population-based cohort study

The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3) present clinical criteria for the classification of patients with sepsis. We investigated incidence and long-term outcomes of patients diagnosed with these classifications, which are currently unknown. We did a retrospective analysis using data from 30 239 participants from the USA who were aged at least 45 years and enrolled in the Reasons for Geographic and Racial Differences in Stroke (REGARDS) cohort. Patients were enrolled between Jan 25, 2003, and Oct 30, 2007, and we identified hospital admissions from Feb 5, 2003, to Dec 31, 2012, and applied three classifications: infection and systemic inflammatory response syndrome (SIRS) criteria, elevated sepsis-related organ failure assessment (SOFA) score from Sepsis-3, and elevated quick SOFA (qSOFA) score from Sepsis-3. We estimated incidence during the study period, in-hospital mortality, and 1-year mortality. Of 2593 first infection events, 1526 met SIRS criteria, 1080 met SOFA criteria, and 378 met qSOFA criteria. Incidence was 8·2 events (95% CI 7·8-8·7) per 1000 person-years for SIRS, 5·8 events (5·4-6·1) per 1000 person-years for SOFA, and 2·0 events (1·8-2·2) per 1000 person-years for qSOFA. In-hospital mortality was higher for patients with an elevated qSOFA score (67 [23%] of 295 patients died) than for those with an elevated SOFA score (125 [13%] of 960 patients died) or who met SIRS criteria (128 [9%] of 1392 patients died). Mortality at 1 year after discharge was also highest for patients with an elevated qSOFA score (29·4 deaths [95% CI 22·3-38·7] per 100 person-years) compared with those with an elevated SOFA score (22·6 deaths [19·2-26·6] per 100 person-years) or those who met SIRS criteria (14·7 deaths [12·5-17·2] per 100 person-years). SIRS, SOFA, and qSOFA classifications identified different incidences and mortality. Our findings support the use of the SOFA and qSOFA classifications to identify patients with infection who are at elevated risk of poor outcomes. These classifications could be used in future epidemiological assessments and studies of patients with infection. National Institute for Nursing Research, National Center for Research Resources, and National Institute of Neurological Disorders and Stroke.

Shortened Antimicrobial Treatment for Acute Otitis Media in Young Children

BackgroundLimiting the duration of antimicrobial treatment constitutes a potential strategy to reduce the risk of antimicrobial resistance among children with acute otitis media.MethodsWe assigned 520 children, 6 to 23 months of age, with acute otitis media to receive amoxicillin–clavulanate either for a standard duration of 10 days or for a reduced duration of 5 days followed by placebo for 5 days. We measured rates of clinical response (in a systematic fashion, on the basis of signs and symptomatic response), recurrence, and nasopharyngeal colonization, and we analyzed episode outcomes using a noninferiority approach. Symptom scores ranged from 0 to 14, with higher numbers indicating more severe symptoms.ResultsChildren who were treated with amoxicillin–clavulanate for 5 days were more likely than those who were treated for 10 days to have clinical failure (77 of 229 children [34%] vs. 39 of 238 [16%]; difference, 17 percentage points [based on unrounded data]; 95% confidence interval, 9 to 25). The mean symptom scores over the period from day 6 to day 14 were 1.61 in the 5-day group and 1.34 in the 10-day group (P=0.07); the mean scores at the day-12-to-14 assessment were 1.89 versus 1.20 (P=0.001). The percentage of children whose symptom scores decreased more than 50% (indicating less severe symptoms) from baseline to the end of treatment was lower in the 5-day group than in the 10-day group (181 of 227 children [80%] vs. 211 of 233 [91%], P=0.003). We found no significant between-group differences in rates of recurrence, adverse events, or nasopharyngeal colonization with penicillin-nonsusceptible pathogens. Clinical-failure rates were greater among children who had been exposed to three or more children for 10 or more hours per week than among those with less exposure (P=0.02) and were also greater among children with infection in both ears than among those with infection in one ear (P<0.001).ConclusionsAmong children 6 to 23 months of age with acute otitis media, reduced-duration antimicrobial treatment resulted in less favorable outcomes than standard-duration treatment; in addition, neither the rate of adverse events nor the rate of emergence of antimicrobial resistance was lower with the shorter regimen. (Funded by the National Institute of Allergy and Infectious Diseases and the National Center for Research Resources; ClinicalTrials.gov number, NCT01511107.)

Reducing the number of fetuses in a pregnancy: providers' and patients' views of challenges.

How do patients and providers perceive and make decisions about possible reductions of multi-fetal pregnancies? Physicians may be transferring additional embryos, assuming that patients will later undergo reduction if need be; but decisions to reduce pregnancies are difficult for patients, who may agree to undergo the procedure in advance and later renege. Implanting more than one embryo increases the likelihood that at least one embryo will successfully lead to a child but also that the patient may end up with twins or higher-order multiple births. In-depth interviews of ~1 h each were conducted with 37 ART providers and patients (17 physicians, 10 other health providers and 10 patients) and systematically analyzed. The telephone interviews explored the participants' views and decisions regarding pregnancy reduction. The answers were analyzed systematically. Providers may be transferring additional embryos, thinking that doing so will increase the likelihood of a 'take home baby' and that the patients could undergo reductions, if need be, to avoid the risks and complications of twins or multiple births. Yet patients often appear to have difficulty confronting the prospect of fetal reduction and/or renege on prior agreements to undergo the procedure. Providers should thus be wary and exceedingly careful about these situations. The sample size was sufficient for qualitative research designed to elucidate the issues and themes that emerge, but not for statistically analyzing how various groups may differ (e.g. physicians versus patients). Future studies should investigate these issues with larger samples. These data, the first to examine how IVF providers and patients view and approach decisions regarding the reduction of fetuses, suggest several complications and dilemmas. This information has critical implications for future practice, guidelines, research and education of providers, patients, insurers, policymakers and others. Funding was provided by grant #UL1 RR024156 from the National Center for Research Resources, the Greenwall Foundation and the John Simon Guggenheim Memorial Foundation. There are no conflicts of interest to declare.

Could targeted, antibiotic-loaded gold nanoconstructs be a new magic bullet to fight infection?

Could targeted, antibiotic-loaded gold nanoconstructs be a new magic bullet to fight infection?

Efficacy and safety of 3-week response-guided triple direct-acting antiviral therapy for chronic hepatitis C infection: a phase 2, open-label, proof-of-concept study

To shorten the course of direct-acting antiviral agents for chronic hepatitis C virus (HCV) infection, we examined the antiviral efficacy and safety of 3 weeks of response-guided therapy with an NS3 protease inhibitor and dual NS5A inhibitor-NS5B nucleotide analogue. In this open-label, phase 2a, single centre study, Chinese patients with chronic HCV genotype 1b infection without cirrhosis were randomly allocated by a computer program to one of three treatment groups (sofosbuvir, ledipasvir, and asunaprevir; sofosbuvir, daclatasvir, and simeprevir; or sofosbuvir, daclatasvir, and asunaprevir) until six patients in each group (1:1:1) achieved an ultrarapid virological response (plasma HCV RNA <500 IU/mL by day 2, measured by COBAS TaqMan HCV test, version 2.0). Patients with an ultrarapid virological response received 3 weeks of therapy. Patients who did not achieve an ultrarapid response were switched to sofosbuvir and ledipasvir for either 8 weeks or 12 weeks. The primary endpoint was the proportion of patients with a sustained virological response at 12 weeks (SVR12) after treatment completion, analysed in the intention-to-treat population. All patients who achieved an ultrarapid virological response were included in the safety analysis. This trial is registered with ClinicalTrials.gov, number NCT02470858. Between April 5, 2015, and April 15, 2015, 26 eligible patients were recruited. 12 patients were assigned to sofosbuvir, ledipasvir, and asunaprevir; six to sofosbuvir, daclatasvir, and simeprevir; and eight to sofosbuvir, daclatasvir, and asunaprevir. Six patients in each group achieved an ultrarapid virological response (18 [69%]). All patients with an ultrarapid virological response who were given 3 weeks of triple therapy achieved SVR12. The most common adverse events were fatigue (one [17%] of six patients receiving sofosbuvir, ledipasvir, and asunaprevir; one [17%] of six patients receiving sofosbuvir, daclatasvir, and simeprevir; and two [33%] of six patients receiving sofosbuvir, daclatasvir, and asunaprevir) and headache (one [17%] patient in each group). No patients experienced any serious adverse events. In this proof-of-concept study, all patients with chronic HCV without cirrhosis who achieved an ultrarapid virological response on triple direct-acting antiviral regimens by day 2 and received 3 weeks of treatment were cured, with excellent tolerability. By shortening the duration of therapy from the currently recommended 12 weeks to 3 weeks, we could drastically reduce the cost of therapy and the rate of adverse events. Further large-scale studies should be done to confirm our findings. Center for AIDS Research, National Institutes of Health, US Department of Energy, National Center for Research Resources and the Office of Research Infrastructure Programs, Cheng Si-Yuan (China-International) Hepatitis Research Foundation, and Humanity and Health Medical Group.

Eulogy for the clinical research center.

The extramural General Clinical Research Center (GCRC) program has been funded for more than 50 years, first by the National Center for Research Resources, NIH, and more recently as part of the Clinical Translational Science Award (CTSA) program through the newly formed National Center for Advancing Translation Sciences (NCATS). The GCRCs represent the federally funded laboratories that employ a highly trained cadre of research nurses, dietitians, and other support staff and in which generations of clinical investigators trained and performed groundbreaking human studies that advanced medical science and improved clinical care. Without the opportunity for adequate discussion, NCATS has now stopped funding these Research Centers. In this "eulogy," we review the origins and history of the GCRCs, their contributions to the advancement of medicine, and the recent events that have essentially defunded them. We mourn their loss.

Increased Post-exercise Lung Volume Does Not Affect Exercise-induced Bronchoconstriction In Asthmatic Adults

PURPOSE: Due to force coupling between lung parenchyma and intrapulmonary airways, lung volume (VL) has a marked impact on airway caliber. Increased VL has also been shown to disrupt airway smooth muscle actomyosin cross-bridge formation in healthy and asthmatic persons. We hypothesized that elevated operating VL and regular deep lung inflations following an asthmogenic exercise bout would decrease the severity of exercise-induced bronchoconstriction (EIB) in asthmatic adults. METHODS: Nine asthmatic adults completed three experimental trials. During all three trials, subjects completed six minutes of cycling exercise at 85% of the peak workrate reached during a graded exercise test. Following exercise, subjects remained on the ergometer with the mouthpiece in place, and performed a maximal volitional flow-volume maneuver every even minute following exercise for a total of 20 minutes. During the first trial, subjects breathed spontaneously during the 20 minute period following exercise (SPON). The second and third trials were randomized and balanced. During one of the trials, minute ventilation was gradually decreased in a stepwise manner during the first ten minutes following exercise (GRAD); during the other trial, subjects performed an inspiratory capacity every odd minute during the recovery period (IC). Forced expiratory volume 1.0 second (FEV1.0) was compared among the three trials. RESULTS: Nadir post-exercise FEV1.0 (% change baseline) was similar among the three trials (SPON, -27.7 ± 5.3%; GRAD, -22.5 ± 5.2%; IC, -23.3 ± 5.1%; P=0.52). Nadir FEV1.0 also occurred at a similar time after exercise during the three trials (SPON, 11.1 ± 1.8 min; GRAD, 12.9 ± 1.5 min; IC, 9.1 ± 1.6 min, P=0.10). Area under the curve for FEV1.0 was the same during the three trials (SPON, 47.9 ± 15.1 min·l; GRAD, 50.5 ± 12.7 min·l; IC, 50.5 ± 13.8 min·l; P=0.49). CONCLUSIONS:Contrary to our hypothesis, these data suggest that increased VL and regular deep lung inflations after exercise do not attenuate EIB in asthmatic patients. Thus, maintenance of the bronchodilatory influence of increased VL after exercise cessation does not override the opposing stimuli that cause airway narrowing in the asthmatic. FUNDING: Vermont Genetics Network, P20 RR16462 from the INBRE Program of the National Center for Research Resources, NIH.

Vitamin D Supplementation and DNA Methylation Patterns during Pregnancy and Lactation in Mothers and Infants

The purpose of this study was to identify the temporal influence of maternal vitamin D supplementation during pregnancy and lactation on DNA methylation in mothers and infants. In this double blind randomized control trial, pregnant women planning to exclusively breastfeed were randomized to control (400 IU vitamin D3 daily, n=8) or experimental (3800 IU vitamin D3 daily, n=8) groups from 28 weeks gestation through 4 weeks postpartum. Genome‐wide DNA methylation (Illumina Infinium 450K) was analyzed in white blood cells from mothers at 28 weeks gestation (baseline), 2 days postpartum and 4 weeks postpartum and in their fully breastfed infants at birth and 4 weeks of age. Significant DNA methylation was determined by at least 20% difference in individual CpG methylation between control and experimental groups. In the experimental group, differential methylation in &gt;400 individual maternal CpGs in 280 known genes across the genome was identified with methylation loss present in 2/3 of individual CpGs. Among the infants, differential methylation in 143 CpGs (91 genes) was identified with common methylation loss patterns as in the mothers. These findings provide evidence for the direct influence of vitamin D supplementation on DNA methylation in pregnant and lactating women, in utero on the fetus and via breastmilk in newborns.Support or Funding Information1P20GM104360‐01 National Institutes of Health, National Center for Research Resources (NCRR) Centers of Biomedical Research Excellence (COBRE). Center of Excellence in Epigenetics/Epigenomics of Development and Disease. COBRE Epigenomics Jumpstart Pilot Grant, Epigenomic gestational responsiveness to vitamin D Association of Women's Health, Obstetric and Neonatal Nursing (AWHONN). Maternal vitamin D supplementation to correct deficiency in mothers and breastfed infants.

Suramin Protects From Cisplatin‐Induced Acute Kidney Injury

Cisplatin, a commonly used cancer chemotherapeutic, has a dose limiting side effect of nephrotoxicity. According to the Risk, Injury, Failure, Loss of Function, and End Stage Renal Disease (RIFLE) criteria of kidney injury, approximately 30% of patients administered cisplatin suffer from kidney injury and there is currently no treatment for cisplatin‐induced kidney injury other than palliative care. Suramin, FDA‐approved for the treatment of trypanosomiasis, improves kidney function following various forms of kidney injury in rodent models. We hypothesized that suramin would attenuate cisplatin‐induced kidney injury. Suramin treatment prior to cisplatin administration reduced cisplatin‐induced decreases in kidney function and injury. Furthermore, suramin attenuated cisplatin‐induced expression of inflammatory cytokines and chemokines, endoplasmic reticulum stress, and apoptosis in the kidney cortex. Treatment of mice with suramin 24 hours after cisplatin improved kidney function, suggesting that the mechanism of protection is neither via inhibiting tubular cisplatin uptake nor its metabolism to nephrotoxic species. If suramin is to be utilized in the context of cancer, then suramin cannot prevent cisplatin‐induced cytotoxicity within tumors. Mice harboring lung adenocarcinomas were pretreated with suramin and then administered cisplatin. Lung histology and markers of kidney function indicated that suramin protected mice from cisplatin‐induced acute kidney injury, but did not inhibit cisplatin induced cytotoxic effects within the tumor. These results provide evidence that suramin has great potential as a renoprotective agent for the treatment/prevention of cisplatin‐induced acute kidney injury.Support or Funding InformationSupport for these studies was provided by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant R01‐ DK093462, to L.J.S]. This work was also supported by the National Institutes of Health National Institute of General Medical Sciences [Grant R01‐GM084147 to R.G.S.]; the National Institutes of Health National Center for Research Resources [Grant UL1‐RR029882 to R.G.S.]; the Biomedical Laboratory Research and Development Program of the Department of Veterans Affairs [1BX000851 to R.G.S.]; the South Carolina Clinical and Translational Research Institute, with an academic home at the Medical University of South Carolina (to R.G.S.); and the National Institute of Environmental Health and Sciences (SBIR/STTR ES023767 to R.G.S.).