“
“A method for an Agrobacterium-mediated transformation with phosphomannose isomerase-based selection system was

investigated for cotyledons explants of Lotus corniculatus L. cultivar Leo. The effects of mannose on shoot formation and rooting was evaluated to choose appropriate selection pressure for Lotus corniculatus L. transformation. Complete inhibition in Murashige and Skoog medium supplemented with 20 g/L mannose 3-MA cost in the presence of 20 g/L sucrose was observed, indicating that Lotus corniculatus L. is incapable of utilizing mannose as a carbon source. The mannose-resistant shoots from individual transformed lines selected in early developmental stage were identified by PCR analysis, Quisinostat in vitro resulting in a transformation frequency of 16.2 %.

Six randomly chosen PCR-positive plantlets were further confirmed by Southern-blotting and RT-PCR analysis to demonstrate integration of the phosphomannose isomerase (PMI) gene. Chlorophenol red assays were performed to determine PMI enzyme activity. These results demonstrate that the PMI-based selection is a highly efficient system for Lotus corniculatus L. transformation without requiring the use of conventional antibiotics or herbicide marker genes.”
“Kukke SN, Sanger TD. Contributors to excess antagonist activity during movement in children with secondary dystonia due to cerebral palsy. J Neurophysiol 105: 2100-2107, 2011. First published this website February 16, 2011; doi:10.1152/jn.00998.2009.-Children with secondary dystonia due to cerebral

palsy exhibit abnormal upper extremity postures and slow voluntary movement. However, the interaction between abnormal postures and abnormal movement in dystonia is still unclear. Some mechanisms by which postures are maintained in dystonia include stretch reflexes, overflow of muscle activation to other muscles, and direct coactivation of antagonist muscles. This study explored the independent contributions of each of these postural mechanisms to abnormal biceps brachii (antagonist) activity during elbow extension, which slows movement. A linear model of biceps activation as a function of velocity-dependent reflexes, triceps-dependent overflow, and direct drive to the biceps was fitted to experimental data from 11 children and young adults with secondary dystonia due to cerebral palsy and 11 age-matched control subjects. Subjects performed elbow extension movements against each of four levels of resistance without perturbations or in each of two perturbation conditions. Results show that biceps activity in children with dystonia consists of significant contributions of reflex activation, overflow from triceps, and direct muscular drive. Additionally, stretch reflexes during movement are shown to be elevated at three latencies after stretch.

We have established quantitative SPECT/computed tomography (CT) in vivo imaging protocols for determination of liver tumor burden based on the known role of learn more Kupffer cells in cancer of the liver. As it is also known that functional Kupffer cells accumulate particulate material contained in the arterial blood of liver supply, we used radiolabeled macro-aggregated albumin

particles ([Tc-99m]-MAA) injected intravenously to image liver disease. Quantification of cold spot liver lesion imaging was also a general objective. We examined a healthy control group (BALB/C mice, n = 6) and group of induced hepatocellular carcinoma (HCC, matrilin-2 transgenic KO mice, n = 9), where hepatocellular carcinoma was induced by diethylnitrosamine. We used [Tc-99m]-MAA as radiopharmaceutical for liver SPECT imaging in a small animal SPECT/CT system. A liver radioactivity overview map was generated. Segmentation of the liver was calculated by Otsu thresholding method. Based on the segmentation the radioactivity volume and the summarized liver activity were determined. Tumor burden of the livers was quantitatively determined by creating parametric data from the resulting volumetric maps. Ex vivo liver mass data were applied for the validation of in vivo measurements. An uptake with cold spots as tumors was observed in all diseased

animals in SPECT/CT scans. Isotope-labeled particle uptake (standardized uptake concentration) of control (median 0.33) and HCC (median 0.18) groups was selleck products significantly different (p = 0.0015, Mann Whitney U test). A new potential application of [Tc-99m]-MAA was developed and presents a simple and very effective means to quantitatively characterize liver cold spot lesions resulting from Kupffer cell dysfunctions as a consequence of tumor burden.”
“Mycobacterium tuberculosis is the major causative agent of tuberculosis (TB). The gamma interferon (IFN-gamma) release assay (IGRA) has been widely used to diagnose TB by testing

cell-mediated immune responses but has no capacity for distinguishing between active TB and latent TB infection (LTBI). This study aims to identify a parameter that will help to discriminate active TB and LTBI. Whole-blood samples from 33 active TB patients, Selleck LY3023414 20 individuals with LTBI, and 26 non-TB controls were applied to the commercial IFN-gamma release assay, QuantiFERON-TB Gold In-Tube, and plasma samples were analyzed for interleukin-2 (IL-2), IL-6, IL-8, IL-10, IL-13, tumor necrosis factor-alpha (TNF-alpha), IFN-gamma, monokine induced by IFN-gamma (MIG), interferon gamma inducible protein 10 (IP-10), interferon-inducible T cell alpha chemoattractant (I-TAC), and monocyte chemoattractant protein 1 (MCP-1) by using a commercial cytometric bead array. The Mycobacterium tuberculosis antigen-specific production of most of the assayed cytokines and chemokines was higher in the active TB than in the LTBI group. The mitogen-induced responses were lower in the active TB than in the LTBI group.

Given this scenario, the CX-6258 concentration growth potential remained constant within the studied salinity gradient, as did the oxygen:nitrogen ratio. The results suggest that the increased energy losses at low salinity due to respiration and excretion are compensated by an increment in the ingestion rate, contributing

to the success of H. crenulatus in dynamic habitats such as estuaries.”
“Most cases of Type 2 diabetes are attributable to excess weight and physical inactivity. We investigated trends in mortality based on doctors certification of diabetes and obesity.\n\nAnalysis of a national data set of all certified causes of death, i.e. underlying cause and contributing causes (mentions), in England 19952010.\n\nDiabetes exhibited divergent trends for mortality based on underlying cause and mentions. Underlying cause rates were 107.2 per million population [95 confidence interval (CI): 105.7108.6] in 1995, but only 68.9/10(6)

(CI: 67.969.9) in 2010. Mortality rates for mentions of diabetes were 403.1/10(6) (CI: 400.4405.8) in 1995, increasing to 478.4/10(6) (CI: 475.7481.0) in 2010. Underlying cause mortality for obesity was 3.7/10(6) (CI: 3.24.1) in 1995 and 7.5 (CI: 7.08.0) in 2010. The corresponding rates for mentions of obesity were 13.2/10(6) (CI: 12.613.9) and 34.5/10(6) (CI: 33.635.4), respectively. 24.0 of death certificates with a mention of obesity also had diabetes selleck chemical recorded on the same certificate.\n\nMultiple-cause mortality statistics provide a more accurate picture than underlying cause of the total mortality burden 4SC-202 attributed on death certificates to diabetes and obesity. Rates for both increased substantially: analysis by underlying cause alone would have missed

this for diabetes.”
“Most important breeding goals in ornamental crops are plant appearance and flower characteristics where selection is visually performed on direct offspring of crossings. We developed an image analysis toolbox for the acquisition of flower and petal images from cultivated carnation (Dianthus caryophyllus L.) that was validated by a detailed analysis of flower and petal size and shape in 78 commercial cultivars of D. caryophyllus, including 55 standard, 22 spray and 1 pot carnation cultivars. Correlation analyses allowed us to reduce the number of parameters accounting for the observed variation in flower and petal morphology. Convexity was used as a descriptor for the level of serration in flowers and petals. We used a landmark-based approach that allowed us to identify eight main principal components (PCs) accounting for most of the variance observed in petal shape. The effect and the strength of these PCs in standard and spray carnation cultivars are consistent with shared underlying mechanisms involved in the morphological diversification of petals in both subpopulations.

ESX-1 consists of at least 10 genes (Rv3868-Rv3877) encoding the T-cell. antigens ESAT-6 and CFP-10 as well as AAA-ATPases, chaperones, and membrane proteins which probably form a novel export system. To better understand the mode of action of the ESX-1 proteins, as a prelude to drug development, we examined systematically the interactions between the various proteins using the two-hybrid system in Saccharomyces cerevisiae. Interestingly, ESAT-6 and CFP-10 formed

both hetero- and homodimers. Moreover, Rv3866, Rv3868, and CFP-10 interacted with Rv3873 which also homodimerized. The data were summarized in a protein linkage map that is consistent with the model for the secretion apparatus and can be used as a basis to identify inhibitors of specific interactions. (C) 2007 Elsevier GmbH. All rights reserved.”
“Autism spectrum disorder (ASD) is increasingly being recognized as an important issue in adult psychiatry and psychotherapy. High www.selleckchem.com/products/a-769662.html intelligence indicates overall good brain functioning and might thus present a particularly good opportunity Silmitasertib datasheet to study possible cerebral correlates of core autistic features in terms of impaired social cognition, communication skills, the need

for routines, and circumscribed interests. Anatomical MRI data sets for 30 highly intelligent patients with high-functioning autism and 30 pairwise-matched control subjects were acquired and analyzed with voxel-based morphometry. The gray matter volume of the pairwise-matched patients and the controls did not differ significantly. When correcting for total brain volume influences, the patients with ASD exhibited smaller left superior

frontal volumes on a trend level. Heterogeneous volumetric findings in earlier studies might partly be explained by study samples biased by a high inclusion rate of secondary forms of ASD, which often go along with neuronal abnormalities. Including only patients with high IQ scores might have decreased the influence of secondary forms of ASD and might explain the absence see more of significant volumetric differences between the patients and the controls in this study. (C) 2014 Elsevier Ireland Ltd. All rights reserved.”
“Use of Hirshfeld surfaces calculated from crystal structure determinations on various transition metal ion complexes of three terpyridine ligands carrying trimethoxyphenyl substituents has enabled an assessment of the contribution made by the ligand components to the interactions determining the lattice structures, interactions expected also to be present in metallomesogens derived from similar ligands. The form of the link joining the trimethoxyphenyl substituent to the 4 position of 2,2;6,2-terpyridine is of some importance. In the case of the Co(II) complexes of two of the ligands, their spin-crossover characteristics can be rationalised in terms of the different interactions seen in their lattices.

72-0.92), meconium aspiration syndrome (aOR, 0.30; 95% CI, 0.19-0.48), and admission to neonatal intensive care unit (aOR, 0.87; 95% CI, 0.78-0.97). Similar findings were seen for women who were induced at 40 weeks compared to delivery later.\n\nCONCLUSION: Induction of labor in low-risk women at term is not associated with increased risk of cesarean delivery compared to delivery later.”
“Objectives: The epidemiology of the systemic inflammatory response syndrome (SIRS) in children is poorly understood. We sought to determine national estimates of the incidence of pediatric SIRS and its corresponding clinical etiologies presenting to US emergency

departments (EDs) using current definitions.\n\nMethods: We analyzed ED visits by children younger than 18 years from 2007 to 2010 in the National Hospital Ambulatory Medical Care Survey. We used a Bayesian logical framework of prior probability SBI-0206965 Autophagy inhibitor distributions for white blood cell count result to make minimum, moderate, and maximum estimates for pediatric SIRS.\n\nResults:

Taking the minimum and maximum estimates as modified credible intervals, we report an overall incidence of pediatric SIRS presenting to the ED to be 21.7% (95% modified credible interval, 18.1%-25.4%). The national moderate estimate of pediatric ED visits Wnt activity presenting with SIRS was approximately 6.2 million per year. Children with SIRS and without SIRS had similar baseline characteristics, but SIRS patients were younger (2.9 vs 5.5 years; P < 0.0001), had higher triage acuity (emergent, 9.0 vs 6.3%; P < 0.0001), and were more often admitted (7.0 vs 2.4%; P < 0.0001) than children without SIRS. Based on the moderate estimate, infection was the most common (53%) associated etiology, followed by trauma (10%). Other traditional categories of SIRS were extremely rare. Of note, 35% of children with SIRS did not fall into any of the previously established categories.\n\nConclusions: Pediatric SIRS is common;

its associated clinical contexts include potentially dangerous etiologies; many cases of pediatric SIRS can be recognized in triage; and there is significant heterogeneity in the etiology of pediatric SIRS.”
“We present a web-based pediatric CT dose tool that estimates effective dose based on dose length product, Citarinostat patient age and region of body scanned. The tool also provides an estimate of additional lifetime risk of cancer from CT exams. These estimations are based on the interpolation of factors from published methods. The calculator serves as an educational tool that can be used by radiologists and clinicians to better understand CT dose and its associated risks.”
“Chicken broodiness is a polygenic trait controlled by autosomal genes. Prolactin gene is a candidate of great interest in molecular studies of broodiness. However, another candidate dopamine D2 receptor (DRD2) gene has not been studied extensively.

Supporting data are largely from mixed populations of adolescents and adults, although ICS/LABA combinations

are not approved for adolescents in all countries. This analysis evaluates overall asthma control in asthma patients aged >= 16 years receiving ICS/LABA combinations.\n\nMethods: This was a post hoc analysis of asthma patients aged >= 16 years in a randomized, double-blind/open-label extension, parallel-group study. Patients received fixed maintenance-dose budesonide/formoterol (Symbicort (R) Turbuhaler (R)), fixed maintenance-dose salmeterol/fluticasone propionate (Seretide/Advair/Adoair (TM) Diskus (TM)) or adjustable maintenance-dose P005091 concentration budesonide/formoterol. Patients used terbutaline or salbutamol for as-needed reliever medication. The primary efficacy variable was the odds of having a well controlled asthma week during the randomized treatment period.\n\nResults: ICS/LABA regimens were well tolerated and efficacious, and the odds for achieving a well controlled asthma week did not differ between groups in this sub-analysis.

The number of exacerbations was similar between fixed-dose regimens; however, there were trends toward fewer exacerbations requiring hospitalization/emergency room treatment in the fixed- and adjustable maintenance-dose budesonide/formoterol groups (three and two events, respectively) than in the fixed-dose salmeterol/fluticasone propionate group (eight events). Improvements in forced expiratory volume in 1 second (FEV(1)) were small but significantly greater with fixed-dose budesonide/formoterol FK506 datasheet versus fixed-dose salmeterol/fluticasone propionate.\n\nConclusions: This post hoc analysis

supports the use of ICS/LABA combinations in adults aged >= 16 years.”
“Lymphotoxin alpha (LTA) is a member of the TNF cytokine superfamily, produced principally by lymphocytes. It plays an important role in immune and inflammatory responses. Many TNF superfamily members have functionally important isoforms generated by learn more alternative splicing but alternative splicing of LTA has never been studied. The known LTA protein is encoded by a transcript containing four exons. Here we report seven new LTA splice variants, three of them evolutionary conserved. We demonstrate their presence in cytoplasmic RNA suggesting that they could be translated into new LTA isoforms. We observed that their expression is differentially regulated upon activation of peripheral blood mononuclear cells and lymphocyte subpopulations (CD4+, CD8+, and CD19+). Our data suggest that the new LTA splice variants might play a role in the regulation of the immune response. (C) 2007 Elsevier Ltd. All rights reserved.”
“Disturbance of cholesterol homeostasis in the brain is coupled to age-related brain dysfunction.

We observed correlations between TBX5 and CAV1 and atrial fibrillation (P = 4.0 x 10(-5) and P = 0.00032, respectively), between TBX5 and advanced atrioventricular block (P = 0.0067), and between SCN10A and pacemaker implantation

(P = 0.0029). We also replicated previously described associations with the QT interval.”
“It is of practical interest to investigate the effect of nitrates on bacterial metabolic regulation of both fermentation and energy generation, as compared to aerobic and anaerobic growth without nitrates. Although gene level regulation has previously been studied for nitrate assimilation, it is important to understand this metabolic regulation in terms of global regulators. In the present study,

Galardin manufacturer therefore, we measured gene expression using DNA microarrays, intracellular metabolite concentrations using CE-TOFMS, and metabolic fluxes using the C-13-labeling technique for wild-type E. coli and the Delta arcA (a global regulatory gene 3-MA research buy for anoxic response control, ArcA) mutant to compare the metabolic state under nitrate conditions to that under aerobic and anaerobic conditions without nitrates in continuous culture conditions at a dilution rate of 0.2 h(-1). In wild-type, although the measured metabolite concentrations changed very little among the three culture conditions, the TCA cycle and the pentose phosphate pathway fluxes were significantly different under each condition. These results suggested that the ATP production rate was 29% higher under nitrate conditions than that under anaerobic conditions, whereas the ATP production rate was 10% lower than that under aerobic conditions. BMS-777607 solubility dmso The flux changes in the TCA cycle were caused by changes in control at the gene expression level. In DarcA mutant, the TCA cycle flux was significantly increased (4.4 times higher than that of the wild type) under nitrate conditions. Similarly, the intracellular ATP/ADP ratio increased approximately two-fold compared to that of the wild-type strain.”
“Three beta-glucosidase- and two endoglucanase-encoding genes were cloned from Aspergillus

oryzae, and their gene products were displayed on the cell surface of the sake yeast, Saccharomyces cerevisiae GRI-117-UK. GRI-117-UK/pUDB7 displaying beta-glucasidase AO090009000356 showed the highest activity against various substrates and efficiently produced ethanol from cellobiose. On the other hand, GR1-117-UK/pUDCB displaying endoglucanase AO090010000314 efficiently degraded barley beta-glucan to glucose and smaller cellooligosaccharides. GRI-117-UK/pUDB7CB codisplaying both beta-glucosidase AO090009000356 and endoglucanase AO090010000314 was constructed. When direct ethanol fermentation from 20 g/l barley beta-glucan as a model substrate was performed with the codisplaying strain, the ethanol concentration reached 7.94 g/l after 24 h of fermentation.

From these, 13 proteins with the greatest expression differences were identified. One of these proteins, peroxiredoxin-6 (Prdx6), has never before been described in mitochondria. In hepatocytes

from sham-operated mice, Prdx6 expression was found exclusively in the cytoplasm. Small molecule library After ischemia or I/R, Prdx6 expression disappeared from the cytoplasm and appeared in the mitochondria, suggesting mitochondrial trafficking. To explore the functional role of Prdx6 in hepatic I/R injury, wild-type and Prdx6-knockout mice were subjected to I/R injury. Prdx6-knockout mice had significantly more hepatocellular injury compared with wild-type mice. Interestingly, the increased injury in Prdx6-knockout mice occurred despite reduced inflammation and was associated with increased mitochondrial generation of H(2)O(2) and dysfunction. The mitochondrial dysfunction appeared to be related to complex I of the electron transport chain. These data suggest that hepatocyte Prdx6 traffics to the mitochondria during I/R to limit mitochondrial dysfunction

as a protective mechanism against hepatocellular injury.”
“A Putative laccase gene was cloned from Shigella dysenteriae W202 and expressed in Escherichia coli as a soluble fusion protein with high yield. The purified product (Wlac) Smad inhibitor was characterized as the CueO-like laccase from E. coli, a monomer of molecular mass 55 kDa, with a maximum activity of 24.4 U/mg (K(m) = 0.086) and a pH optimum of 2.5. in a standard assay using ABTS (2,2′-azino-di(3-ethyl-benzthiazoline-6-sulfonate) as the substrate. Activity was stable at 0-25 degrees C but inhibited above 40 C. Purified Mac was completely inhibited by 200 mM EDTA and partially by selleck inhibitor 32 mM SDS. 50 mM NaN(3) and 60 mM thioglycolic acid. Activity was stimulated by Cu(2+); other metal ions had only slight

or negative effects. Two mutated variants. WlacS and WlacD, were obtained by Substituting Glu 106 with Phe 106, and adding a deletion of an alpha-helix domain (from Leu 351 to Gly 378). WlacS had a 2.2-fold (52.9 U/mg) and WlacD a 3.5-fold (85.1 U/mg) higher enzyme activity than the wild-type laccase and WlacD showed greater thermostability at higher temperatures. Sce VMA intein-associated fusion proteins maintained similar to 80% of total enzyme activity. Thus. deletion and site-directed mutagenesis of laccases are capable of promoting both enzymatic activity and thermostability. (C) 2009 Elsevier Inc. All rights reserved.”
“Increased evidence indicates an association between psychiatric disorders and dietary pattern. The objective of this study is to describe the differences in food consumption between suicide attempters and non-attempters. We analyzed the dietary information retrospectively collected from 6803 adults, aged 17 to 39 years, who also completed a mental disorder diagnostic interview as a part of the Third National Health and Nutrition Examination Survey, 1988-1994.

“
“Objectives: To explore undergraduate students’ expectations and teachers’ views of written feedback.\n\nDesign: Narrative literature review.\n\nData Sources: Seven electronic databases were searched for primary research published in PHA-739358 concentration English with additional manual searches and reference tracking.\n\nReview Methods: Systematic approach to search strategy,

selection and appraisal of papers, data extraction and synthesis following Hawker et al.’s (2002) guidelines.\n\nResults: 21 studies met the inclusion criteria. Four student themes were identified concerning written feedback: quality, quantity and location of feedback, feed-forward and timeliness. Teachers reported that time pressures, institutional selleck policies, and administrative issues affect feedback provision.\n\nConclusions: Rigorous research is needed to gain a better understanding of students’ expectations of written feedback. Strategies need to be adopted to meet students’ expectations and educate students to take an active role and reflect on the feedback received. (C) 2013 Elsevier Ltd. All rights reserved.”
“Introduction: Physiological resistance of Aedes aegypti is a major threat to

effective control programs in the transmission of dengue virus. Objective: To determine the status of susceptibility to insecticides used in public health, in natural populations of A. aegypti from three endemic dengue localities of Casanare. Materials and methods: Adult mosquitoes were recovered from A. aegypti immature stages from seven natural populations PLX3397 in vivo collected for three municipalities. The first filial generation was

used to assess the biochemical mechanisms associated with loss of susceptibility: nonspecific esterase (NSE) and enzyme cytochrome P450 monooxygenases group. The second filial generation allowed us to evaluate the susceptibility to insecticides from bioassays using the CDC 1998 methodology for adult mosquitoes and WHO 1981 technique for larvae. Results: In the seven adult populations recorded loss of susceptibility to organochlorine DDT and pyrethroids lambda-cyhalothrin and permethrin. Two populations showed susceptibility to deltamethrin and five populations showed susceptibility to cyfluthrin. There was a susceptibility to organophosphates temephos, malathion and fenitrothion in all populations. No population showed increased NSE but an increase of P450 in two populations of Yopal. Conclusions: It appears that the P450 may play an important role in resistance to pyrethroids and DDT, still other resistance mechanisms may be acting in populations. Susceptibility to organophosphate allows continued use of this chemical group to interrupt transmission of dengue in Casanare.”
“BACKGROUND: Oncotype DX is a 21-gene assay that calculates a risk of distant recurrence in women with estrogen-receptor-positive, lymph node-negative breast cancer.

Single pure colonies of bacteria were transferred into fresh BHI broth and incubated overnight. Bacterial counting was done using hemocytometer. Antibacterial activities were determined using bacteria grown on Mueller Hinton II agar and antimicrobial disc diffusion susceptibility testing with paper discs impregnated with ClO(2) and Hyaluronate gels as well as by minimum inhibition concentration (MIC) test. Bacterial morphological

alterations following treatment with ClO(2) and Hyaluronate gels were viewed under Scanning Electron Microscope (SEM) at 3500x, 10000 x and 20000x magnification. Positive results were obtained Cl-amidine cell line with disc diffusion technique whereby both agents exhibited antibacterial action against the microorganisms tested. ClO(2) gel produced large diameter PD173074 clinical trial inhibition zones while Hyaluronate gel resulted in smaller diameter inhibition zones. In MIC test the lowest MIC value of ClO2 gel (0.02% w/v) was obtained for S. aureus, S. mitis and S. constellatus. The other bacteria and pool samples of dental biofilm indicated slightly higher MIC values (0.2% w/v) for ClO(2) gel. However, MIC values for Hyaluronate gel could not be determined. Under SEM, ClO(2) gel produced obvious alterations to the bacterial morphology while no changes were observed after treatment with hyaluronate gel. Chlorine dioxide gel demonstrated

stronger and obvious antibacterial activity.”
“It is well established that the reinforcing properties of nicotine (NIC) depend on its action on nicotinic acetylcholine receptors expressed by brain neurons. However,

when administered systemically, NIC first phasically activates nicotinic receptors located on the afferents of sensory nerves at the sites of drug administration before reaching the brain and directly interacting with central neurons. While this peripheral action of NIC has been known for years, it is usually neglected in any consideration of the drug’s reinforcing properties and experiencedependent changes of its behavioral and physiological effects. The goal of this work was to review our recent behavioral, electrophysiological, and physiological VS-4718 solubility dmso data suggesting the critical importance of peripheral actions of NIC in mediating its neural effects following acute drug exposure and their involvement in alterations of NIC effects consistently occurring following repeated drug exposure. Because NIC, by acting peripherally, produces a rapid sensory signal to the central nervous system that is followed by slower, more prolonged direct drug actions in the brain, these two pharmacological actions interact in the central nervous system during repeated drug use with the development of Pavlovian conditioned association.