Flux control of sulphate assimilation in Arabidopsis thaliana : adenosine 5′-phosphosulphate reductase is more susceptible than ATP sulphurylase to negative control by thiols

The effect of externally applied l-cysteine and glutathione (GSH) on ATP sulphurylase and adenosine 5′-phosphosulphate reductase (APR), two key enzymes of assimilatory sulphate reduction, was examined in Arabidopsis thaliana root cultures. Addition of increasing l-cysteine to the nutrient solution increased internal cysteine, γ-glutamylcysteine and GSH concentrations, and decreased APR mRNA, protein and extractable activity. An effect on APR could already be detected at 0.2 mm l-cysteine, whereas ATP sulphurylase was significantly affected only at 2 mm l-cysteine. APR mRNA, protein and activity were also decreased by GSH at 0.2 mm and higher concentrations. In the presence of l-buthionine-S, R-sulphoximine (BSO), an inhibitor of GSH synthesis, 0.2 mm l-cysteine had no effect on APR activity, indicating that GSH formed from cysteine was the regulating substance. Simultaneous addition of BSO and 0.5 mm GSH to the culture medium decreased APR mRNA, enzyme protein and activity. ATP sulphurylase activity was not affected by this treatment. Tracer experiments using 35SO42– in the presence of 0.5 mm l-cysteine or GSH showed that both thiols decreased sulphate uptake, APR activity and the flux of label into cysteine, GSH and protein, but had no effect on the activity of all other enzymes of assimilatory sulphate reduction and serine acetyltransferase. These results are consistent with the hypothesis that thiols regulate the flux through sulphate assimilation at the uptake and the APR step. Analysis of radioactive labelling indicates that the flux control coefficient of APR is more than 0.5 for the intracellular pathway of sulphate assimilation. This analysis also shows that the uptake of external sulphate is inhibited by GSH to a greater extent than the flux through the pathway, and that the flux control coefficient of APR for the pathway, including the transport step, is proportionately less, with a significant share of the control exerted by the transport step.

Negative perceptions about self-control and identification with gender-role stereotypes related to binge eating, problem drinking, and to co-morbidity among adolescents

Purpose
To examine the role of both positive and negative styles of self-control, and gender-role stereotypes in binge eating and problem drinking.

Method
Participants were 428 adolescent boys and 555 girls from predominantly Anglo-Australian backgrounds who attended regional state schools in New South Wales, Australia. Students completed standardized questionnaires that assessed problem drinking, binge eating, self-control styles, and identification with gender-role stereotypes. ANOVA and post hoc Tukey tests were conducted to examine differences among adolescents who reported problems in binge eating, drinking, and both domains.

Results
Adolescents who reported eating and drinking problems also reported a high negative and a low positive sense of self-control coupled with self-identification with the traits that typically describe negative dimensions of gender-role stereotypes. Regardless of gender, problem drinking was mainly related to traits of negative masculinity (bossy, noisy aggressive, etc.) whereas binge eating was mainly related to negative femininity (shy, needs approval from others, etc.). Participants who reported eating and drinking symptoms recorded low scores on positive control, high scores on negative control, and also high scores on the negative dimensions of masculinity and femininity.

Summary
A negative and passive style of self-control coupled with an identification with negative dimensions of gender summarizes the type of self-regulation that is implicated in both binge eating and problem drinking, and co-morbid symptoms. There is a need for interventions working toward a more balanced gender self-concept and a positive sense of self-control.

Role of the N-terminal region in G protein-coupled receptor functions : negative modulation revealed by 5-HT2B receptor polymorphisms

The putative role of the N-terminal region of rhodopsin-like 7 transmembrane biogenic amine receptors in agonist-induced signaling has not yet been clarified despite recent advances in 7 transmembrane receptor structural biology. Given the existence of N-terminal nonsynonymous polymorphisms (R6G;E42G) within the HTR2B gene in a drug-abusing population, we assessed whether these polymorphisms affect 5-hydroxytryptamine 2B (5-HT2B) receptor in vitro pharmacologic and coupling properties in transfected COS-7 cells. Modification of the 5-HT2B receptor N terminus by the R6G;E42G polymorphisms increases such agonist signaling pathways as inositol phosphate accumulation as assessed by either classic or operational models. The N-terminal R6G;E42G mutations of the 5-HT2B receptor also increase cell proliferation and slow its desensitization kinetics compared with the wild-type receptor, further supporting a role for the N terminus in transduction efficacy. Furthermore, by coexpressing a tethered wild-type 5-HT2B receptor N terminus with a 5-HT2B receptor bearing a N-terminal deletion, we were able to restore original coupling. This reversion to normal activity of a truncated 5-HT2B receptor by coexpression of the membrane-tethered wild-type 5-HT2B receptor N terminus was not observed using a membrane-tethered 5-HT2B receptor R6G;E42G N terminus. These data suggest that the N terminus exerts a negative control over basal as well as agonist-stimulated receptor activity that is lost in the R6G;E42G mutant. Our findings reveal a new and unanticipated role of the 5-HT2B receptor N terminus as a negative modulator, affecting both constitutive and agonist-stimulated activity. Moreover, our data caution against excluding the N terminus and extracellular loops in structural studies of this 7 transmembrane receptor family

EGFR signalling as a negative regulator of Notch1 gene transcription and function in proliferating keratinocytes and cancer.

The Notch1 gene has an important role in mammalian cell-fate decision and tumorigenesis. Upstream control mechanisms for transcription of this gene are still poorly understood. In a chemical genetics screen for small molecule activators of Notch signalling, we identified epidermal growth factor receptor (EGFR) as a key negative regulator of Notch1 gene expression in primary human keratinocytes, intact epidermis and skin squamous cell carcinomas (SCCs). The underlying mechanism for negative control of the Notch1 gene in human cells, as well as in a mouse model of EGFR-dependent skin carcinogenesis, involves transcriptional suppression of p53 by the EGFR effector c-Jun. Suppression of Notch signalling in cancer cells counteracts the differentiation-inducing effects of EGFR inhibitors while, at the same time, synergizing with these compounds in induction of apoptosis. Thus, our data reveal a key role of EGFR signalling in the negative regulation of Notch1 gene transcription, of potential relevance for combinatory approaches for cancer therapy.

Studies on arbuscular mycorrhiza (AM) in the Alentejo (Portugal) using pea mutants resistant to AM fungi as a control tool for field conditions

The utilization and management of arbuscular mycorrhiza (AM) symbiosis may improve production and sustainability of the cropping system. For this purpose, native AM fungi (AMF) were sought and tested for their efficiency to increase plant growth by enhanced P uptake and by alleviation of drought stress. Pot experiments with safflower (Carthamus tinctorius) and pea (Pisum sativum) in five soils (mostly sandy loamy Luvisols) and field experiments with peas were carried out during three years at four different sites. Host plants were grown in heated soils inoculated with AMF or the respective heat sterilized inoculum. In the case of peas, mutants resistant to AMF colonization were used as non-mycorrhizal controls. The mycorrhizal impact on yields and its components, transpiration, and P and N uptake was studied in several experiments, partly under varying P and N levels and water supply. Screening of native AMF by most probable number bioassays was not very meaningful. Soil monoliths were placed in the open to simulate field conditions. Inoculation with a native AMF mix improved grain yield, shoot and leaf growth variables as compared to control. Exposed to drought, higher soil water depletion of mycorrhizal plants resulted in a haying-off effect. The growth response to this inoculum could not be significantly reproduced in a subsequent open air pot experiment at two levels of irrigation and P fertilization, however, safflower grew better at higher P and water supply by multiples. The water use efficiency concerning biomass was improved by the AMF inoculum in the two experiments. Transpiration rates were not significantly affected by AM but as a tendency were higher in non-mycorrhizal safflower. A fundamental methodological problem in mycorrhiza field research is providing an appropriate (negative) control for the experimental factor arbuscular mycorrhiza. Soil sterilization or fungicide treatment have undesirable side effects in field and greenhouse settings. Furthermore, artificial rooting, temperature and light conditions in pot experiments may interfere with the interpretation of mycorrhiza effects. Therefore, the myc- pea mutant P2 was tested as a non-mycorrhizal control in a bioassay to evaluate AMF under field conditions in comparison to the symbiotic isogenetic wild type of var. FRISSON as a new integrative approach. However, mutant P2 is also of nod- phenotype and therefore unable to fix N2. A 3-factorial experiment was carried out in a climate chamber at high NPK fertilization to examine the two isolines under non-symbiotic and symbiotic conditions. P2 achieved the same (or higher) biomass as wild type both under good and poor water supply. However, inoculation with the AMF Glomus manihot did not improve plant growth. Differences of grain and straw yields in field trials were large (up to 80 per cent) between those isogenetic pea lines mainly due to higher P uptake under P and water limited conditions. The lacking N2 fixation in mutants was compensated for by high mineral N supply as indicated by the high N status of the pea mutant plants. This finding was corroborated by the results of a major field experiment at three sites with two levels of N fertilization. The higher N rate did not affect grain or straw yields of the non-fixing mutants. Very efficient AMF were detected in a Ferric Luvisol on pasture land as revealed by yield levels of the evaluation crop and by functional vital staining of highly colonized roots. Generally, levels of grain yield were low, at between 40 and 980 kg ha-1. An additional pot trial was carried out to elucidate the strong mycorrhizal effect in the Ferric Luvisol. A triplication of the plant equivalent field P fertilization was necessary to compensate for the mycorrhizal benefit which was with five times higher grain yield very similar to that found in the field experiment. However, the yield differences between the two isolines were not always plausible as the evaluation variable because they were also found in (small) field test trials with apparently sufficient P and N supply and in a soil of almost no AMF potential. This similarly occurred for pea lines of var. SPARKLE and its non-fixing mycorrhizal (E135) and non-symbiotic (R25) isomutants, which were tested in order to exclude experimentally undesirable benefits by N2 fixation. In contrast to var. FRISSON, SPARKLE was not a suitable variety for Mediterranean field conditions. This raises suspicion putative genetic defects other than symbiotic ones may be effective under field conditions, which would conflict with the concept of an appropriate control. It was concluded that AMF resistant plants may help to overcome fundamental problems of present research on arbuscular mycorrhiza, but may create new ones.

Revascularization and periapical repair after endodontic treatment using apical negative pressure irrigation versus conventional irrigation plus triantibiotic intracanal dressing in dogs` teeth with apical periodontitis

Objective. The objective of this study was to evaluate in vivo the revascularization and the apical and periapical repair after endodontic treatment using 2 techniques for root canal disinfection (apical negative pressure irrigation versus apical positive pressure irrigation plus triantibiotic intracanal dressing) in immature dogs` teeth with apical periodontitis. Study design. Two test groups of canals with experimentally induced apical periodontitis were evaluated according to the disinfection technique: Group 1, apical negative pressure irrigation (EndoVac system), and Group 2, apical positive pressure irrigation (conventional irrigation) plus triantibiotic intracanal dressing. In Group 3 (positive control), periapical lesions were induced, but no endodontic treatment was done. Group 4 (negative control) was composed of sound teeth. The animals were killed after 90 days and the maxillas and mandibles were subjected to histological processing. The sections were stained with hematoxylin and eosin and Mallory Trichrome and examined under light microscopy. A description of the apical and periapical features was done and scores were attributed to the following histopathological parameters: newly formed mineralized apical tissue, periapical inflammatory infiltrate, apical periodontal ligament thickness, dentin resorption, and bone tissue resorption. Intergroup comparisons were done by the Kruskal-Wallis and Dunn`s tests (alpha = 0.05). Results. Although statistically significant difference was found only for the inflammatory infiltrate (P < .05), Group 1 presented more exuberant mineralized formations, more structured apical and periapical connective tissue, and a more advanced repair process than Group 2. Conclusion. From the histological observations, sodium hypochlorite irrigation with the EndoVac system can be considered as a promising disinfection protocol in immature teeth with apical periodontitis, suggesting that the use of intracanal antibiotics might not be necessary. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010; 109: 779-787)

Anticoccidial effects of coumestans from Eclipta alba for sustainable control of Eimeria tenella parasitosis in poultry production

The aim of this study was to evaluate the anticoccidial efficacy of a product containing coumestans from Eclipta alba. Experimental conditions were set up as to reproduce the environment conditions for husbandry adopted in commercial broiler farms. Broilers were raised in broiler chicken shed provided with feeders, drinkers, illumination and temperature control systems and floor covering to afford an adequate nourishing environment. Male Cobb broilers (240) were assigned to four experimental groups being each experimental group set apart in rice straw-covered shed isolated with wire mesh. One-day-old broilers were reared in a coccidian-free environment with ad libitum supply of filtered water and freely available standard feed, from the 1st to the 35th day of life. The T1 group received standard feed (negative control); T2 was treated with standard feed supplemented with 66 ppm of salinomycin (positive control); groups T3 and T4 had standard feed supplemented with the ethyl acetate fraction from methanolic extract of E. alba aerial parts, which contains the coumestans WL and DWL (120 and 180 ppm, respectively). The chicken broilers were individually infected with 2 x 104 oocysts of Eimeria tenella when they were 14 days old and were monitored weekly to evaluate zootechnical parameters such as weight gain and food conversion ratio. Counting of coccidial oocyst in chiken feces was assessed from random samples, from the 21st to 28th days of life, which corresponded to 7-14 days after the infection. Five chickens selected at random from each experimental group were subsequently euthanized at 21, 28 or 35 days of life to determine the lesion score in the cecal region and to excise a cecum portion for histopathological evaluation. The group treated with coumestans from E. alba presented an average weight gain and food conversion ratio higher than the negative control group and similar to the mean value of the positive control group. Coumestan-treated groups showed a significant decrease in the oocyst counting since the 21th day of life and displayed a reduced number of macroscopic lesions. Histopathological evaluations of cecum fragments showed that both treatments induced the migration of defense cells at the site of infection. A severe destruction of the cecal lining was found in the intestinal tract of broilers fed with a coumestans dose of 180 ppm. Overall, our results validate the use of a phytotherapy containing E. alba coumestans at a dose of 120 ppm as a therapeutic or prophylactic agent against avian coccidiosis. (C) 2010 Elsevier B.V. All rights reserved.

Botanical extracts: alternative control for silverleaf whitefly management in tomato

Bemisia tabaci (Hemiptera: Aleyrodidae) biotype B is one of the most limiting pests of tomato crops in the world. Tomato yield is currently dependent on the use of pesticides, which are problematic to farmers, consumers and the environment. A promising alternative to reduce the harmful effects caused by the indiscriminated use of synthetic insecticides is the use of insecticides of botanical origin. This study aimed to evaluate the effect of 3% (w/v) aqueous extracts from different structures of thirteen botanical species on the behavior of B. tabaci biotype B adults, as well as insecticidal activity of such aqueous extracts on B. tabaci eggs, nymphs, and adults infesting tomato plants. A distilled water solution was used as a negative control, and thiamethoxam insecticide (18 g/100 L of water) as a positive control. Leaf extract of Toona ciliata was observed to have the most efficient inhibitory effect in tests of extracts on whitefly behavior. Furthermore, the use of leaf extract of Toona ciliata led to the most drastic reduction in the number of adults and eggs on tomato leaflets. Leaf extract of Piper aduncum led to the greatest observed ovicidal effect (78.00% of non-hatched nymphs); however it was not effective against nymphs and adults. The leaf extracts of Trichilia pallida, Trichilia casaretti, and Toona ciliata showed the highest control indexes (67.9, 60.3, and 55.1%, respectively). For adults mortality, T. pallida was the most effective (72.8%). Our results indicate that application of extracts of T. pallida, T. ciliata, and T. casaretti are promising strategies to manage B. tabaci biotype B on tomato.

Application of Cymbopogon winterianus Jowitt and Azadirachta indica A. Juss in the control of Rhipicephalus (Boophilus) microplus

Parasitism constitutes one of the main problems that affect livestock. Commercial acaricides have been used to control tick, but these chemicals have been responsible for the development of drug resistance and residues in the environment. Changes in the concept of production determined the search for natural measures, which guarantees animal sanity along with safety of the produced food. This assay had as objective to evaluate the citronella oil and neem oil in the control of bovine ticks. By the technique of adult ticks immersion, 280 ticks were evaluated, distributed in equal number throughout four treatments: negative control group, positive control (ivermectin), neem oil and citronella oil. It was analyzed the mortality index, estimated reproduction, product efficiency, index of eggs production and the hatchability rate. The efficiency of the product was verified by the mortality index just for the positive control group (100%) and citronella oil (97.14%). Also, the citronella oil inhibited the eclosion of eggs in 100%. The other treatments did not presented the minimum eclosion inhibition level of 95%. Under the conditions of the present assay citronella oil was efficient against Rhipicephalus (Boophilus) microplus. This result was not observed in relation to the neem oil.

Formulations of Melia azedarach to control diabrotica speciosa (germar) (coleoptera: chrysomelidae) larvae in corn and plant enhancement

We evaluated oil and powder formulations of Melia azedarach for controlling larvae of Diabrotica speciosa (Germar) in corn and plant enhancement. Five concentrations of each formulation were evaluated and compared to fipronil (negative control) and distilled water (positive control). After treatment, the number of surviving insects (larvae, pupae, and adults), the adult body weight, the sex ratio, and the longevity were recorded, while the height, dry weight of aerial part and roots, and number of leaves of plants were measured. The oil formulation at 4.0 mL reduced the larvae population of D. speciosa similarly to the insecticide fipronil, which resulted in greater height, dry weight of the root system, and number of leaves. Powder formulation at concentrations of 40, 80, and 160 mg caused larval mortality above 80%; however, these concentrations did not prevent reduction of plant height and dry weight of aerial part. Further studies assessing the residual period of M. azedarach control against D. speciosa larvae and its phytotoxicity, which are common traits associated with azadirachtin application, are necessary to subsidize the next steps of this alternative control strategy.

Actividad biológica de Lonchocarpus guaricensis Pittier en el control de larvas de Tuta absoluta (Meyrick)

La actividad biológica de Lonchocarpus guaricensis Pittier fue evaluada utilizando dos dosis de Tecnona® en el control de larvas de Tuta absoluta (Meyrick), Valle de Azapa, Chile, mediante una pulverización sobre plantas de tomate cv. Naomi en macetas, ubicadas aleatoriamente en un invernáculo dentro de un vivero. Semanas previas a la pulverización, las macetas se infestaron artificialmente con adultos del fitófago para obtener larvas en los foliolos. Los tratamientos evaluados fueron los siguientes: T1 (0,21 g de IA de L. guaricensis·L-1), T2 (0,43 g de IA de L. guaricensis·L-1), T3 (control positivo a base de spinosad 0,048 g de IA·L-1) y T0 (control negativo a base de agua de pozo). De acuerdo con el porcentaje de mortalidad acumulada de larvas contabilizadas a las 24, 48, 120 horas y 9no día post aplicación, no hay diferencias estadísticas entre los tratamientos T0 y T1, a su vez, T2 alcanza una media de 53,05% de mortalidad, no diferenciándose de T3 que logra un 73,9%. Se concluye que la dosis experimental L. guaricensis de 0,43 g de IA∙ L-1 puede constituir una alternativa interesante de utilizar en el Manejo Integrado de Plagas del cultivo de tomate en el Valle de Azapa.

Discovery and characterization of IGFBP-mediated endocytosis in the human retinal pigment epithelial cell line ARPE-19

Insulin-like growth factor binding proteins (IGFBPs) are prime regulators of IGF-action in numerous cell types including the retinal pigment epithelium (RPE). The RPE performs several functions essential for vision, including growth factor secretion and waste removal via a phagocytic process mediated in part by vitronectin (Vn). In the course of studying the effects of IGFBPs on IGF-mediated VEGF secretion and Vn-mediated phagocytosis in the RPE cell line ARPE-19, we have discovered that these cells avidly ingest synthetic microspheres (2.0 μm diameter) coated with IGFBPs. Given the novelty of this finding and the established role for endocytosis in mediating IGFBP actions in other cell types, we have explored the potential role of candidate cell surface receptors. Moreover, we have examined the role of key IGFBP structural motifs, by comparing responses to three members of the IGFBP family (IGFBP-3, IGFBP-4 and IGFBP-5) which display overlapping variations in primary structure and glycosylation status. Coating of microspheres (FluoSpheres®, sulfate modified polystyrene filled with a fluorophore) was conducted at 37 °C for 1 h using 20 μg/mL of test protein, followed by extensive washing. Binding of proteins was confirmed using a microBCA assay. The negative control consisted of microspheres treated with 0.1% bovine serum albumin (BSA), and all test samples were post-treated with BSA in an effort to coat any remaining free protein binding sites, which might otherwise encourage non-specific interactions with the cell surface. Serum-starved cultures of ARPE-19 cells were incubated with microspheres for 24 h, using a ratio of approximately 100 microspheres per cell. Uptake of microspheres was quantified using a fluorometer and was confirmed visually by confocal fluorescence microscopy. The ARPE-19 cells displayed little affinity for BSA-treated microspheres, but avidly ingested large quantities of those pre-treated with Vn (ANOVA; p < 0.001). Strong responses were also observed towards recombinant formulations of non-glycosylated IGFBP-3, glycosylated IGFBP-3 and glycosylated IGFBP-5 (all p < 0.001), while glycosylated IGFBP-4 induced a relatively minor response (p < 0.05). The response to IGFBP-3 was unaffected in the presence of excess soluble IGFBP-3, IGF-I or Vn. Likewise, soluble IGFBP-3 did not induce uptake of BSA-treated microspheres. Antibodies to either the transferrin receptor or type 1 IGF-receptor displayed slight inhibitory effects on responses to IGFBPs and Vn. Heparin abolished responses to Vn, IGFBP-5 and non-glycosylated IGFBP-3, but only partially inhibited the response to glycosylated IGFBP-3. Our results demonstrate for the first time IGFBP-mediated endocytosis in ARPE-19 cells and suggest roles for the IGFBP-heparin-binding domain and glycosylation status. These findings have important implications for understanding the mechanisms of IGFBP actions on the RPE, and in particular suggest a role for IGFBP-endocytosis.

Ovine Enzootic Abortion (OEA): a comparison of antibody responses in vaccinated and naturally-infected swiss sheep over a two year period

Background Prevention and control of ovine enzootic abortion (OEA) can be achieved by application of a live vaccine. In this study, five sheep flocks with different vaccination and infection status were serologically tested using a competitive enzyme-linked immunosorbent assay (cELISA) specific for Chlamydophila (Cp.) abortus over a two-year time period. Results Sheep in Flock A with recent OEA history had high antibody values after vaccination similar to Flock C with natural Cp. abortus infections. In contrast, OEA serology negative sheep (Flock E) showed individual animal-specific immunoreactions after vaccination. Antibody levels of vaccinated ewes in Flock B ranged from negative to positive two and three years after vaccination, respectively. Positive antibody values in the negative control Flock D (without OEA or vaccination) are probably due to asymptomatic intestinal infections with Cp. abortus. Excretion of the attenuated strain of Cp. abortus used in the live vaccine through the eye was not observed in vaccinated animals of Flock E. Conclusion The findings of our study indicate that, using serology, no distinction can be made between vaccinated and naturally infected sheep. As a result, confirmation of a negative OEA status in vaccinated animals by serology cannot be determined.

Porous scaffold architecture guides tissue formation

Critical-sized bone defect regeneration is a remaining clinical concern. Numerous scaffold-based strategies are currently being investigated to enable in vivo bone defect healing. However, a deeper understanding of how a scaffold influences the tissue formation process and how this compares to endogenous bone formation or to regular fracture healing is missing. It is hypothesized that the porous scaffold architecture can serve as a guiding substrate to enable the formation of a structured fibrous network as a prerequirement for later bone formation. An ovine, tibial, 30-mm critical-sized defect is used as a model system to better understand the effect of the scaffold architecture on cell organization, fibrous tissue, and mineralized tissue formation mechanisms in vivo. Tissue regeneration patterns within two geometrically distinct macroscopic regions of a specific scaffold design, the scaffold wall and the endosteal cavity, are compared with tissue formation in an empty defect (negative control) and with cortical bone (positive control). Histology, backscattered electron imaging, scanning small-angle X-ray scattering, and nanoindentation are used to assess the morphology of fibrous and mineralized tissue, to measure the average mineral particle thickness and the degree of alignment, and to map the local elastic indentation modulus. The scaffold proves to function as a guiding substrate to the tissue formation process. It enables the arrangement of a structured fibrous tissue across the entire defect, which acts as a secondary supporting network for cells. Mineralization can then initiate along the fibrous network, resulting in bone ingrowth into a critical-sized defect, although not in complete bridging of the defect. The fibrous network morphology, which in turn is guided by the scaffold architecture, influences the microstructure of the newly formed bone. These results allow a deeper understanding of the mode of mineral tissue formation and the way this is influenced by the scaffold architecture. Copyright © 2012 American Society for Bone and Mineral Research.