Previous studies have shown that many multigene families, including proteins of the immune system, evolved according to a mechanism defined as the birth-and-death

process (Nei and Rooney, 2005). MK-1775 clinical trial This process was reported for mammalian β-defensin genes (Morrison et al., 2003), bovine defensin genes (Liu et al., 2009) and α-defensin genes (Das et al., 2010), and may explain the degree of diversity amongst the sequences in Anolis carolinensis ( Dalla Valle et al., 2012). The unusually high degree of sequence variation in the mature peptide produced by the paralogous and in some cases orthologous genes implies extensive specialization and species-specific adaptation ( Semple et al., 2006). Comparative studies are important in determining patterns of evolution and function of the innate immune system. In this work, we describe new β-defensin-like genes in Brazilian pitvipers of the Bothrops and Lachesis genera, where we analyzed them phylogenetically and Fulvestrant reconciled the species tree with gene tree to infer duplication/speciation

nodes of these β-defensin-like genes. The snakes studied in this work were Bothrops alternatus (Estiva – MG, IBSP 77.198), B. atrox (Rio Branco – AC, IBSP 79.765), B. diporus (Blumenal – SC, IBSP 60.323), B. insularis (Queimada Grande Island – SP), B. erythromelas (Ibitira – BA, IBSP 79.766), B. jararaca (Embu Guaçu – SP), B. jararacussu (Ubatuba – SP), B. leucurus (Porto Seguro – BA, IBSP 79.100), B. mattogrossensis (N. Sra do Livramento – MT, IBSP 77.705), B. neuwiedi (Baependi – MG, IBSP 74.566), B. pauloensis (Frutal – MG, IBSP 71.111), Crotalus durissus, Lachesis muta (Northeast Brazil). We used livers and scales from snakes deposited in the Tissue Collection of Alphonse Hoge Herpetological Collection at the Butantan Institute and the blood from B. insularis snakes, kept alive in the Ecology and Evolution Laboratory, and from L. muta, kept in the Herpetology Laboratory, both at the Butantan Institute.

The DNA was purified from liver tissues (Ausubel et al., 2000), scales (Fetzner, 1999) or blood (ZR Genomic DNA Tissue kit, ZymoResearch), which was then quantified at 260 nm using the NanoDrop ND-2000c spectrophotometer. The forward and reverse primers H010 (5′-AAGCAGTCTCAGCATGAAGAT-3′) and 3′UTRas (5′-GGCACTCTCAGGTCCTTGGCCAT-3′) were designed on the basis of crotamine (Rádis-Baptista et al., 2003) and crotasin Cyclin-dependent kinase 3 (Rádis-Baptista et al., 2004) gene sequences to amplify β-defensin-like sequences. A 50 μl reaction mix contained 100–1000 ng DNA sample, 0.1 μM each primer, 1.25 U Taq DNA Polymerase Platinum (Invitrogen), buffer with the addition of 1.5 mM MgCl2, and 0.2 mM dNTPs mix. The amplification process used an initial denaturation step of 4 min at 94 °C, followed by 30 cycles of 45 s at 94 °C, 45 s at 52.5, 55 or 58 °C and 45 s at 72 °C, and finally 1 min at 72 °C. The amplified DNA was purified, after electrophoresis on a 1% agarose gel, using the Zymoclean Gel DNA Recovery kit (ZymoResearch).

This suggests that these proteins play an important role in the modulation of host response. Understanding the role of SOCS proteins in the negative regulation of cytokine signaling, especially in the JAK/STAT pathway, may provide novel information on the susceptibility to periodontal diseases and also for therapeutic strategies NVP-BKM120 solubility dmso based on the modulation of the inflammatory process. The authors want to express their gratitude to the research technician Ana Claudia Gregolin da Costa Miranda (Department of Diagnosis and Surgery, UNESP at Araraquara) for her technical assistance. Grant support was provided by grants #2007/06658-7

and #2007/06332-4 awarded by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) to C.R.J. and transferred to J.A.C. Funding: Sao Paulo State Research Support Foundation: FAPESP. A research support foundation

from the State of São Paulo, Brazil. Grants #2007/06658-7 KU-60019 datasheet and #2007/06332-4. Conflict of interest: None. Ethical approval: By the Ethical Committee on Animal Experimentation of the School of Dentistry at Araraquara – UNESP (protocol number 23/2007), where the in vivo part of the study was conducted. “
“The number of individuals with diabetes mellitus has been increasing worldwide. In Brazil, 5.2% of the adult population has this disease and in the United States 72,507 of deaths are related to this hyperglycaemic condition, with diabetes being amongst the 10 leading causes of death in that country.1, 2, 3 and 4 These data are important since diabetes is an irreversible disease and is associated with hormone alterations that result in various complications in the patient, including involvement of the salivary glands.5, 6, 7, 8, 9, 10, 11, 12 and 13 In general, glandular tissues are under the influence of hormones that regulate cell activity. These biological Isotretinoin effects are mediated by the interaction between hormones

and cellular receptors.14, 15, 16, 17, 18 and 19 In this respect, insulin is the main hormone involved in the regulation of cell growth and its action is mediated by receptors (INS-R).20, 21 and 22 Understanding the relationship between functional proteins and tissues and the impaired interaction in hyperglycaemic conditions, some studies have tried to reverse this damage by treatment with hypoglycaemic agents. For example, various investigators studied the effect of insulin on the salivary glands. The results showed that, despite beneficial metabolic effects, doubts exist regarding the true recovery of tissues in response to this type of treatment.

Ongoing research on alternative experimental administration strategies includes ballistic delivery to skin (the gene gun), the transdermal patch and

other intradermal methods, plus sublingual, aerosol, rectal and vaginal mucosal vaccines. The main advantages of alternative delivery strategies are the potential to induce immune responses at the common portals of pathogen entry (eg oral DAPT research buy polio vaccine replicating in the gut), potential convenience (eg ease of use of the transdermal patch), potential combination of vaccines to reduce or simplify the vaccination schedule, and reduction or elimination of administration via standard hypodermic needle injection. Despite the intuitive

value of these approaches, few vaccines today are administered via non-IM routes. This is for several reasons including feasibility, lack of proven efficacy and limited safety data. Some problems have been observed selleck compound with new routes of delivery, for example, after the 2000 launch of an inactivated intranasal influenza vaccine (a virosome formulation adjuvanted by heat labile enterotoxoid of Escherichia coli), post-licensure data indicated a significantly increased risk of Bell’s palsy in vaccinees and forced its withdrawal from the market. This experience led to a higher level of caution in the development of intranasal vaccines. Transdermal microneedle patch vaccine administration utilises an array of microneedles (Figure 6.6) to deliver the vaccine to the epidermis, which is rich in innate and adaptive immune response elements. Aerosol delivery: ‘Mass immunization of almost all susceptible children in a short period

of time, has the potential of rapidly eliminating measles as a public health problem. Immunization by inhalation of aerosolised measles vaccine provides a procedure that could make such a mass programme possible, especially in parts of the world where measles continues to be a serious problem…’ CHIR-99021 in vitro (Sabin et al., 1983). Administering the measles vaccine as an aerosol, either as nebulised vaccine or as a dry powder, provides a promising alternative to subcutaneous administration, particularly in countries with concerns over inadequately safe injection practices. Numerous clinical trials with aerosolised measles vaccine have been performed in populations of various ages and appear to be equally or more immunogenic than subcutaneous vaccination in adults and children over 9 months old (data from younger children are inconclusive, possibly because of administration difficulties).

, 2008). Leukocyte–endothelial

interactions are the initial and fundamental events for the migration of circulating leukocyte to an inflammatory buy PD0332991 focus. This highly coordinated process depends on the sequential expressions of selectins, integrins and immunoglobulin superfamily adhesion molecules, influenced by actions of inflammatory chemical mediators. E-, P- and L-selectins control the initial interaction of leukocytes into vessel wall, and β integrins and intracellular (ICAM-1), vascular (VCAM-1) and platelet-endothelial (PECAM-1) cell adhesion molecules mediate their subsequent adhesion to the microvascular endothelium and transmigration into inflamed tissues (Wong et al., 2010 and Ley et al., 2007). Vascular,

metabolic, and immune diseases, as well as environmental and selleck chemical occupational pollutants, can modify the physiological expression pattern of adhesion molecules, leading to altered host defense (Khan et al., 2010, Barreiro et al., 2010, Etzioni, 2010, Lino dos Santos Franco et al., 2009 and Lino dos Santos Franco et al., 2010). We have previously shown that in vivo HQ exposure alters leukocyte migration to inflammatory sites during the development of acute innate and acquired responses in rats. While the effects on acquired immunity are related to reduced anaphylactic immunoglobulin production, the mechanisms involved in the acute innate inflammation have not been clearly elucidated ( Ferreira et al., 2007, Macedo et al., 2007 and Macedo et al., 2006). Little http://www.selleck.co.jp/products/sorafenib.html is known about the in vivo HQ toxicity ( McGregor, 2007), and more specifically, on leukocyte recruitment to the inflammatory site. Therefore, in this study we show that lower levels of systemic HQ exposure impairs neutrophil migration during a LPS-induced lung inflammation in mice

and highlights specific intracellular pathways in circulating neutrophils as important target of HQ action. Lipopolysaccharide (LPS) from Escherichia coli (serotype 026:B6), N-formyl-methionyl-leucyl-phenylalanine (fMLP), hydroquinone (99%), n-butanol, 1,1,3,3-tetramethoxypropane (99%), hexadecyltrimethylammonium bromide, ortho-dianizidine, acetonitrile, butylated hydroxytoluene, potassium iodide, Triton X100, propidium iodide and RNAse A were purchased from Sigma–Aldrich (St. Louis, MO, USA); hexane, ethanol (99%), hydrogen peroxide, acetic acid, trichloroacetic acid, sodium chloride, monobasic and dibasic sodium phosphate, ammonium chloride and acetone were obtained from Synth (Sao Paulo, SP, Brazil); DCFH was obtained from Molecular Probes (Carlsbad, CA, USA); ketamine (1.16 g/10 ml) and xylazine (2.

473, regarding exposure conditions, doses with precipitation but no cytotoxicity should be used as the highest dose in the in vitro chromosomal aberration test. In the present buy GSK458 study, all three concentrations assessed resulted in precipitation of the styrene oligomers out of the culture medium, confirming that the concentration of oligomers used in the present study contained high concentrations of styrene oligomers. In addition,

there was no cytotoxicity at the three doses assessed. It is likely that the present results obtained by using an extracted solution of styrene oligomers are comparable to those that would be obtained by using a pure oligomer solution. Our findings show the availability of acetone instead of 50% ethanol aqueous solution, which is recommended as a fatty-food simulant for polystyrene by FDA and EFSA, to extract styrene oligomers from polystyrene intended for use in contact with food to allow the evaluation of genotoxicity in vitro. Even if high concentrations were applied the Ames test and the chromosomal aberration test, styrene oligomers extracted from GPPS did not induce gene mutation nor chromosomal

aberration, suggesting that the risk of the genotoxicity of styrene oligomers migrated from polystyrene food packaging into food is likely very low. The authors declare that there are no conflicts www.selleckchem.com/products/ly2157299.html of interest. [20] and [21] “
“Senile dementia, such as Alzheimer’s disease, is a serious global public health crisis as there is no effective therapy for it currently. Neurohealth,

is thus a major concern of the predicted Silver Tsunami—the growing wave of people who will reach the age of 65 over the next two decades and may be affected by geriatric cognitive disorders— which will greatly impact society in the next 40 years as the number of dependent older people is estimated to increase three-fold, from 101 million in 2010 to 277 million in 2050 [1]. It has been shown that dysregulation of nerve growth factor (NGF) signaling is linked to early stages of neurological diseases [2] and [3]. The absence of NGF has shown to cause an Alzheimer-like symptom in the brains of 15 to 17 months old anti-NGF transgenic mice [4], but such symptoms could be ameliorated by the intranasal administration IMP dehydrogenase of NGF in transgenic anti-NGF mice (AD11 mice) that have a progressive neurodegenerative phenotype resembling Alzheimer’s disease [5]. Although there is a widespread interest in NGF as a potential therapeutic agent, the high molecular weight of NGF makes it unable to cross the blood–brain barrier. Alternatively, there are new approaches being developed which focus on low-molecular weight compounds that can cross the brain-blood and promote NGF biosynthesis [6]. Hericium erinaceus (H. erinaceus), a culinary and medicinal mushroom, has been extensively studied for its neurohealth properties.

TIQ score ≥70 was defined “normal”. Moreover we calculated the difference between Verbal and Performance Intelligence Quotient (VIQ-PIQ). A VIP-PIQ score ≥ than 8 represents an abnormal development of Verbal ability in comparison to Performance ability and a score ≤ than −8 represent an abnormal development of Performance ability in comparison to Verbal ability. selleck inhibitor All patients underwent a TCD evaluation of the main intracranial arteries in order to detect

any increase of TAMM velocities (normal <170 cm/s, altered ≥170 cm/s according to the STOP protocol); TCD was performed by an experienced neurosonographer, in a quiet atmosphere and without pharmacological sedation, using a 2 MHz probe (Viasys Healthcare Sonara). All patients underwent brain magnetic resonance imaging (MRI) by means of a 1.5 T MR scanner (Achieva,

Philips, Best, The Netherlands). The study protocol included axial Fluid Attenuated Inversion Recovery (FLAIR) sequence (repetition time 11,000 ms; echo time 140 ms; inversion time: 2800; echo train length 53; flip angle 90°; field of view 230 mm; matrix 256 × 256; slice thickness 5 mm; interslice gap 0.5 mm; number of averages 2) to disclose ischemic lesions. Regarding the neuropsychological evaluation, 29/35 (82.8%) patients (Group 1) had a normal (≥70) TIQ, while 6/35 (17.2%) patients (Group 2) were defined intellectually impaired (TIQ <69). TCD detected altered velocities in 8/35 (22.8%)

patients: Androgen Receptor antagonist 6 in Group 1 and 2 in Group 2. No significant differences were found in the percentage of altered TAMM velocities between the two groups (Fisher’s exact test: p = 0.42). MRI detected silent ischemic lesions in 14/35 patients (40.0%): 12 in Group 1 and 2 in Group 2. No significant differences were found in silent stroke frequencies (Fisher’s exact test: p = 0.25) between Group 1 and Group 2. VIQ-PIQ was normal in 16/35 (45.7%) patients and altered in 19/35 (54.2%) patients. TCD detected altered TAMM in 5 patients with normal VIQ-PIQ and in 3 patients STK38 with altered VIQ-PIQ. No significant differences were found in the percentage of altered TAMM velocities between these two groups (Fisher’s exact test: p = 0.28). MRI detected silent ischemic lesions in 6 patients with normal VIQ-PIQ and in 8 patients with altered VIQ-PIQ. No significant differences were found in silent stroke frequencies (Fisher’s exact test: p = 0.52) between these two groups. According to our results, altered TAMM values and silent strokes do not seem to predict cognitive impairment in SCD patients. Our results do not seem to confirm the data found in literature, particularly the association between cognitive impairment and silent strokes [5] and [6]. The relationship between brain tissue injury and cognitive impairment in SCD is not well understood.

Nervous systems, however, have evolved as information processing systems and information transmission plays only a minor role. Then the more important question is how does sparse coding benefit brain computation? We

consider three related arguments. In a spatially sparse code, single elements represent highly specific stimulus features. A complex object can be formed only through the combination of specific features at the next level, a concept that is often referred to as the binding hypothesis (Knoblauch et al., 2001). In this scheme, attentional mechanisms could mediate a perceptual focus of objects with highly specific features by enhancing co-active units and suppressing Venetoclax mouse background activity. In a dense coding scheme, enhanced silencing of individual neurons would have an unspecific effect. A spatially sparse stimulus representation can facilitate the formation of associative memories (Palm, 1980). A particular object in stimulus space activates a highly selective set of neurons. Using an activity-dependent mechanism of synaptic plasticity allows the formation of stimulus-specific associations in this set of neurons.

This concept is theoretically and experimentally well studied in the insect mushroom body where the sparse representation of olfactory stimuli at the level of the Kenyon cells (Perez-Orive et al., 2002 and Honegger Ceritinib et al., 2011) is thought to underlie associative memory formation during classical conditioning (Huerta et al., 2004, Huerta and Nowotny, 2009, Cassenaer and Laurent,

2012 and Strube-Bloss et al., 2011). This system has been interpreted in analogy to machine learning techniques that employ a strategy of transforming a lower dimensional input space into a higher dimensional feature space to improve stimulus classification (Huerta and Nowotny, 2009, Huerta, 2013 and Pfeil et al., 2013). Theories of temporal coding acknowledge the importance of the individual spike and they receive support from accumulating experimental evidence (e.g. Riehle et al., 1997, Maldonado et al., 2008 and Jadhav et al., 2009). Coding schemes that rely on dynamic formation of cell assemblies and exact spike timing work best under conditions of spatially and a temporally sparse stimulus representations and low background activity. Endonuclease To develop the Temporal Autoencoding training method for Temporal Restricted Boltzmann Machines used in this work, we have extended upon existing work in the field of unsupervised feature learning. Two unsupervised learning methods well known within the Machine Learning community, Restricted Boltzmann Machines (RBMs) and Autoencoders (AEs) (Larochelle and Bengio, 2008 and Bengio et al., 2007) form the basis of our temporal autoencoding approach. Both are two-layer neural networks, all-to-all connected between the layers but with no intra-layer connectivity.

To successfully select those residues in the active site, a theoretical model of RgPAL was constructed through homology modeling using RtPAL (PDB ID: 1T6J) as the template. As shown in Fig. 2, all of the residues that were

the superimposed with RtPAL showed an RMSD of 0.224 Å ( Fig. 2A), and the Ramachandran plot suggests that 94.9%, 3.2%, and 1.9% of the residues in derived model are in acceptable region, marginal mTOR inhibitor region and disallowed region, respectively ( Fig. 2B), These finding indicated that the model is reasonable and could be used in further molecular docking simulation. Using the AutoDock global–local evolutionary algorithm, we searched for those sites with the lowest free energy of binding between the ligand and the enzyme. As shown in Fig. 3, the active site cavity of RgPAL was bisected into

two regions ( Fig. 3A): one binds the amide group adjacent to the aromatic ring and the other binds the carboxyl group of the substrate. The phenyl ring of the substrate is roughly orthogonal to the plane of the MIO, and the methylidene of the MIO points to C2 of the aromatic ring ( Fig. 3A and B). In the carboxyl group binding pocket, the Arg361 residue is 3.2 Å from the carboxyl group of the substrate, and this residue might play a role in check details the binding of the carboxylate moiety of the substrate through a salt bridge. The Tyr358 residue is 2.7 Å from the β-H of substrate, which is close enough to act as the β-H abstracted base ( Fig. 3C). The Glu491 residue is the closet residue to the amino group of the substrate (2.8 Å, Fig. 3C) and might accept the amino group of substrate as the enzyme base, which is consistent with the results reported by Bartsch [1]. The Tyr358, Arg361 and Glu491 are highly conserved in PAL ( Fig. 1). In the aromatic ring binding pocket, the His136 residue points to the phenyl ring of the substrate. The imidzaole group

of His136 is parallel to the phenyl ring and might generate a π–π interaction. Moreover, the imidazole of His136 and the adjacent amide group of Gln137 which points to the phenyl ring within a distance of 4.5 Å, form a hairpin motif to clamp the phenyl ring ( Fig. 3B and C). To verify the function of the hairpin, the His136, Gln137 were deleted (RgPAL-Δ136H, RgPAL-Δ137Q) and mutated to negative (RgPAL-H136E, Chlormezanone RgPAL-Q137E) and positive charges (RgPAL-H136K, RgPAL-Q137K) as well as uncharged amino acids (RgPAL-H136F, RgPAL-Q137L), respectively. The mutant and wild type RgPAL proteins appeared a single band of about 75 kDa on SDS-PAGE ( Fig. 4). The activities of RgPAL-Δ136H and RgPAL-Δ137Q were not detected (data not shown), suggesting that the residues at the two sites were essential for catalysis. The RgPAL-H136K, RgPAL-Q137K and RgPAL-H136E lost the enzymatic activity (data not shown), and the RgPAL-H136F, RgPAL-Q137L sharply decreased the activity ( Fig. 5). Compared with those mutants, the activity of RgPAL-Q137E decreased slightly ( Fig. 5).

CC and CXC chemokines Y-27632 datasheet seem the most relevant subfamilies in cerebral ischemia, since they recruit neutrophils and monocytes, which present an important phagocytic activity [4]. A wide number of studies focused on the analysis of chemokines evidence their relevant role in cerebral ischemia and show an increased expression of these molecules within the ischemic brain regions. However, non-concluding remarks can be obtained regarding its plausible role as biomarkers in the diagnosis or prognosis of stroke (Table 1). The response to inflammation within the brain involves all the cellular components of the neurovascular unit as both, producers of and responders to inflammatory molecules.

As examples, endothelial cells express cell adhesion molecules that facilitate leukocytes infiltration in response to chemokines; glial cells can secrete chemokines after ischemic stimulus; and neurons suffer the deleterious selleck chemicals llc effects of inflammation in the injured tissue (reviewed in [5]). On the other hand, chemokines are also involved in other biological functions affecting neurovascular unit components, such as angiogenesis or neuronal survival [6]. Considering all these precedents, we aimed to study the expression of chemokines by several

components of the neurovascular unit after stroke. For that purpose, we have combined two precise techniques: a multiple ELISA array of nine chemokines from CC and CXC families and laser microdissection to obtain neurons and blood brain vessels from

patients who died following an ischemic stroke. Moreover, in order to assess the plausible use of chemokines as biomarkers or therapeutic targets in stroke field, we evaluated their temporal profile in blood samples and their association with stroke severity and outcome. Four deceased patients who had an ischemic stroke secondary to Protein tyrosine phosphatase middle cerebral artery (MCA) occlusion within the previous 4 days (range, 40–100 h) were included in this part of the study (Supplementary Table 1). Brain tissue sampling from infarcted core and healthy contralateral areas was performed within the first hours after death according to our previously published procedure [7]. All samples were snap frozen in liquid nitrogen and immediately stored at −80 °C until use. Differential diagnosis of stroke was based on clinical examination by an expert neurologist and supported by computed tomography. In all cases, stroke onset was defined as the last time the patient was known to be asymptomatic. Description of demographic and clinical factors of patients that were included in this study is shown in Supplementary Table 2. Patients from the placebo arm of the MISTICS study [8] were considered for exploring blood temporal profile. From that cohort, 20 patients with a cortical ischemic stroke admitted to the emergency department within the first 3–12 h after symptoms onset were included in the study.

, 2012). In the Selleckchem KU57788 current study, we were able to distinguish if individual infected birds were vaccinated or not, since the vaccinated group possessed higher specific responses than unvaccinated birds. Our results suggest that infection of CKC with recombinant virus containing transgenes for an epitope of interest could be used to increase the sensitivity of assays

to detect antigen and epitope specific T cells. In summary we have developed a sensitive method for the detection of antigen specific T cells, which will be important in the analysis of immune responses to both vaccines and pathogens. The assay provides greater sensitivity than the use of inactivated or live virus in ELISpot, Vorinostat cost and reduced background compared with peptide library ELISpot. Our method is also more accessible to a wider community than methods employing expensive peptide libraries, the interpretation of which data is rendered problematic due to an incomplete knowledge of avian MHC binding specificities. While we have demonstrated its efficacy for influenza, this technique can be applied to the study of T cell responses for many avian pathogens. We also demonstrated that the use of recombinant virus to infect CKC can further define antigen specificity, and additionally

reduce the requirement to handle live zoonotic pathogen, an important safety consideration. We thank Dr. Mike Skinner for providing the Fowlpox construct and Dr. Sarah Gilbert for providing the MVA constructs. We thank John R. Young for his comments and help in analyzing data. We would like to acknowledge the expert and dedicated help of the Animal and Media Services staff at the Pirbright Institute. This work was funded by the BBSRC (Biotechnology and Biological Sciences Research Council) under grant number BB/E011691/1. Ureohydrolase The

funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. “
“Monitoring antigen-specific T-cell immunity is central in clinical trials aiming to develop innovative preventative and therapeutic vaccines (Seder et al., 2008). In order to compare the immunogenicity of different vaccine candidates between multiple clinical trials, the standardization of the procedures used for blood collection, processing, preservation and blood cell analysis is of utmost importance (Maecker et al., 2005, Britten et al., 2008 and Mallone et al., 2011). Intracellular cytokine staining (ICS) is a flow cytometry-based assay increasingly used to identify, quantify and qualify antigen-specific T-cell mediated immune (CMI) responses in vaccine clinical trials (Kierstead et al., 2007, Boaz et al., 2009, Olemukan et al., 2010 and Kutscher et al., 2013).