This revealed a hublike structure in which the right posterior parietal cortex synchronized most prominently with other nodes of the

network. Thus, in contrast to the widespread stimulus-related decrease in local AG 14699 beta-band activity (compare Figure 2B), long-range beta-synchrony was enhanced in a highly structured network during stimulus presentation. If beta-band synchronization within this network was functionally relevant for processing of the sensory stimulus, intrinsic fluctuations of synchrony may predict the subjects’ alternating perception of the constant physical stimulus. Indeed, we found that beta-synchrony was not only enhanced during stimulus processing but also predicted the subjects’ percept of the stimulus. We compared coherence within the identified network for trials in which the subjects perceived the stimulus as “bouncing” or “passing.” This yielded a highly significant difference (Figure 3D, permutation-test, p < 0.0001) with enhanced beta-coherence for bounce trials. Receiver operating characteristic (ROC) analysis revealed that, even on a single-trial

level, the strength of beta-coherence significantly predicted the subjects’ percept (permutation-test, p < 0.0001). In other words, when large-scale beta-band synchronization was enhanced between frontal, parietal, and extrastriate areas, subjects were more likely to perceive the same sensory stimulus as bouncing rather than passing. Although this percept-predictive difference in synchronization overall had a network structure similar to the stimulus-related increase in synchrony,

we found the strongest perception-related effects Estrogen antagonist for synchronization with frontal regions (Figure 3E). In principle, differences in neural activity between bounce and pass trials may either reflect neural processes directly causing the subjects’ percepts or, alternatively, may reflect only secondary processes ensuing from the alternating percept. The time course of neural activity relative to the perceptual ambiguity provides critical evidence to resolve this question. We GPX6 thus exploited the temporal resolution of EEG and tested whether the difference in coherence temporally preceded the time when the stimulus became ambiguous (t = 0 s). Indeed, we found that already before the time of bar overlap (time < −0.125 s; accounting for the size of the analysis window) coherence significantly predicted the subjects’ percepts (ROC analysis, permutation-test, p = 0.0002). This provides strong evidence that, rather than merely being a consequence of the different percepts, fluctuations of large-scale beta-synchrony in fact determined the perceptual interpretation of the stimulus. Modulations of neural synchronization in the beta-network could not simply be explained by changes in signal power. We first compared power within the identified beta-synchrony network between bounce and pass trials (Figure 3F).

Trichoid sensilla ( Figure 1C) in many insects, including vinegar flies and most (if not all) moths, house OSNs tuned to pheromones ( van der Goes van Naters

and Carlson, 2007 and Kaissling et al., 1989). However, whether the trichoid structure itself is advantageous for the detection of this type of chemicals is uncertain. Likewise, OSNs housed in coeloconic sensilla ( Figure 1G) respond BVD-523 clinical trial mostly to water-soluble amines and acids (see below), but what role, if any, the actual coeloconic architecture play is unknown. The peripheral olfactory system of insects shows a remarkable morphological diversity at all levels. The role of this diversity remains unclear but probably reflects selection pressures for high sensitivity, phylogenetic and/or developmental constraints, and imposed by the physical environment, rather than adaptations to detect specific volatile chemicals. The odor molecules pass through pores or slits in the sensillum cuticle and enter the sensillum lymph (Steinbrecht, 1997). From here on, the typically hydrophobic chemicals that constitute odor ligands on land interact

with members from multiple gene families, of which only two will be discussed here. The MLN8237 supplier odor molecules initially bind to so-called odorant binding proteins (OBPs, Vogt and Riddiford, 1981). OBPs are secreted in large quantities by support cells surrounding the OSNs and show specific binding properties (Swarup et al., 2011). Although their exact function remains to be elucidated (but see Laughlin et al., 2008, for their role in pheromone communication), these proteins are probably involved in transporting the odor ligands to the receptor sites, situated in the dendritic membrane of the OSNs. The OBPs form a large insect-exclusive gene family with conserved structure, but which otherwise shows

a high degree of sequence diversity. The OBP family is possibly as old as the insects themselves, having evolved in response to demands imposed by the conquest of land (Vieira and Rozas, 2011, but see Forêt and Maleszka, 2006). So-called odorant binding proteins are also found in vertebrates; these, however, belong to the lipocalin family and show no structural similarity to the insect OBPs (Bianchet et al., 1996). The OBP family in the vinegar fly comprises 51 Calpain members (Hekmat-Scafe et al., 2002), and similar numbers have been found in other insects so far investigated. Although subfamilies can be discerned within the OBP family, examination of these genes across broader taxonomic range reveals that the OBPs largely cluster according to phylogeny, with groupings representing independent, lineage specific radiations of specific OBPs. Clear orthologs, present across different insect orders, are hence essentially lacking (Vieira and Rozas, 2011). Analyses of the OBP repertoires from the 12 complete Drosophila spp.

, 2008). In this respect, delay activity could be thought of as the response function of the network, rather than active maintenance of a static firing state. Here, we highlight short-term synaptic dynamics as an attractive putative mechanism for rapid adaptive coding Selleckchem INK1197 in PFC (see also Buzsáki, 2010; Deco et al., 2010). However, other phenomena

that systematically shift the response properties of a population could also contribute to adaptive coding. For example, temporary activity-dependent changes in membrane potentials could also shift the tuning profile of the network (Buonomano and Maass, 2009). Moreover, a systematic shift in the baseline activity state of the network could reroute processing

via conditional logic gates (McCulloch and Pitts, 1943) and/or exploiting nonlinear dynamics (Izhikevich, 2007). Finally, neural synchrony might be especially important for temporary shifts in effective connectivity (Fries, 2009). Phase synchrony has been implicated in WM (Axmacher et al., 2010; Buschman et al., 2011; Fell and Axmacher, 2011), and a recent study has further shown how rapid configuration of synchronized networks in PFC is specific to different rules states (Buschman et al., 2012). These mechanisms might also PD173074 ic50 be able to implement the functional change we describe here—a context-dependent shift in network dynamics, altering the mapping of sensory inputs to final behavioral decisions. Subjects were two male rhesus monkeys (Macaca mulatta), weighing 11 and 12 kg. All experimental procedures were approved by the UK Home Office and were in compliance with the guidelines of the European Community for the care and use of laboratory animals (EUVD, European Union directive 86/609/EEC). The cued target detection task is schematized in Figure 1B. Each trial commenced with a 500 ms baseline

period, during which the monkey held fixation on a red central fixation point accompanied by two dim gray circles (location markers) 6° to left and right on the horizontal meridian. Next, Sclareol one of three cue stimuli was presented for 500 ms at either the left or right (randomized) location marker. The cue determined the spatial location of all subsequent stimuli within that trial and also the direction of the eventual saccade response at the end of the trial. Most importantly, the cue identity also instructed which choice stimulus would be the target stimulus for the current trial. During initial training sessions, monkeys learned to associate three specific cue stimuli with three specific target stimuli. An additional stimulus served as a neutral nontarget item. All pictures were randomly drawn from the same set of images (2° × 2°). New stimulus pairs and neutral pictures were occasionally introduced and maintained for a number of sessions.

, 1992; Evans et al., 1992; Silinsky et al., 1992). check details By this time, ATP receptors had also been subdivided as belonging to two major families: metabotropic P2Y receptors and ionotropic P2X receptors. With the subsequent cloning of the genes encoding P2X receptors came a new era. In this review, we focus on P2X receptor mediated ATP signaling in the brain, discussing general themes pertinent to mechanisms and neuromodulation at the molecular, cellular,

systems and disease levels. ATP activates a family of metabotropic P2Y and ionotropic P2X receptors. Collectively, the actions of ATP and its breakdown products produce responses that last from milliseconds to minutes, and even longer time scales through changes in gene regulation via second messengers. Signaling diversity is further increased by the fact that ATP receptors display a very broad range of ATP sensitivities, ranging from nanomolar in the case of P2Y receptors, to hundreds of micromolar for P2X7 receptors (Surprenant et al.,

1996). Thus, ATP receptors respond over remarkably broad spatiotemporal scales, and ATP signaling is very dynamic. The first P2X receptor genes were identified in 1994 (Brake et al., 1994; Valera et al., 1994) and within 2 years the whole family had been identified (Buell et al., 1996; Chen et al., 1995; Collo et al., 1996; Lê et al., 1997; Lewis et al., 1995; Soto et al., 1996; Surprenant et al., 1996). This was an exciting period that culminated with the realization that P2X receptors defined a unique protein family. Each of the homomeric P2X receptors also displays distinct functional FK228 mw properties (Figure 1; Table 1). The available data on the subunit composition and properties of heteromeric P2X receptors (Coddou et al., 2011) are not considered in depth here. P2X receptors are nonselective

cation channels with high Ca2+ permeability that carry a depolarizing current under standard physiological conditions. In some cells, P2X channels are also significantly permeable to anions. For example, the full length P2X5 receptor is permeable to Cl− (North, 2002). This remains the exception rather than the rule. Thus at its most fundamental level, P2X receptor mediated below neuromodulation starts with chemistry: ATP rapidly gates P2X receptor pores, triggering transmembrane fluxes of selected ions. The seven mammalian P2X subunits range from 379 (P2X6) to 595 (P2X7) amino acids in length. Each subunit contains two hydrophobic membrane-spanning segments (TM1 and TM2) separated by an ectodomain which contains ten conserved cysteine residues that form disulfide bonds. Representing the simplest known architecture for ligand-gated ion channels, P2X receptors adopt a relatively simple fold, with intracellular N and C termini and most of the molecule forming an extracellular loop.

nivale DNA correlated both with increasing malt friability and laboratory wort colour, S3I-201 in vivo since the release of amino acids

and reducing sugars from the breakdown of protein and starch respectively, increases with the extent of modification and friability of malt. There have been several reports on the changes in the diversity of composition of the FHB complex in cereals within different geographical and climatic locations. In the past ten years in Europe, F. culmorum has been replaced by F. graminearum ( Waalwijk et al., 2003, Jennings et al., 2004, Stepien et al., 2008, Xu et al., 2008, Isebaert et al., 2009 and Nielsen et al., 2011) and furthermore F. poae has been shown to replace F. graminearum in Southern Europe ( Pancaldi et al., 2010 and Shah et al., 2005). In contrast, in Central Europe and in North America and China, DON is the main trichothecene associated

predominantly with F. graminearum and species of the F. graminearum clade ( O’Donnell et al., 2004). In these studies we describe the impact of newly emerging species of importance, M. nivale and F. langsethiae, on the malting and brewing quality of naturally infected barley. The results clearly indicate that the pathogen populations of the FHB complex in barley in the UK are diverse and dominated by non-toxigenic Microdochium species and toxigenic Fusarium species such as F. poae, F. avenaceum and F. langsethiae. Future research efforts should focus on elucidating the impact of these newly emerging species and their mycotoxins, for example, enniatins produced by F. avenaceum and F. tricinctum on barley and Selleckchem ABT-263 HT-2 and T-2 produced by F. langsethiae. The authors wish to thank Velcourt, Syngenta Seeds, Syngenta Crop Protection, Openfield, SABMiller plc, BBSRC and the Technology Strategy Board for their financial support of the most SAFEMalt project grant number 100882. Samples from 2007 to 2009 were collected and mycotoxin analysis was completed as part of HGCA-AHDB project RD-2007-3401. “
” The death of Professor Dr. Tibor Deák on March 3rd, 2013 in Budapest, Hungary has saddened colleagues, friends, and the international scientific community.

Those who knew Tibor had great respect for him, enjoyed his company, and felt privileged to be influenced by his remarkable knowledge of microbiology in general and food microbiology in particular. He is survived by his wife, Mary, and daughter, Susanne for whom he was a very caring husband and father. Tibor Deák, Ph.D., D. Sc., Professor Emeritus left an enormously rich and substantial life work for future generations. Over 350 peer-reviewed publications, books, and chapters are only the printed proof of his legacy. After receiving a teaching degree in biology–chemistry in 1957 at the University of Szeged, he chose to work in the industry and became a specialist in fermentation microbiology. He earned his Ph.D. on The Microbiology of Lactic Acid Fermentation at Eötvös Loránd University in Budapest in 1963.

g., male/female, cycling/swimming). Reliability coefficients were much more uniform in the type calculated/cited. Most often researchers who calculated a reliability coefficient did so with internal consistency (n = 24) whereas two studies also reported test-retest reliability. Researchers choosing to cite the reliability used in previous studies did so more often with internal consistency (n = 46) while nine studies also referenced test-retest reliability

of the eating disorder measure used. Three major findings were unveiled as a result of this learn more review. Although not surprising, the first finding of this review revealed that the number of studies (14% of the 50 studies reviewed) completed on exclusively male

athletes was much lower in comparison to those conducted on exclusively female athletes. Secondly, this review found eight different measures were used in the assessment of ED in athletes. Specifically, the use of the EAT, EDI, QEDD, BULIT-R, and EDE-Q questionnaires, developed for non-athlete PD173074 populations, was much more prevalent than the use of psychometric measures assessing the same ED constructed specifically for athletes—namely the WPSS-MA, AQ, and AMDQ. Finally, this review found a majority of the literature available examining ED in athletes to cite the validity and reliability of ED questionnaires reported in previous studies but fewer calculated their own validity and reliability coefficients with the athletic population they studied. This review found research on ED in exclusively male athlete populations is less prevalent than research examining these same behaviors in female athlete samples. Hudson et al.43 found Mannose-binding protein-associated serine protease rates of anorexia and bulimia to be significantly higher in non-athlete males than previously thought. The same is true within male athlete populations as the prevalence of eating disorder behaviors has also been increasing in this population.4, 6, 39,

43, 44, 45, 46 and 47 This increase in the prevalence of eating disorder behaviors indicates the need for sports psychologists to validate eating disorder assessments in this population to gain further knowledge of predisposing factors that might be unique to the development of ED in male athletes. Despite the limited amount of research on exclusively male athlete samples and ED, the QEDD has been found to be a valid psychometric measure for the assessment of these abnormal behaviors in this population.4 and 6 One barrier to studying ED in male athletes might be that ED have largely been considered a “woman’s problem” and, therefore, the development of psychometric measures for ED has been tailored toward the “thinness” ideal some women engage in eating disorder behaviors to achieve.48 Male athletes are more often concerned with increasing muscularity than with losing body fat, as doing so projects the epitome of masculinity/male athletes in contemporary culture.

001 by Fisher’s PLSD post hoc analysis; Figure 6D) and rTgWT neurons (∗p < 0.05 and ∗∗p < 0.01 by Fisher's PLSD post hoc analysis; Figure 6D). As reported previously, (Liao et al., 1999), synaptic GluR1 receptor expression detected on fixed neurons (Figure 6A) strongly corresponded to

surface N-GluR1 receptor expression detected on living neurons (Figure 5D). Labeling of total NR1 receptors with a rabbit polyclonal antibody against the N terminus of NR1 (Liao et al., 1999) in fixed neurons cultured from these three lines of mice revealed a similar pattern of expression (Figure 6C). In TgNeg and rTgWT neurons, NR1 receptors are clustered in spines as identified by the strong overlap between PSD95 and NR1 expression (small arrows in upper panels of Figure 6C). In contrast, the large arrows in the lower panels of Figure 6C indicate the reduced colocalization of ABT 199 PSD95 and NR1 in rTgP301L neurons. Normalization of www.selleckchem.com/products/ve-822.html NR1 fluorescence intensity in spines to that in the dendritic shafts demonstrated that the synaptic expression of NR1 receptors are decreased in rTgP301L mice compared to TgNeg and rTgWT neurons (∗∗∗p < 0.001 by Fisher's PLSD post hoc analysis; Figure 6D). Our results indicate that tau mislocalization to dendritic spines damages

these spines profoundly by disrupting the synaptic targeting or anchoring of both AMPA and NMDA receptors. We postulated that the mislocalization of tau in spines depends upon abnormal htau hyperphosphorylation, which occurs to a greater extent in rTgP301L than rTgWT mice (Figure 7). Tau consists

Adenosine of at least three structurally distinct regions, including an amino-terminal projection domain and a carboxyl-terminal domain that contains repetitive microtubule-binding motifs flanked by proline-rich regions (Buée et al., 2000 and Avila et al., 2004). Hyperphosphorylation of htau at the 14 serine (S) and threonine (T) residues that can be phosphorylated by proline (P)-directed S/T kinases has been shown to modulate tau neurotoxicity, control tau binding to F-actin and regulate the effect of htau on the viability of retinal neurons in the fruit fly (Fulga et al., 2007, Steinhilb et al., 2007a and Steinhilb et al., 2007b). The neurons of fruit flies, like other lower organisms, lack dendritic spines (Hering and Sheng, 2001). Therefore, the relevance of this finding to mammalian neurons, where F-actin concentrates in dendritic spines, has not been established. To validate that P301L htau is hyperphosphorylated in vitro on SP/TP residues previously implicated in tau neurotoxicity, we examined htau phosphorylation levels in our rat neuron cultures expressing WT or P301L htau. Changes in tau phosphorylation underlying tau pathology have a temporally specific sequence (Maurage et al., 2003, Luna-Muñoz et al., 2007 and Bertrand et al.

(2011) analyzed Vegfa120/120 mice, which cannot produce the Npn-1-binding isoforms VEGF164 or VEGF188, but do express VEGF120, which does not bind Npn-1 and supports blood vessel formation. Similar to Npn-1 null mice, Vegfa120/120 mice display increased ipsilateral projections and decreased contralateral projections, supporting the idea that VEGF/Npn-1 interactions promote RGC axon Selleck RO4929097 crossing at the optic chiasm. Vegfa120/120 mice survive to birth, so retrograde DiI labeling was employed to independently assess ipsilaterally projecting RGC axons and determine the origin of misrouted axons within the retina. In wild-type mice, ipsilateral RGCs are primarily restricted to the ventrotemporal region

of the retina ( Figure 1B). In Vegfa120/120 mice, however, retrogradely labeled INK1197 datasheet RGCs were found throughout the temporal and nasal retina. To directly test whether VEGF functions as a chemoattractant, RGC growth cones were exposed to a VEGF164 gradient. Consistent with a previous study

showing that VEGF promotes regenerative growth of axotomized RGCs in culture ( Böcker-Meffert et al., 2002), VEGF164 was found to act as a selective attractant for dorsotemporal RGC growth cones, neurons that give rise to contralateral projections, but not for ventrotemporal RGC growth cones, neurons that give rise to ipsilateral projections. Collectively, these studies show that VEGF164 functions as a chemoattractant to promote midline crossing of Npn-1-expressing RGC axons at the optic chiasm in vivo. VEGF also functions as an attractant for spinal commissural axons, as reported in the study by Ruiz de Almodovar et al. (2011). VEGF is expressed at the floor plate at the time when spinal commissural axons cross the midline (Figure 1A). Mice lacking function of a science single VEGF allele specifically in the floor plate (Vegf FP+/−) secrete less VEGF and exhibit concomitant abnormal pathfinding of precrossing

commissural axons. While most Robo3-positive commissural axons reach the floor plate in Vegf FP+/− mice, labeled commissural axons in embryonic spinal cord sections are observed to be defasciculated, and they often project to the lateral edge of the ventral spinal cord. Important control experiments show that the defects observed are not secondary to altered expression of Netrin-1 or Shh in the floor plate of Vegf FP+/− mice. In vitro, an attractive response by commissural axons to a gradient of VEGF-A was observed in the Dunn chamber assay. Interestingly, VEGF-A attraction was completely abolished in the presence of a function blocking anti-Flk1 (KDR/VEGFR2) antibody or by pharmacological inhibition of Src family kinases. Anti-Npn1 in this same assay had no effect on VEGF-A attraction. Immunolabeling of precrossing commissural axons revealed coexpression of Flk1 and Robo3, and in vivo, conditional ablation of Flk1 in commissural neurons (Flk1CN-ko) phenocopies defects observed in the Vegf FP+/− mice.

Another possibility is

that superlinear release does not represent synaptic vesicle fusion, but rather, endosomal fusion or fusion of vesicles at some distant site (Coggins et al., 2007 and Zenisek et al., 2000). Direct tests of this possibility are lacking; however, the ability of afferent fibers to operate spontaneously at rates of more than 100 spikes/s and to sustain release in the face of stimulation at rates more than 400 spikes/s argue for the requirement of rapid vesicle replenishment (Liberman and Brown, 1986 and Taberner and Liberman, 2005). The maximal release rate reported here for mammalian inner hair cells when the superlinear component SCH772984 order is included is about 307 vesicles/s/synapse (assuming 15 synapses and 50 aF/vesicle)(Meyer et al., 2009), probably underestimating the release required to sustain these large firing rates. Prepulse experiments further illustrate that under more physiological

stimulation conditions, Ibrutinib in vitro release is linear and sustained; neither of these properties would occur without the superlinear component summing with the first release component. Finally, previous experiments have imaged vesicle release in mammalian hair cells at rates higher than reported here for superlinear component of release and also suggested trafficking must be rapid (Griesinger et al., 2005). Data suggest that low-frequency cells release at faster rates per synapse than high-frequency cells, though the release rate per cell was similar for both components. In turtle, largely one fiber innervates one hair cell, but with multiple synapses it may be the overall release Amisulpride rate that is more significant than release per synapse, in contrast to the mammalian system in which one fiber innervates one synapse. The underlying mechanisms responsible for differences in release per synapse remain to be determined. In contrast, work in mammalian systems (Johnson et al., 2008) has shown a difference in the Ca2+ dependence of release. In turtle there was an apparent difference in Ca2+ dependence associated

with the ability of low-frequency cells to recruit superlinear release with less Ca2+ than high-frequency cells. Comparable experiments are needed to test this in mammalian hair cells. Our data are consistent with the existence of multiple vesicle pools, with the first linear saturable release component including both the RRP and recycling vesicle pools and the superlinear release component corresponding to the reserve pool (Figure 8A). Based on release measurements, we estimated vesicle pool sizes of 600 vesicles in the RRP (0.6%), 8000 vesicles in the recycling pool (7.4%), and 100,000 vesicles in the reserve pool (92%) (Figure 8A). These sizes are consistent with data from other synapse types (Rizzoli and Betz, 2005), the major difference being the ability of vesicles to be recruited for release from each pool.

5 ml extract solution and observed for white precipitation which indicates presence of tannin. 0.2 g of the extract was Modulators shaken with 5 ml of distilled water and then heated to boil. Frothing shows the presence of saponin. 0.2 g of the extract was dissolved in 10% NaOH solution, yellow colouration indicates the presence of flavonoid. To 2 ml of extract solution, added 2 ml of alcohol and few drops of ferric chloride solution and observed for colouration. Five ml of each extract was treated with 2 ml of glacial acetic acid containing one drop of ferric chloride solution. This was under layered with 1 ml of conc. sulphuric selleck screening library acid. A brown ring at the interface indicated

the present of cardiac glycoside. (A violet ring may appear below the ring while in the acetic acid layer, a greenish ring may formed). 0.5 g extract was boiled with conc. HCl and filtered. 0.5 ml of picric

acid and Mayer’s reagent was added separately to about 1 ml of the filtrate in a different test tube and observed for coloured precipitate or turbidity. To 0.2 g of extract, added 5 ml of chloroform and 5 ml of 105 ammonia solution. The presence of bright pink colour in the aqueous layer indicated the presence of anthraquinone. Five ml of extract solution was mixed in 2 ml of chloroform, and 3 ml of conc. sulphuric acid was added to form a layer. A reddish brown colouration of the interface buy BVD-523 was formed to indicate the presence of terpenoids. Red colour at the lower surface indicates presence of steroid. To 0.5 ml of extract solution, 1 ml of water and heated after adding 5–8 drops of Fehling’s solution. Brick red precipitation indicated the presence of reducing sugar. Antioxidants react with 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) radical and convert it to 1, 1-diphenyl-2-picryl hydrazine. The degree of change in colour from purple to yellow can be used as a measure of the scavenging potential of antioxidant extracts. Aliquots of ethanol extract solutions (1 mg/ml) were taken and made up the volume to 3 ml with methanol. 0.15 ml of freshly prepared DPPH 3-mercaptopyruvate sulfurtransferase solution

was added, stirred and left to stand at room temperature for 30 min in dark. The control contains only DPPH solution in methanol instead of sample while methanol served as the blank (negative control). Absorbance was noted at 517 nm using the Systronics make spectrophotometer (Visiscan 167). The capacity of scavenging free radicals was calculated as scavenging activity (%) = [(Abscontrol−Abssample/Abscontrol)] × 100 where Abscontrol is the absorbance of DPPH radical + methanol; Abssample is the absorbance of DPPH radical + sample extract/standard. The ABTS assay was carried out following the method of Re et al.9 The stock solution included 7 mM ABTS solution and 2.4 mM potassium persulfate solution and mixed them in equal proportion then allowed to react for 12 h at room temperature in the dark and diluted by mixing 1 ml ABTS solution with 60 ml methanol to obtain an absorbance of 0.706 ± 0.