Of primary interest were major seasonal differences in the effect of location and distance. Two seasons were considered,

nominally referred to here as winter and summer reflecting water temperature (less than 10 °C and more than 10 °C respectively). Season was, therefore, also considered fixed. For each sampling time (Month) two individual reef modules from each group of six (Group) were randomly selected. Avasimibe nmr At each reef-distance 10 redox measurements were taken, the locations of which were randomly allocated by the diver swimming for a pre-selected random time of between 1 and 15 s around the reef perimeter (0 m stations) or guided to 1 and 4 m stations using a marked rope. The objective was to take 180 measurements per time interval (3 groups, 2 modules from each group, 30 readings per module). However, during periods of poor weather this sampling programme was not completed and the following numbers of modules per group (A, B and D) were measured on the following dates: March 2005 A: zero, B: one and D: two; September 2005 A: zero, B: one and D: one and October 2005 A: two, B: one and find more D: one. At all other occasions the full sampling programme was achieved.

Two dives were permissible per day resulting in a minimum of three consecutive days to visit the six modules (two modules on each of three groups). During poor weather the period over which a single time-period’s data were collected was extended up to seven days. These data were considered to represent one time period. Visual assessments of the reefs and the surrounding environment were made, particularly in reference to any accumulations of organic material and the nature of the sediment. The bottom-water temperature was recorded using an integral old depth gauge and thermometer during each dive. The mean temperature for each month is reported. Pre-analysis data exploration (checking outliers, homogeneity, normality) followed the protocol of Zuur et al. (2010). Model development and selection in mixed models can be relatively complex (and iterative) and the guidance given in Zuur et al. (2009), detailed

below, was followed: 1. The beyond optimal (all fixed effects and interactions) model was initially fitted using generalised least-squares regression and the residuals examined for homoscedasticity. If any residual trends were identified a range of variance structures were tested and compared on the basis of their Akaike information criteria (AIC) score (where the lowest AIC was considered the optimal model). The goal was to identify, and allow for, differences in variance as a function of either one or more categorical predictors. Residuals from the model with the lowest AIC were reassessed to check that any heteroscedasticity had been incorporated into the model. All model predictions, and 95% confidence intervals (shown graphically) relate only to the fixed factors.

Wśród tych zakażeń 75,7% (n = 610) wystąpiło u chorych poniżej 20. r.ż. (421 izolatów, 189 materiałów PCR+), hospitalizowanych w szpitalach reprezentujących wszystkie polskie województwa. Dalsza, szczegółowa analiza dotyczy chorych poniżej 20. r.ż. Wśród analizowanych 610 zakażeń meningokokowych materiał do badań w 314 przypadkach (51,5%) stanowiła krew, w 282 (46,2%) płyn mózgowo-rdzeniowy, w 7 tkanki pobrane post mortem i w 7 wymazy z nosogardła lub gardła od pacjentów AZD2281 z objawami typowymi dla IChM. Pomimo że najwyższy odsetek izolatów wyhodowano z krwi, u największej liczby pacjentów rozpoznano ZOMR lub ZOMR z bakteriemią/posocznicą

(w sumie 369 przypadków, 60,5%). Rozpoznanie posocznicy i bakteriemii, bez umiejscowienia zakażenia, stwierdzono odpowiednio u 203 (33,3%) i 19 (3,1%) pacjentów. W 19 (3,1%) przypadkach nie podano rozpoznania. Wśród pacjentów przeważała płeć męska (55,4%). Zapadalność na IChM w grupach wiekowych w analizowanych latach przedstawiono w tabeli I. Wysoką zapadalność odnotowano u dzieci poniżej 5. r.ż. (średnio dla trzech

lat 6,98/100 000), w tym zwłaszcza u dzieci poniżej Etoposide molecular weight 1. r.ż. (13,99/100 000). Wyższą zapadalność niż średnia obserwowano również u osób w wieku 15–19 lat (1,34/100 000). Grupę serologiczną meningokoków odpowiedzialnych za zakażenia w badanym okresie ustalono w 553 przypadkach (90,7%). Większość zakażeń wywołały meningokoki należące do serogrupy B (MenB, n = 330; 59,7%), następnie serogrupy C (MenC, n = 209, 37,8%), Y (n = 9, 1,6%) i W-135 (n = 5, 0,9%). U dzieci poniżej 2. r.ż. 73,1%, a u dzieci poniżej 1. r.ż. 76,4% przypadków

Casein kinase 1 było wywołanych przez meningokoki serogrupy B, podczas gdy u dzieci powyżej 10. r.ż. nieznacznie częściej występowały zakażenia szczepami MenC (53,3%). Liczbę zakażeń i zapadalność na inwazyjną chorobę meningokokową wywołaną przez określone grupy serologiczne w grupach wiekowych w Polsce przedstawiono w tabeli II. W tabeli III umieszczono wartości współczynników zapadalności na IChM wraz z procentowym udziałem w zakażeniach grup serologicznych w poszczególnych województwach. Zaobserwowano znaczne różnice w wartościach współczynników zapadalności pomiędzy województwami. Ogólny współczynnik śmiertelności (case fatality ratio; CFR) wyniósł 13,3%, gdyż wśród 406 przypadków IChM, dla których uzyskano informację na temat zejścia zakażenia, 54 zakończyły się zgonem pacjentów <20. r.ż. Najwyższy współczynnik CFR odnotowano u dzieci w wieku 24.–35. m.ż. (20,9%), a nieco niższy u niemowląt (16,7%) ( Tab. IV Ryc. 1). Biorąc pod uwagę rozpoznanie, najwyższy CFR odnotowano u pacjentów z sepsą (23,3%) w porównaniu z pacjentami z ZOMR/sepsą (5,9%) i ZOMR (1,2%). Współczynniki śmiertelności dla zakażeń wywoływanych przez serogrupę B i C w badanej grupie wiekowej były podobne i wynosiły odpowiednio 15,2% i 12,9%.

To normalize mineral homeostasis in VDR∆/∆ and Kl−/−/VDR∆/∆ mice [11], all genotypes were fed a rescue diet (Ssniff, Soest, Germany) which contained 2.0% calcium, 1.25% phosphorus, 20% lactose and 600 IU vitamin D/kg, starting from 16 days of age. Kidney cryosections were prepared from snap-frozen tissues. Proximal and distal tubules were harvested using a Veritas (Arcturus) LCM system. RNA was extracted using the PicoPure RNA isolation kit (Qiagen). RNA purity and integrity were determined with a bioanalyzer (Agilent). Reverse transcription was performed using iScript™ cDNA Synthesis Kit (Bio-Rad). Real-time

PCR was performed in duplicate on a Rotor-Gene™ 6000 cycler (Corbett Life Science) using QuantiFast™ SYBR® Green PCR Kit (Qiagen). RT-minus samples served as a control to exclude amplification from genomic DNA, learn more and amplicon melting analysis was performed to exclude primer dimerization and unspecific amplification. β2‐Microglobulin was used as house-keeping gene for normalization of the mRNA expression data. N-fold change in gene expression

was calculated using the Pfaffl model [12]. Primary mouse proximal tubular epithelial cells were isolated from C57BL/6 mice according to previously established protocols by collagenase digestion and density gradient centrifugation, selleckchem and cultured in serum-free, hormonally defined culture medium [13] and [14]. Purity of proximal tubular cells was examined by qRT-PCR analysis of calbindin D28k, transient receptor potential vanilloid-5 (TRPV5),

and NaPi2a mRNA. Renal proximal tubules were isolated as reported previously [15], [16] and [17]. In brief, murine kidneys were perfused with sterile culture medium (Ham’s F12; GIBCO) containing 1 mg/ml collagenase (type II; Sigma) and 1 mg/ml pronase E (type XXV, Sigma) at pH 7.4 and 37 °C. The cortical tissue was dissected in small pieces and placed at 37 °C in sterile Ham’s F12 medium containing 0.5 mg/ml collagenase II and 0.5 mg/ml pronase E for 15 min with vigorous shaking. After centrifugation at 3000 rpm for 4 min, the enzyme-containing solution was removed, and Exoribonuclease tubules were resuspended in ice-cold medium containing 1% antibiotic/antimycotic and placed on ice. Individual proximal tubule segments were identified based on morphology in a dissection microscope at × 25–40 magnification by their appearance and dimensions. In vitro experiments with cultured proximal tubular cells and dissected tubular segments were performed in serum-free, hormonally defined culture medium at 37 °C in 5% CO2 [13] and [14]. Proximal tubular cells were incubated with 1–100 ng/ml of recombinant human FGF23 R176/179Q (rFGF23) [18] for 0.5, 1, 2, and 4 h.

Most such cases of bilateral basal ganglia infarction reported previously have no known established cause. The patient denied using 3,4-methylenedioxymethamphetamine (MDMA or “Ecstasy”), a substance which has very rarely been reported to be associated with basal ganglia infarction (Hanyu et al., 1995). Healthy volunteers, [19 male, non-colour blind, mean age = 41 (SD 5.7); 12 right-handed] were recruited Selleck PD 332991 by

website advertisement and from the UCL Psychology Department’s subject pool, with local ethics committee approval. They completed both experimental tasks during a 1 h testing session. On the Barratt Impulsiveness Scale [BIS-11 (Patton et al., 1995)] their mean total score find more was 65.3 (SD 11.6). Written consent was obtained from all test subjects, according to the Declaration of Helsinki. The research studies reported here with KD started 9 months after his initial strokes. T1-weighted MR acquisitions of KD’s brain were obtained at 1 × 1 × 1 mm resolution (Fig. 2A and B) on a 1.5 T Sonata Scanner (Siemens). Diffusion-weighted imaging (DWI) was performed with an echo

planar sequence comprising a double spin-echo module to reduce the effect of eddy currents (Reese et al., 2003). Each data volume consisted of 40 axial slices of 2.3 mm thickness with no interslice gaps and an acquisition matrix of 96 × 96 in a field of view (FoV) of 220 × 220 mm, resulting in 2.3 mm3 isotropic voxels [echo time (TE), 90 msec; flip angle, 90°; fat saturation; bandwidth, 2003 Hz/pixel]. Each dataset consisted of 61 high-diffusion-weighted images (b = 1000 sec/mm2), with diffusion gradients applied Dolutegravir in vitro along 61 evenly distributed diffusion directions obtained from a previously reported optimization procedure ( Jansons and Alexander, 2003) and seven additional images with minimal diffusion weighting (b = 100 sec/mm2) and

evenly distributed directions. The diffusion tensor was fitted using a standard linear least squares fit to the log measurements ( Basser et al., 1994). Additionally, the fitting provides an effective b = 0 image. We also acquired high-resolution T1-weighted structural data using the modified driven equilibrium Fourier transform sequence [176 slices; 1 mm3 isotropic voxels; sagittal, phase encoding in anterior/posterior; FoV, 224 × 256 mm; matrix, 224 × 256; repetition time, 20.66 msec; TE, 8.42 msec; inversion time, 640 msec; flip angle, 25°; fat saturation; bandwidth, 178 Hz/pixel] ( Deichmann, 2006). Several recent human atlases were used to establish the extent of KD’s lesions. Note that atrophy secondary to neuronal degeneration means that there is distortion of normal anatomy, in addition to the lesions themselves. It is therefore important to be familiar with such changes when interpreting these images. KD’s lesions largely involved the GPi, more prominently on the left.

These analyses were done considering three key variables, i.e. gear, habitat see more (where fishing took place) and time (northeast monsoon, dry, southeast monsoon). Two 3-way ANOVAs with the above variables and their respective interactions were performed; one for biomass and one for income (Appendix III, Supplementary Information). When significant differences occurred (p < 0.05) the Bonferroni correction (BC) was applied to determine the final significant differences between habitats. For each ANOVA pairwise tests were performed summing up to 72 pairwise tests totally ( Appendix III, Supplementary Information). The significance level for the pairwise tests was determined by the critical p-value based

on the BC, i.e. 0.05/36 = 0.00139. To better fulfill the ANOVA assumptions on normality and variance homogeneity the analysis was performed on log-transformed values. All the statistical analyses were performed with the statistical program

Stata version 12. Fish species composition was calculated using the relative abundance of the species found in each “batch” brought to the market belonging to the selected three habitats, i.e. mangroves, seagrasses and corals. GW-572016 in vitro Data was then aggregated by time (season) and pooled for all habitats to determine the most common species found in the bay. This analysis, although lacking details, provides a clear indication of what type of fish dominates the catches in Chwaka Bay (Table 2). The study limitations are acknowledged in the sense that only the biggest market in the bay was sampled and that there is no replication over time. However, the choice was based on the fact that the Chwaka market is the largest and most important within the bay but also in Zanzibar where seagrass associated fish is very common in catches for the whole Island (DFMR, 2007). Spatial replication is considered acceptable since we are using a case study approach and each area dominated by the particular habitat within the bay was composed of numerous fishing grounds. All these grounds were mapped those and all fish harvested in those

areas was sampled (see above). The restrictions in sampling were due to logistical reasons since sampling in these rural developing areas is highly resource demanding. However, the results are considered reliable and valid enough to illustrate the arguments and to promote better management. The data analysis showed that fishing takes place in the three investigated habitats (mangroves, seagrasses and corals) in Chwaka Bay (Table 1, Fig. 2). However, compared to mangroves and coral dominated fishing grounds, seagrass dominated grounds were the most visited places for fish harvesting (Fig. 2). The dominating gears in the area were basket traps, drag-nets and spears. The fishing pressure (No. fishers km−2 day−1) varied a lot between the three habitats, but with seagrasses showing the highest (Table 1, Fig. 2).

Beside the therapeutic effects reviewed above, there are some other kinds of chronic pain that can be treated. In 2010, Santamato et al. reported the treatment of the neck pain that was related to nocturnal bruxism with BoNT/A. In this study, each masseter muscle was injected with a dose of about 40 units and the temporal muscle was bilaterally injected with 25 units. After three days of treatment with BoNT/A, a decrease in bruxism symptoms was noted (Santamato et al., 2010). Furthermore, Jason Abbott also used BoNT/A in women with chronic pelvic pain in 2009. They indicated

that BoNT/A (20–40 units) used in the vulva may have a continued benefit for 3–6 months after injection with limited Nutlin-3a manufacturer side effects (Abbott, 2009). The LC in the type E BoNT gives rise to a more extensively truncated SNAP-25 product that is unable to form functional complexes with its SNARE partners. Therefore, it offers a more fast acting effect compared to that of BoNT/A. Besides, it can also pseudo-irreversibly abolish release of neurotransmitters. Generally speaking, BoNT/E blocks the neurotransmission more quickly and more potently compared to BoNT/A. However, the clinical application of BoNT/A is restricted by its neuromuscular paralytic

action being transient (less than 4 weeks) in contrast to BoNT/A (more than 4 months). In the past few years, Meng J reported the construction Alectinib of a chimera of BoNT/A and/E by introducing a nucleotide sequence encoding the acceptor binding Hc domain of type A into the BoNT/E gene (Fig. 3). The recombinant EA chimeric protein can then be expressed in Escherichia coli and be purified. They found that it cleaved SNAP-25 in the trigeminal neurons and blocked CGRP release triggered by all stimuli tested, including capsaicin ( Wang et al., 2011). After that, some people proved that it was possible to show this dramatic increase in persistence of neuroparalysis ( Dolly and O’Connell, 2012). In these days, a faster and more efficient BoNT-based neurotherapeutics

becomes a possibility considering Plasmin the advances in protein engineering. BoNT/A has been under clinical trials for treatment of migraine and other chronic pain for many years. Therefore, the translation of the encouraging results from preclinical studies in animal pain models to clinical treatments of more various types of chronic pain in human sufferers can be a significant step. However, more in depth studies are necessary to reach to a point where it can be clinically applicable. None of the previous studies have established the exact mechanism responsible for analgesic effects of BoNT/A; which could provide the essential foundation of developing future therapeutic strategies. Besides, there is a lack of precise applicable doses and injecting sites to refer to. Therefore, more studies are required to determine the best and accurate method of using BoNT/A is the goal of many ongoing efforts.

It was possible to compensate for up to 85% of the series resistance without introducing oscillations GSK-3 inhibitor into the recorded currents. Data were displayed on a digital oscilloscope (310, Nicolet Instrument) and stored on the hard disk of the computer (sampling frequency 33.3 kHz) for subsequent off-line analysis. Spontaneous action potentials were displayed on a digital oscilloscope (310, Nicolet Instruments, Madison, WI) and stored on a DAT (DTR-1024, Biologic Science Instruments). For current-clamp experiments, depolarizing current pulses were elicited at 0.5 Hz with a programmable stimulator (SMP 310, Biologic). Evoked action potentials were displayed

and stored on the hard disk of the computer using pClamp as described above. For current-clamp experiments,

the bathing solution contained (mM): NaCl, 200; KCl, 3.1; MgCl2, 4; CaCl2, 5; HEPES buffer, 10; pH was adjusted to 7.4 with NaOH. The recording electrode was filled with the following solution (mM): K-aspartate, 160; KF, 10; NaCl, 10; MgCl2, 1; ATP-Mg, 1; CaCl2, 0.5; EGTA, 10; HEPES buffer, 10; pH was adjusted to 7.4 with KOH. For voltage-clamp experiments, the superfusing solution used to record inward sodium currents contained (mM): NaCl, 80; Tetra-ethylammonium-chloride (TEA-Cl), 120; KCl, 3.1; CaCl2, 2; MgCl2, 7; CdCl2, 1; 4-aminopyridine (4-AP), 5; HEPES buffer, 10; pH was adjusted to 7.4 with TEA-OH. Patch-clamp electrodes were filled with an internal ID-8 solution containing (mM): CsCl, 90; CsF, 70; NaCl, 15; MgCl2, 1; ATP-Mg, 3; EGTA, 5; HEPES buffer, 10; pH was adjusted to 7.4 with CsOH. CX-5461 chemical structure Two different strategies were employed for the purification of μ-TRTX-An1a, i.e., two-dimensional ( Fig. 1) and one-dimensional ( Fig. 2) chromatography, both leading to the purification of μ-TRTX-An1a, as determined by MALDI-TOF analysis (data not shown). The first strategy brought the fraction of interest with eluent B concentrations between 28.8–32.8% and 31.3–32.8% through CIEX and RPC, respectively. The second strategy allowed the elution of the toxin at concentrations between

30 and 31% for the two stages of RPC. Samples of μ-TRTX-An1a purified by means of two-dimensional chromatography were used in the electrophysiological assays, while the samples purified by means of one-dimensional chromatography were used for primary structure determination. μ-TRTX-An1aalq was submitted to N-terminal sequencing by Edman degradation. This yielded the elucidation of the 37 N-terminal residues (Table 1). The remaining residues were elucidated by means of LC-MS-MS (not shown). On MS mode, μ-TRTX-An1aalq was visualized by means of its ions [M + 7H]+7, [M + 8H]+8, [M + 9H]+9 and [M + 10H]+10. The monoisotopic mass was determined as 5718.87 u. This fact suggested the presence of 6 cysteine residues, due to the difference of 348 u [i.e.

One of the powerful multidimensional separation methods in proteomics is ion-exchange chromatography (IEC) in the first dimension. Reversed-phase liquid chromatography (RPLC) most often in the second dimension is due to its compatibility with the downstream mass spectrometry (sample concentration, desalting properties, and used volatile solvents). IEC is very suitable for the separation of proteins and peptides based on their differences on overall charges. IEC’s stationary phase is either anion or cation exchanger, prepared by immobilization of positively or negatively charged

functional groups learn more on the surface of chromatographic column, respectively. Proteins or peptide separation occurs by linear change of the mobile-phase composition (salt concentration or pH) that decreases the interactions with the stationary phase

and finally eluted [17]. For neuroproteomic studies, Gao et al. [26] have described a method for the 2-D differential display of proteins of inflicted vs. non inflicted pediatric TBI cerebrospinal fluid (CSF) study. Also, Kobeissy et al. [27] have used a mixed cation- and anion-exchange chromatography (CAX) and 1-D sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) approach for differential protein separation, differentially expressed protein bands are excised and trypsinized followed by nanoLC and ESI-MS/MS protein identification. With this method, 59 proteins were identified as potential biomarker candidates

(Fig. 1). Protein marker candidates identified Bafilomycin A1 chemical structure include MAP-2, ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1), collapsin response element-2 Monoiodotyrosine (CRMP-2), synaptotagmin and alpha II-spectrin breakdown products UCH-L1 was one of these proteins that was subsequently confirmed to be a good translational biomarker for TBI. Liu et al. [28] first validated that UCH-L1 marker is not only differentially expressed in rat brain tissue, but also in biofluids following brain injury in rodents. CSF is important here as it is proximal to the injured organ, and thus likely to have these candidate markers in high concentrations. Indeed that was the case for UCH-L1 – which is elevated not only in the rat model of TBI (controlled cortical impact; CCI) but also in the rat model of ischemic stroke (using both quantitative immunblotting and sandwich ELISA method) [28]. Secondly with the aid of the two antibody-based sandwich ELISA, they were able to identify elevation of UCH-L1 in serum in both injury models as well. Subsequently, UCH-L1 protein was found to be elevated in human CSF and serum samples in both adult and in pediatric TBI [29], [30] and [31] (Table 1). Others have also used 2-D separation followed by MS/MS to identify candidate protein alterations for SCI [16], [32], [33] and [34]. For example, Yan et al.

Moving beyond the quantitation of information, key qualitative questions remain about how ‘meaning’ is transferred along with information. This is not merely an abstract question; synthetic biology can engineer reliable information transfer, but how would such systems encode or process higher order meaning, such as the difference between to ‘I must’ and ‘I want to’? Simple IF-THEN logic does not suffice. To harness essential features of biology, synthetic biologists

CX-5461 ic50 somehow need to wire components to encode choice and reward, perhaps by including feedbacks in system resource allocation. We still do not know how to engineer higher order meaning, such as desire or fear. While information theory clearly has a part to play in increasing our engineering capability, we also need to develop a functional philosophy of meaning. Papers of particular interest, published within the period of review, have been highlighted as: • of special interest DB and VRS are both selleck funded by La Caixa PhD Fellowships. MI is supported by EC FP7-610730 EVOPROG, and Wellcome Trust UK New Investigator Award WT102944.

We thank Jesper Ferkinghoff-Borg for providing us with original images of information channels inside a single protein. “
“The IC50 value of 3 has been reported as 18(5) μM in MCF-7. It actually is 185(5) μM for MCF-7 and hence the corrected Table 3 is as follows: “
“Indazoles are rare in nature, and so far only three natural products based on an indazole ring have been isolated [1]. These are the indazole alkaloids nigellicine [2], nigeglanine [3], and nigellidine [4]. The total syntheses of nigellicine and

nigeglanine are also well documented [5] and [6]. The indazole ring system is of much current interest as partial structure of a large number of biologically active compounds. Different aspects of pharmaceutical and other useful applications of indazoles Resminostat have been reviewed [7] and [8]. Some substituted indazoles exhibit relevant biological properties for development as anticancer drugs [9], [10], [11], [12], [13], [14] and [15]. One of the tetrasubstituted indazoles, namely, CI-958, entered clinical trials for prostate cancer treatment about a decade ago [16]. From the unsubstituted indazole derivatives the most prominent example is the ruthenium(III) compound (H2ind)[trans-RuIIICl4(Hind)2] (KP1019, Hind = 1H-indazole), which is now in clinical trials as an anticancer agent against metastatic solid tumors [17] and [18]. Of potential interest are also complexes closely related to (H2im)[trans-RuIIICl4(DMSO)(Him)] (NAMI-A, Him = imidazole) [19], an investigational drug which is currently evaluated in a clinical phase II trial for its capacity of inhibiting the process of metastasis, namely (H2ind)[trans-RuIIICl4(DMSO)(Hind)] [20] and its osmium counterpart [21].

Peptides were deprotected and released from the resin by TFA treatment in the presence of appropriate scavengers. The peptides were lyophilized and their purity was assessed by both HPLC (Akta Explorer 100) and mass spectrometry (MALDI-TOF/TOF, Autoflex III, Bruker Daltonics Inc.). Peptides were covalently coupled through their C-terminal

click here cysteine to lysine residues of BSA (Capelli-Peixoto et al., 2011). Briefly, BSA, previously diluted in 20 mM sodium phosphate buffer (pH 7.4) containing 0.15 M sodium chloride, was activated by sulfo-SMCC (10 mg/ml; Pierce Chemical Co., Rockford, IL). After 1 h of constant stirring at room temperature, the excess reagent was removed by elution through a PD-10 column. The activated BSA was reacted for 2 h with the cysteine-containing peptide learn more at room temperature under constant stirring and while being protected from light. A buffer containing reduced cysteine (1 mM) was added. The peptides coupled to BSA were separated into aliquots and stored at −20 °C. An analysis of variance (ANOVA) for a factorial experiment design was used for the analyses of the ELISA results. The significance level was set at p < 0.01. The Tukey test was used for the pairwise comparison between the factors; p < 0.05 was considered to be statistically significant. The analyses were performed using the ASSISTAT-7.2 program ( Silva and Azevedo,

2009). The sequences of LiD1 (GenBank: AAQ16123.1) from

L. intermedia, SMase I (GenBank: AAM21154.1) from L. laeta, and A1H-LoxGa (GenBank: AAY42401.1) from L. gaucho were aligned using ClustalW ( Larkin et al., 2007). The epitopes were analyzed in the three dimensional structures of the SMase I (PDB accession code: 1XX1) ( Murakami et al., 2005) using the PyMOL Molecular Graphics System (Version 1.2r3pre, Schrödinger, LLC). The molecular weight and theoretical isoelectric point of the peptides were calculated using the program PEPTIDES MASS (Wilkins et al., 1997). For the solvent accessibility calculation of the LiD1, SMase I, and A1H-LoxGa proteins we used the PSA program Cytidine deaminase and implemented the algorithm by Lee and Richards (1971). Residues were categorized as inaccessible by comparing them to an extended conformation; a 7% relative accessibility cut-off was applied (Hubbard and Blundell, 1987). The amino acid accessibility (in percentage) for the epitope regions was calculated. The amino acid hydrophobicity of each peptide was determined according to the Kyte and Doolittle scale (1982) and as described by Alvarenga et al. (2010a). We assessed whether there was a correlation between the neutralizing potency of anti-Loxosceles horse antisera (measured in vivo) and their ELISA reactivity. Nine Loxosceles antisera and a pre-immunized horse serum were tested by ELISA for reactivity using venoms from three species of Loxosceles (L.