Typical animal tissues have background concentrations of ferromagnetic materials in the 1–1000 ng/g range, with average levels of ∼4 ng/g. A recent high-resolution study of magnetoreceptor cells containing biological magnetite in fish by Eder et al. [12] demonstrated that the individual cells are surprisingly magnetic (up to 100 fAm2), with magnetite concentrations often 100 times greater than typical cells of magnetotactic DAPT bacteria. These cells have

interaction energies of up to 1500 times larger than the background thermal noise (kT, where k is the Boltzmann constant and T the absolute temperature) in the geomagnetic field, which would be on the order of 4500 times larger than kT in the typical magnetic fields (0.15 mT) used in the CAS freezers [18]. In our work on human Enzalutamide concentration tissues [21], we reported the presence of ∼4 ng/g of magnetite in the cortex and cerebellum (with a factor of 10× larger in the meninges), values similar to that measured with superconducting magnetometry in a variety of other

animal tissues [20]. With these measured Vertebrate cell concentrations, this yields minimum estimates of nearly 100,000 of these magnetic clusters per gram of typical tissue. In turn, this implies that the average distance of any cell within a magnetite-bearing tissue would on the order of 20 μm from a ferromagnetic cluster. Smaller particle sizes would imply correspondingly more particles, and shorter distances, from the nearest cluster. It seems most likely that the electrostatic enhancement observed during the CAS freezing process is a simple disruption of the surface boundary effect of inert air, and a more efficient heat transport process. The enhanced removal pheromone of heat from the tissues may be one factor in producing the supercritical cooling observed. In their attempt to test components of the CAS hypotheses, Suzuki et al. [38] were able to refute

claims that the magnetic treatment was involved with heat transport. We concur with their analysis, but suggest that the electric exposure, not the magnetic exposure, is responsible for that aspect. If the oscillation of sub-micron ferromagnetic particles distributed through tissues is involved in the reported action of CAS freezers, then we see two possible mechanisms for this inhibition of ice crystal nucleation. First, and most obvious, is the possibility that these particles normally act as some of the nucleation sites for the formation of ice crystals. Oscillations would then tend to inhibit the aggregation of the few hundred water molecules involved in the early crystal growth (e.g., [32]). This could certainly be tested experimentally. Second, the low-frequency acoustic waves from the oscillating particles will radiate outwards from the magnetite-containing cells.

15 (Table 2) (Gundersen et al., 1999). The estimation of DG microglia mean body cell volume, microglia mean body cell number, and DG volume, was assisted by Stereologer™ software (Stereology Resource Center, Chester, MD). The software was installed on a Dell Optiplex tower computer and connected to a Nikon Eclipse E600 microscope

(Nikon, Melville, NY) fitted with an X–Y–Z motorized stage controller (Prior Scientific, Rockland, MA), linear encoder microcator (z-axis gauge) (Heidenhain, Schaumburg, IL), high resolution color video camera (IMI Tech, Inc., Encinitas, CA) CP-673451 cost and .50 C-mount (Nikon, Melville, NY). DG volume was estimated at 4× (Nikon Plan 4× 0.10); mTOR inhibitor DG microglia mean cell volume and mean cell number were estimated at 60× (Nikon Plan APO 1.40 Oil). The camera image was processed with a high resolution video card and displayed on a 21 in. high resolution Dell monitor. One experimenter (C.S.) collected all of the stereological data without knowledge of the blood Pb level of each subject;

the experimenter was not blind to treatment group. An unbiased estimate of the number of microglia in the DG was obtained using the optical fractionator method (West et al., 1991) as reported previously for quantification of total number of microglia in mouse models of aging and neuropathology (Mouton et al., 2002). For each section the software randomly sampled virtual 3-D counting frames (disector) at 60× magnification with a 2 μm guard area. Using thin-focal

plane optical scanning, microglia were counted when they fell within the central depth of the counting frame and/or touched the inclusion lines. The total number of microglia was estimated with the following Megestrol Acetate formula: Nobj = ΣQ− × 1/SSF × 1/ASF × 1/TSF; where ΣQ− = sum of the objects sampled; SSF = sampling interval; ASF = total area sampled/total area on all sampled sections; and TSF = the height of the sample/total section thickness. For each frame, mean cell volume was quantified on microglia counted with the disector probe. The dentate gyrus reference volume (V(ref)) was determined at 4× magnification using the Cavalieri-point counting approach ( Gundersen and Jensen, 1987): V(ref) = ([k × t] × ∑P × [a(p)/M2]); where: k = sampling interval; t = post-processing section average thickness; and thus [k × t] = distance between planes; ∑P = sum of points counted; [a(p)/M2] = test grid area per point (μm2) divided by the magnification factor squared. Examples of microglia images are provided in Fig. 4. SAS Version 9.2 statistical software was used for all analyses. All data were entered and checked for accuracy and distribution properties prior to analysis. No extreme outliers were identified, and all data were included for analysis.

These data suggest that LEF did not induce vascular effect ISRIB concentration as observed by exposure to the lectins from Canavalia brasiliensis (ConBr), Canavalia ensiformis (ConA), Dioclea guianensis (DguiL) and Vatairea macrocarpa ( Teixeira et al., 2001, Havt et al., 2003 and Martins et al., 2005). As LEF has different carbohydrate specificity compared to ConBr, ConA, DguiL and Vatairea macrocarpa lectin, it might not have interacted with the target site that triggers changes on perfusion pressure and renal vascular resistance. The increase in glomerular filtration rate (Fig. 4) and decrease in the percentage

of Na+/K+/Cl− tubular transport (Fig. 5), both induced by LEF-perfusion, produced a tubuloglomerular feedback alteration which is a complex process that regulates the glomerular filtration rate. Interference in Na+/K+/Cl− transport and increase in glomerular filtration rate was also observed in ConBr-perfused rat kidney (Teixeira et al., 2001). However, ConA affected only K+ reabsorption (Havt et al., 2003) and V. macrocarpa lectin had no interference with electrolyte transport, but increased the glomerular filtration rate and the urinary flow ( Martins et al., 2005). Nevertheless, these above lectin-associated effects suggest the possible involvement of carbohydrate specific target receptors on the animal cell

recognized selleck chemical by lectins. In conclusion, the toxic effects observed in the various models used in this study when cAMP exposed to LEF strongly suggest that one of the toxic principles of I. asarifolia is a sialic acid binding lectin present in its leaves. The authors declare that there are no conflicts of interest. We thank EMBRAPA (Empresa Brasileira de Pesquisa Agropecuária) for partially support this research and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for doctoral

scholarship (Grant no. 081408/2003-05) to H.O. Salles. We also thank to Centro Nordestino de Aplicação e uso da Ressonância Magnética Nuclear (CENAUREMN) of Federal University of Ceará for NMR analyze, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Programa Nacional de Cooperação Acadêmica (PROCAD) and Fundação de Amparo à Pesquisa do Estado do Ceará (FUNCAP). “
“Thalassophryne nattereri (niquim) is a venomous fish of the Batrachoididae family and, in Brazil, it is known by the severity of the accidents provoked in fishermen and bathers ( Fonseca and Lopes-Ferreira, 2000 and Faco et al., 2003). Its venomous apparatus is composed of two dorsal and two lateral canaliculated spines covered by a membrane connected to venomous glands at the base of the fins. The venom displays proteolytic and myotoxic activities, but it is devoid of phospholipase A2 activity ( Lopes-Ferreira et al., 1998).

These various measures would apply in different ways, depending

on the nature of the vessel and the voyage. In practice, a regulatory regime could begin with voluntary measures and, depending on the success of those measures and a need for more formal actions, evolve towards mandatory standards of care established by the U.S. or Russia, in the case of domestic regulations, and the IMO, for international regulations. The measures themselves may be similar in nature and intent, with the main difference being the way they are implemented and enforced. Voluntary safety and environmental protection measures may be recommended by regulators, including government agencies as well as the IMO. These measures can include all of the regulatory measures, such as voluntary vessel speed limits, reporting recommendations, routing selleck screening library recommendations, or other actions. Although commercial shippers do not have to adhere to voluntary SCH727965 datasheet measures, compliance with some voluntary measures can be high [71], though variable [72] and may be low or negligible for some measures, as was found for speed restrictions off the coast of California [73]. Compliance is likely due to a desire to operate responsibly

to reduce risk, or requirements by insurers that vessels follow appropriate guidelines whether mandatory or not. If regulators recommend pragmatic voluntary measures to which commercial shippers are likely to adhere, regulators may be able to significantly increase on-the-water safety

and environmental protections in a relatively short period of time. In addition to encouraging voluntary compliance in the short-term, these measures may facilitate adoption of binding measures in the long run. Under UNCLOS, coastal states have Fossariinae authority to regulate vessels that fly the flag of the coastal state (Part VII, Articles 92 and 94) or that are going to or from a port of that state, and can enact a broad range of safety and protective measures. They can also regulate foreign-flagged vessels that in transit passage so long as such regulation does not discriminate among foreign ships or impair the right of transit passage (Part III, Article 42). Accordingly, domestic regulation by the United States or Russia could have a significant impact on safety and environmental protection in the region and could set the stage for international regulation. Such actions could be, but do need to be, done cooperatively by the two countries—although full coverage of the transboundary Bering Strait region would clearly require bilateral cooperation. These domestic regulations can cover the types of measures described in Section 5. International regulation of vessel traffic is done through the IMO, which has established a variety of instruments designed to promote safety and prevent marine pollution by vessels.

5 mg/mL) for 4 h at 37 °C in a 5% CO2 atmosphere.

The plate was centrifuged at 1500 rpm for 10 min. The medium was removed and replaced by 100 μL of dimethyl sulfoxide (DMSO), followed by mixing to dissolve the formazan Z-VAD-FMK concentration crystals. Absorbance was measured at 570 nm on a microenzyme-linked immunosorbent assay (ELISA) reader (Spectramax, Molecular Devices®) and the reduction of cell viability was expressed as the percentage compared with the negative control group designated as 100%. A control experiment carried out using only PAMAM in the culture medium did not induced cytotoxicity (data not shown). Nanoparticle-induced DNA damage was performed by the comet assay (also referred to as the single-cell gel electrophoresis – SCGE analysis) under alkaline conditions (Singh et al., 1988). The negative control was

exposed without AuNps under the same conditions. HepG2 cells and PBMC were cultured in 12-well culture plates as described above, and then pretreated for 3 h with 1.0 and 50.0 μM of AuNps-citrate and AuNps-PAMAM. Microscope slides were prepared in duplicate and coated with 1% normal melting point agarose (NMA). 60 μL of each cell suspension with 300 μL of low melting point agarose 1% (LMPA) were placed on these microscope slides containing NMA, deposited over the agarose layer. Coverslips were placed on the gels, which were left to set on ice. After gently removing the coverslips, the slides were immediately submersed in cold lysis solution (2.5 M NaCl, 100 mM EDTA,

10 mM Tris, 1% Tritron selleck compound Elongation factor 2 kinase X-100, pH 10) for 12 h in the dark. DNA was then allowed to unwind for 20 min in alkaline electrophoresis solution (300 mM NaOH, 1 mM EDTA, pH > 13). Electrophoresis was performed under 25 V and 300 mA for 20 min. Subsequently, the slides were placed in a cold neutralizing buffer (400 mM Tris buffer, pH 7.5) for 15 min, dried in 100% methanol for 5 min, and stained with 50 μL of 20 μg/mL ethidium bromide in the dark. At least 50 comets per slide were analyzed under a fluorescence microscope (Nikon Eclipse E200, Japan) equipped with an excitation filter of 515–560 nm and a barrier filter of 590 nm, connected to a digital camera (Nikon DS Qi1, Japan). The classical visual analysis scoring of the comet assay was analyzed by a single analyst, in order to minimize scoring images variation. Data were based on 150 cells for each test or control that were visually scored as belonging to one of five classes, according to tail size and intensity. Classes 0, 1, 2, 3, or 4 were given, with 0 = no detectable damage and 4 = maximum damage. The damage index was obtained by the formula, damage index = (0 × n0) + (1 × n1) + (2 × n2) + (3 × n3) + (4 × n4). The variables n0–n4 represent the number of nucleoids with 0–4 damage level, and each experiment was performed in triplicate. The AuNps cellular uptake was investigated using flow cytometry (Suzuki et al., 2007).

The indirect detection methods must be MK-8776 in vitro sensitive enough that even small amounts of product can trigger a signal from the coupled system. In other words, the secondary detection system cannot be rate-limiting or the kinetics of detection will be observed, not the kinetics

of the reaction. Alternatively, the detection reagents must be in sufficient quantity to detect generated product amounts without being consumed completely. For instance, in two-component detection systems such as HTRF, high amounts of product can saturate the detection components, leading to an artificial plateau in the reaction curve. This can be mistakenly interpreted as having reached equilibrium, when in fact, allowing the reaction to continue will actually generate a decreasing curve. This “hook effect” is common and can be observed, for example,

when titrating a biotinylated peptide which is recognized by an antibody-linked to a donor fluorophore to create a FRET signal when an appropriate acceptor fluorophore is in close proximity ( Figure 5). The “hook effect” can be identified by generating a product standard curve and testing various concentrations of detection components. Finally, the interference by the compounds being assayed with the coupled system must be considered. With the many caveats of indirect detection systems, there are still many situations in which an indirect detection method is superior to a direct Enzalutamide supplier detection method. Particularly for use in HTS, many

direct detection techniques (radioactive substrates/products, Western blots, HPLC, NMR) cannot be adapted for the throughput and automation required to efficiently process large numbers of compounds. The cost of reagents and supplies must also be weighed when considering a detection technique and the cheapest option in the short term may be the least cost effective over the course of an entire screen. Many of the enzyme assays used in HTS that are discussed in the next section involve indirect detection methods. As an example of direct detection, mass spectrometry is often an ideal method for assays involving post-translational modifications such as hydroxylation, phosphorylation or acetylation of substrate peptides, limitations on maximum BCKDHA throughput capabilities may preclude the use of this technique in favor of an indirect detection method such as time-resolved-fluorescence energy transfer (TR-FRET) or Amplified Luminescent Proximity Homogenous Assay (AlphaScreen™, see below). For instance, a multiplexed LC/MS detection protocol can process samples at 30 s per well, or about 3 h per 384w plate. At 8 plates per day, it would take 47 non-stop weeks to screen a deck of 1 million compounds, not counting controls. However using HTRF detection and a ViewLux which can read a 1536-well plate in approximately 2 min, the same screen can be accomplished in 22 h of total read time, saving both time and money.

6) and Methanoregula boonei 6A8 (Score 480, Genome Id 456442.10) were the closest relatives of Methanoculleus sp. MH98A. Digital DNA–DNA hybridization, performed as described by Auch et al. (2010), revealed only 54.2 ± 2.7% similarity between MH98A

and M. marisnigri JR1 indicating distinct delineation between the two species. M. boonei 6A8 was found to be an even more distant relative of MH98A with a genome to genome similarity of only 16.8 ± 2.2%. Further analysis revealed that the draft genome of strain MH98A was 2,542,436 bp in size with 2317 coding sequences, 1 copy of 5S, 16S and 23S rRNA genes and 50 tRNA genes. 32% of the predicted coding sequences were assigned to subsystem categories. The MH98A genome sequence analysis revealed 22 unique genes associated with subsystems when compared to its closest phylogenetic relative, M. marisnigri JR1. Among these are genes associated with carbohydrate metabolism, cell wall component synthesis GSI-IX ic50 and capsule ( Leahy et al., 2010 and Poli et al., 2011), cofactor synthesis, vitamin synthesis, prosthetic group synthesis, pigment synthesis, DNA metabolism, membrane transport, protein metabolism and potassium metabolism. A unique gene associated with subsystem of cofactor synthesis, namely the gene encoding coenzyme gamma-F420-2:l-glutamate ligase was detected. Coenzyme F420 is essential for methane synthesis

via the hydrogenotrophic ABT-199 chemical structure pathway from carbon dioxide and hydrogen. Formate dehydrogenase, a key enzyme for formate utilization in the methanogenesis pathway, was also detected. These observations were consistent with the substrate utilization profile of MH98A which consumes H2/CO2 and formate as substrates for methane production. Genes coding for different enzymes

in hydrogenotrophic methanogenesis which include, formylmethanofuran dehydrogenase, formylmethanofuran–tetrahydromethanopterin N-formyltransferase, N5, N10-methenyltetrahydromethanopterin cyclohydrolase, F420-dependent methylenetetrahydromethanopterin dehydrogenase, F420-dependent Osimertinib mw N5, N10-methylenetetrahydromethanopterin reductase, N5-methyltetrahydromethanopterin: coenzyme M methyltransferase and Methyl coenzyme M reductase were detected. Further comparative genome analyses are ongoing to better elucidate the methanogenesis pathway in Methanoculleus sp. MH98A and improve understanding of the evolutionary relationship between Methanoculleus strains associated with submarine sediments from distant geographical locations. The draft genome sequence of Methanoculleus sp. MH98A was deposited in the DDBJ/EMBL/GenBank database under the Accession number JMIO00000000.1. The strain Methanoculleus sp. MH98A is available from Dr. P.K. Dhakephalkar (Agharkar Research Institute, G.G. Agarkar Road, Pune 411004. India). The strain is also available at MACS Collection of microorganism (Registration No. WDCM 561) under the Accession No. MCMB-889.

Then the absorbance was measured at 515 nm. The capability to scavenge the DPPH radical was calculated using the following equation: DPPH scavenging activity(%)=Acontrol−AsampleAcontrol×100,where Acontrol

was the absorbance of the reaction in the presence of water and Asample the absorbance of the reaction in the presence of the extract. The extract concentration producing 50% inhibition (EC50) was calculated from the graph of the Atezolizumab datasheet DPPH scavenging effect against the extract concentration. Gallic acid, syringic acid and pyrogallol were used as standards. The 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid (ABTS)) assay was done as previously described (Soares et al., 2009). Briefly, the stock solutions were 7.4 mmol/L ABTS + and 2.6 mmol/L potassium persulfate. The working solution was then prepared by mixing the two stock solutions in equal quantities and allowing them to react for 12 h at room temperature in the dark. selleck chemicals llc The solution was then diluted by mixing 1 mL ABTS + solution with 60 mL methanol to obtain an absorbance of 1.1 at 734 nm. A fresh ABTS + solution was prepared for each assay. A volume of 150 μL of each extract (final concentrations from 5 to 100 μg/mL) was allowed to react with 2850 μL of the ABTS + solution (final concentration of 0.02 mmol/L) for 2 h in the dark.

Finally, the absorbance at 734 nm was measured. Distilled water was used instead of mushroom extracts as a control. The capability to scavenge the ABTS radical was calculated using Etofibrate the following equation: ABTS scavenging activity(%)=Acontrol−AsampleAcontrol×100,where Acontrol

was the absorbance of the reaction in the presence of water and Asample the absorbance of the reaction in the presence of the extract. The extract concentration producing 50% inhibition (EC50) was calculated from the graph of the ABTS scavenging effect against the extract concentration. Gallic acid, syringic acid and pyrogallol were used as standards. The ferrous ion chelating ability of extracts was determined as described previously described (Soares et al., 2009). Briefly, a sample (0.7 mL) of each extract was diluted in 0.7 mL of distilled water and mixed with 0.175 mL of FeCl2 (0.5 mmol/L) and the absorbance (A0) was measured at 550 nm. After, the reaction was initiated by the addition of 0.175 mL ferrozine (0.5 mmol/L). The mixture was shaken vigorously for 1 min and left standing at room temperature for 20 min when the absorbance (A1) was again measured at 550 nm. The percentage of inhibition of the ferrozine–Fe2+ complex formation was calculated as follows: chelating ability(%)=A0−A1A0×100. A lower absorbance indicates higher chelating ability. The extract concentration producing 50% chelating ability (EC50) was calculated from the graph of antioxidant activity percentage against the extract concentration. Gallic acid, syringic acid and pyrogallol were used as standards.

All these steps were carried out in 20 μL microdrops at 39 °C under mineral oil. Afterwards the embryos were cultured individually in CR2aa medium under 5% CO2 and 39 °C for 120 min (T120). Pictures of embryos from each culture media were captured at 0, 5, 10 and 120 min (T0, T5, T10 and T120, respectively), with a CCD camera connected to an inverted microscope and saved in a computer using the Pinnacle Studio software, v. 7.11 (Pinnacle, Mountain View, CA, USA). The images were analyzed by the ImageJ software v. 1.40 (National Institute of Health, USA). For embryo area measurement,

the zona pellucida MAPK Inhibitor Library and periviteline space were excluded. For area measurement, images were previously calibrated using a graduated glass slide. Measures of T0 area (T0 = 1) were used as a reference for further T5, T10 and T120 relative area determination. Dehydration was considered the T5 data and indicates the reduction in area immediately after embryo exposure to hypertonic medium (T5). T10 and T120 show the area recovery after 10 (T10) and 120 (T120) min in isotonic medium. Vitrification was performed by OPS method as first described by Vajta et al. [35]. Expanded blastocysts at 168 hpi, morphologically classified as good or excellent, were vitrified

using DMSO and EG as CPAs. The embryos were equilibrated into 10% DMSO plus 10% EG in PBS medium supplemented with 5% FCS (HM2) for 1 min followed by 30 s into 20% DMSO plus 20% EG, loaded into OPS and Sitaxentan plunged into liquid nitrogen. Warming was performed by immersing OPS

into HM2 with 0.25 M sucrose at 39 °C for 1 min, this website followed by two-step rehydration in 0.25 M and 0.15 M of sucrose for 5 min each one. All steps were at 39 °C. Afterwards, the embryos were washed in HM2. Vitrified-warmed embryos were cultured in CR2aa medium with granulosa cells monolayer for 72 h. Control group embryos were cultured simultaneously. Survival rate was assessed by blastocyst re-expansion and hatching at 72 h. Samples obtained from experiments 2 and 3 were used for RNA extraction and PCR analysis. Total RNA was extracted from pools of five embryos using the RNeasy Micro Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions and treated with DNase. Messengers RNA were amplified (one round) using the MessageAmp™II aRNA Amplification Kit (Ambion, Austin, TX, USA) according to the manufacturer’s instructions, in order to get enough material for transcript analysis. This procedure generated a final volume of 20 μL with concentration of ∼70 ng/μL of anti-sense amplified RNA (aRNA). The aRNA samples were reverse transcribed (RT) using the SuperScript III First-Strand Synthesis Supermix (Invitrogen, Carlsbad, CA, USA) and a random hexamer primer, according to the manufacturer’s instructions.

On the other hand, CgNa is a toxin isolated from Condylactis gigantea species and presents a dense negative charge around residues 35–37 (see Table 1). In spite of the presence of such a negative charge its potency is in a similar range such as BgII when tested in dorsal root ganglia (DRG) neurons [28], [29] and [32]. Also, the determination of CgNa three-dimensional structure by NMR exhibits a large negative patch exposed, and

a minor distribution of hydrophobic residues that are important for activity in ApB and ATX-II [29]. As pointed by the authors, this may explain that at least for CgNa the presence of positively charged amino acids and a hydrophobic Z-VAD-FMK patch may not be of utmost importance for its binding on sodium channels, but might contribute to a smaller potency compared

to ATX-II and selleck inhibitor ApB, for instance. Observing the modeled structures shown in Fig. 5, we clearly see that for δ-AITX-Bcg1a peptide an overall charge distribution similar to CgNa may occur. In its primary sequence, we observe a negatively charged amino acid (D37) that is positioned in a correspondent region of D36 and E37 in CgNa, and their contact surface on sodium channels may also be similar. Comparing among the charged molecular surfaces of the three toxins, δ-AITX-Bcg1a has a more intense negatively charged surface when compared to CGTX-II. As for δ-AITX-Bcg1b, the occurrence of an Asp at position 16 may disrupt this possible surface of contact, by increasing the extent of the negative patch and make δ-AITX-Bcg1b much less prone to affect VGSC,

as shown in Fig. 1. This is especially interesting as we consider the only N16D substitution observed between δ-AITX-Bcg1a and δ-AITX-Bcg1b, but the molecular surface of the latter shows that the occurrence of Asp16 drastically increases the negatively charged surface among all three peptides. Consequently, we may suggest that the large negative surface observed in δ-AITX-Bcg1b may be responsible for its mild effects among the eltoprazine assayed Navs. In summary, our results contribute to a better understanding of the selectivity of some sea anemone toxins toward channels Nav1.1–1.7. The presented data demonstrate that the binding sites of these toxins is not restricted to the supposed site 3, between segments S3 and S4 of domain IV. Also, we show that previously assumed critical amino acid positions in this group of peptides may vary and should be carefully considered. By doing this we may avoid misleading interpretations by common generalizations that may arise from site-directed mutagenesis studies. Moreover, subtle variations in primary sequences of toxins may lead to drastic changes in surface charges, such as in the case of δ-AITX-Bcg1a and δ-AITX-Bcg1b, which may contribute to a better understanding of their contact surfaces on the targeted channels.