Five-years post commercial approval monitoring of eucalyptus H421.

Eucalyptus comprises the largest planted area of cultivated production forest in Brazil. Genetic modification of eucalyptus can provide additional characteristics for increasing productivity, protecting plant yield, and potentially altering fiber for various industrial uses. With this objective, a transgenic eucalyptus variety, event H421, received regulatory approval for commercial release after 6 years of approved risk assessment studies by the Brazilian National Technical Biosafety Commission (CTNBio) in 2015, becoming the first approved genetically modified (GM) eucalyptus in the world. GM event H421 enables increased plant biomass accumulation through overexpression of the Arabidopsis 1,4-ß-endoglucanase Cel1, which remodels the xyloglucan-cellulose matrix of the cell wall during development to promote cell expansion and growth. As required, in that time, by the current normative from CTNBio, a post-commercial release monitoring plan for H421 was submitted, incorporating general surveillance for five consecutive years with the submission of annual reports. The monitoring plan was conducted on fields of H421 progenies, with conventional clones as comparators, cultivated in representative regions where eucalyptus is cultivated in the states of São Paulo, Bahia, and Maranhão, representing Southeast, Northeast, and Northern Brazil. Over the course of the five-year general surveillance monitoring plan for the approved GM eucalyptus H421, no adverse effect that could impact the biosafety of the commercially approved event was identified. Additionally, the GM eucalyptus exhibited behavior highly consistent with that of conventional commercial clones. Therefore, there was no need for an extra risk assessment study of a case-specific monitoring plan. The results show the importance of continuously updating the regulation norms of governmental agencies to align with scientific advances.

Survivorship and food consumption of immatures and adults of Apis mellifera and Scaptotrigona bipunctata exposed to genetically modified eucalyptus pollen.

Eucalyptus comprises the largest planted area of cultivated production forest in Brazil. Genetic modification (GM) of eucalyptus can provide additional characteristics for increasing productivity and protecting wood yield, as well as potentially altering fiber for a diversity of industrial uses. However, prior to releasing a new GM plant, risk assessments studies with non-target organisms must be undertaken. Bees are prominent biological models since they play an important role in varied ecosystems, including for Eucalyptus pollination. The main goal of this study was to evaluate whether a novel event (Eucalyptus 751K032), which carries the cp4-epsps gene that encodes the protein CP4-EPSPS and nptII gene that encodes the protein NPTII, might adversely affect honey bees (Apis mellifera) and stingless bees (Scaptotrigona bipunctata). The experiments were performed in southern Brazil, as follows: (i) larvae and adults were separately investigated, (ii) three or four different pollen diets were offered to bees, depending on larval or adult status, and (iii) two biological attributes, i.e., survivorship of larvae and adults and food intake by adults were evaluated. The diets were prepared with pollen from GM Eucalyptus 751K032; pollen from conventional Eucalyptus clone FGN-K, multifloral pollen or pure larval food. The insecticide dimethoate was used to evaluate the sensitivity of bees to toxic substances. Datasets were analyzed with Chi-square test, survival curves and repeated measures ANOVA. Results indicated no evidence of adverse effects of Eucalyptus pollen 751K032 on either honey bees or stingless bees assessed here. Therefore, the main findings suggest that the novel event may be considered harmless to these organisms since neither survivorship nor food consumption by bees were affected by it.

Rheological and Viscoelastic Properties of Chitosan Solutions Prepared with Different Chitosan or Acetic Acid Concentrations.

Chitosan (Ch) is a partially crystalline biopolymer, insoluble in pure water but soluble in acid solutions. It has attracted interest from researchers to prepare solutions using different acid types and concentrations. This research aims to study both the effect of chitosan (Ch) or acetic acid (Ac) concentrations, at different temperatures, on rheological and viscoelastic properties of Ch solutions. To study the effect of Ch, solutions were prepared with 0.5−2.5 g Ch/100 g of solution and Ac = 1%, whereas to study the effect of Ac, the solutions were prepared with 2.0 g of Ch/100 g of solution and Ac = 0.2−1.0%. Overall, all analyzed solutions behaved as pseudoplastic fluid. The Ch strongly affected rheological properties, the consistency index (K) increased and the index flow behavior (n) decreased as a function of Ch. The activation energy, defined as the energy required for the molecule of a fluid to move freely, was low for Ch = 0.5%. The effect of Ac was less evident. Both K and n varied according to a positive and negative, respectively, parabolic model as a function of Ac. Moreover, all solutions, irrespective of Ch and Ac, behaved as diluted solutions, with G" > G'. The relaxation exponent (n") was always higher than 0.5, confirming that these systems behaved as a viscoelastic liquid. This n" increased with Ch, but it was insensitive to Ac, being slightly higher at 45 °C.

In vitro digestion of oil-in-water emulsions stabilized by whey protein nanofibrils.

The effect of pH (3 and 7) and varied energy density of a high-pressure homogenization process on the stability of oil-in-water (O/W) emulsions stabilized by whey protein fibrils was evaluated. A dynamic digestion model comprising the simulation of stomach, duodenum, jejunum and ileum, has been used to evaluate O/W emulsions' behavior under gastrointestinal (GI) conditions. The emulsions did not separate phases during the storage period (7 days). The emulsions stabilized by whey protein fibrils were stable under simulated gastric conditions but were destabilized in the simulated intestinal conditions. In a similar way, the whey protein fibrils' dispersion showed a high resistance to proteolytic in vitro digestion by pepsin (gastric stage) but was more readily degraded by pancreatin (intestinal stage). This fact confirms the significant impact of the interfacial characteristics on emulsions' digestion. The percentage of free fatty acids (FFA) absorbed in the simulated intestinal conditions (jejunum and ileum) was much lower than the total percentage of FFA released due to the use of WPI fibrils as emulsifier. This work contributes to a better understanding about the behavior of O/W emulsions stabilized by whey protein fibrils within the GI tract; this knowledge is fundamental when considering the final application of this protein in food products.

PhTx3-4, a Spider Toxin Calcium Channel Blocker, Reduces NMDA-Induced Injury of the Retina.

The in vivo neuroprotective effect of PhTx3-4, a spider toxin N-P/Q calcium channel blocker, was studied in a rat model of NMDA-induced injury of the retina. NMDA (N-Methyl-D-Aspartate)-induced retinal injury in rats reduced the b-wave amplitude by 62% ± 3.6%, indicating the severity of the insult. PhTx3-4 treatment increased the amplitude of the b-wave, which was almost equivalent to the control retinas that were not submitted to injury. The PhTx3-4 functional protection of the retinas recorded on the ERG also was observed in the neuroprotection of retinal cells. NMDA-induced injury reduced live cells in the retina layers and the highest reduction, 84%, was in the ganglion cell layer. Notably, PhTx3-4 treatment caused a remarkable reduction of dead cells in the retina layers, and the highest neuroprotective effect was in the ganglion cells layer. NMDA-induced cytotoxicity of the retina increased the release of glutamate, reactive oxygen species (ROS) production and oxidative stress. PhTx3-4 treatment reduced glutamate release, ROS production and oxidative stress measured by malondialdehyde. Thus, we presented for the first time evidence of in vivo neuroprotection from NMDA-induced retinal injury by PhTx3-4 (-ctenitoxin-Pn3a), a spider toxin that blocks N-P/Q calcium channels.

Quantitation of infectious myonecrosis virus in different tissues of naturally infected Pacific white shrimp, Litopenaeus vannamei, using real-time PCR with SYBR Green chemistry.

The Pacific white shrimp, Litopenaeus vannamei, is the most important shrimp species in volume in world aquaculture. However, in recent decades, outbreaks of diseases, especially viral diseases, have led to significant economic losses, threatening the sustainability of shrimp farming worldwide. In 2004, Brazilian shrimp farming was seriously affected by a new disease caused by the Infectious myonecrosis virus (IMNV). Thus, disease control based on rapid and sensitive pathogen detection methods has become a priority. In this study, a specific quantitation method for IMNV was developed using real-time PCR with SYBR Green chemistry and viral load of the principal target tissues of chronically infected animals was quantified. The quantitative analysis revealed that mean viral load ranged from 5.08×10(8) to 1.33×10(6)copies/µg of total RNA in the hemolymph, 5.096×10(5) to 1.26×10(3)copies/µg in the pleopods, 6.85×10(8) to 3.09×10(4)copies/µg in muscle and 8.15×10(6) to 3.90×10(3)copies/µg in gills. Different viral loads of IMNV were found with greater values in the hemolymph and muscle, followed by the pleopods and gills.

Mitochondrial calcium regulates rat liver regeneration through the modulation of apoptosis.

UNLABELLED: Subcellular Ca(2+) signals control a variety of responses in the liver. For example, mitochondrial Ca(2+) (Ca(mit)(2+)) regulates apoptosis, whereas Ca(2+) in the nucleus regulates cell proliferation. Because apoptosis and cell growth can be related, we investigated whether Ca(mit)(2+) also affects liver regeneration. The Ca(2+)-buffering protein parvalbumin, which was targeted to the mitochondrial matrix and fused to green fluorescent protein, was expressed in the SKHep1 liver cell line; the vector was called parvalbumin-mitochondrial targeting sequence-green fluorescent protein (PV-MITO-GFP). This construct properly localized to and effectively buffered Ca(2+) signals in the mitochondrial matrix. Additionally, the expression of PV-MITO-GFP reduced apoptosis induced by both intrinsic and extrinsic pathways. The reduction in cell death correlated with the increased expression of antiapoptotic genes [B cell lymphoma 2 (bcl-2), myeloid cell leukemia 1, and B cell lymphoma extra large] and with the decreased expression of proapoptotic genes [p53, B cell lymphoma 2-associated X protein (bax), apoptotic peptidase activating factor 1, and caspase-6]. PV-MITO-GFP was also expressed in hepatocytes in vivo with an adenoviral delivery system. Ca(mit)(2+) buffering in hepatocytes accelerated liver regeneration after partial hepatectomy, and this effect was associated with the increased expression of bcl-2 and the decreased expression of bax. CONCLUSION: Together, these results reveal an essential role for Ca(mit)(2+) in hepatocyte proliferation and liver regeneration, which may be mediated by the regulation of apoptosis.

Phoneutria spider toxins block ischemia-induced glutamate release and neuronal death of cell layers of the retina.

PURPOSE: To investigate the effect of calcium channel blockers, spider toxins, on cell viability and the glutamate content of ischemic retinal slices. METHODS: Rat retinal slices were subjected to ischemia via exposure to oxygen-deprived low-glucose medium for 45 minutes. Slices were either treated or not treated with the toxins PhTx3, Tx3-3, and Tx3-4. After oxygen-deprived low-glucose insult, glutamate content and cell viability were assessed in the slices by confocal and optical microscopy. RESULTS: In the retinal ischemic slices that were treated with PhTx3, Tx3-3, and Tx3-4, confocal imaging showed a decrease in cell death of 79.5 ± 3.1%, 75.5 ± 5.8%, and 61 ± 3.8%, respectively. Neuroprotective effects were also observed 15, 30, 60, and 90 minutes after the onset of the retinal ischemic injury. As a result of the ischemia, glutamate increased from 6.2 ± 1.0 nMol/mg protein to 13.2 ± 1.0 nMol/mg protein and was inhibited by PhTx3, Tx3-3, and Tx3-4 to 8.6 ± 0.7, 8.8 ± 0.9, and 7.4 ± 0.8 nMol/mg protein, respectively. Histologic analysis of the live cells in the outer, inner, and ganglion cell layers of the ischemic slices showed a considerable reduction in cell death by the toxin treatment. CONCLUSION: Spider toxins reduced glutamate content and cell death of retinal ischemic slices.

The effect of spider toxin PhTx3-4, ω-conotoxins MVIIA and MVIIC on glutamate uptake and on capsaicin-induced glutamate release and [Ca2+]i in spinal cord synaptosomes.

In spinal cord synaptosomes, the spider toxin PhTx3-4 inhibited capsaicin-stimulated release of glutamate in both calcium-dependent and -independent manners. In contrast, the conus toxins, ω-conotoxin MVIIA and xconotoxin MVIIC, only inhibited calcium-dependent glutamate release. PhTx3-4, but not ω-conotoxin MVIIA or xconotoxin MVIIC, is able to inhibit the uptake of glutamate by synaptosomes, and this inhibition in turn leads to a decrease in the Ca(2+)-independent release of glutamate. No other polypeptide toxin so far described has this effect. PhTx3-4 and ω-conotoxins MVIIC and MVIIA are blockers of voltage-dependent calcium channels, and they significantly inhibited the capsaicin-induced rise of intracellular calcium [Ca(2+)](i) in spinal cord synaptosomes, which likely reflects calcium entry through voltage-gated calcium channels. The inhibition of the calcium-independent glutamate release by PhTx3-4 suggests a potential use of the toxin to block abnormal glutamate release in pathological conditions such as pain.

Succinate modulates Ca(2+) transient and cardiomyocyte viability through PKA-dependent pathway.

GPR91 is an orphan G-protein-coupled receptor (GPCR) that has been characterized as a receptor for succinate, a citric acid cycle intermediate, in several tissues. In the heart, the role of succinate is unknown. We now report that rat ventricular cardiomyocytes express GPR91. We found that succinate, through GPR91, increases the amplitude and the rate of decline of global Ca(2+) transient, by increasing the phosphorylation levels of ryanodine receptor and phospholamban, two well known Ca(2+) handling proteins. The effects of succinate on Ca(2+) transient were abolished by pre-treatment with adenylyl cyclase and cAMP-dependent protein kinase (PKA) inhibitors. Direct PKA activation by succinate was further confirmed using a FRET-based A-kinase activity reporter. Additionally, succinate decreases cardiomyocyte viability through a caspase-3 activation pathway, effect also prevented by PKA inhibition. Taken together, these observations show that succinate acts as a signaling molecule in cardiomyocytes, modulating global Ca(2+) transient and cell viability through a PKA-dependent pathway.

Phoneutria spider toxins block ischemia-induced glutamate release, neuronal death, and loss of neurotransmission in hippocampus.

The aim of this study was to investigate the effect of spider toxins on brain injury induced by oxygen deprivation and low glucose (ODLG) insult on slices of rat hippocampus. After ODLG insult cell viabilility in hippocampal slices was assessed by confocal microscopy and epifluorescence using the live/dead kit containing calcein-AM and ethidium homodimer and CA1 population spike amplitude recording during stimulation of Schaffer collateral fibers. Spider toxins Tx3-3 or Tx3-4 and conus toxins, omega-conotoxin GVIA or omega-conotoxin MVIIC are calcium channel blockers and protected against neuronal damage in slices subjected to ODLG insult. Confocal imaging of CA1 region of rat hippocampal slices subject to ischemic insult treated with Tx3-3, Tx3-4, omega-conotoxin GVIA or omega-conotoxin MVIIC showed a decrease in cell death that amounted to 68 +/- 4.2%, 77 +/- 3.8%, 32 +/- 2.3%, and 46 +/- 2.9%, respectively. This neuroprotective effect of Tx3-4 was corroborated by eletrophysiological recordings of population spikes amplitudes in CA1. The neuroprotection promoted on hippocampal slices by Tx3-3 or Tx3-4 was also observed when the toxins were applied 10, 20, 30, 60, 90, or 120 min after induction of the ODLG injury. During the ischemic insult, glutamate release from slices was increased by 71% (from 7.0 +/- 0.3 nM/mg of protein control slices not subjected to ischemia to 12 +/- 0.4 nM/mg of protein in slices exposed to ischemia). Tx3-3, Tx3-4, omega-conotoxin GVIA or omega-conotoxin MVIIC inhibited the ischemia-induced increase on glutamate release by 54, 72, 60, and 70%, respectively. Thus Tx3-3 and Tx3-4 provided robust ischemic neuroprotection showing potential as a novel class of agent that exerts neuroprotection in an in vitro model of brain ischemia.

Tx3-4 a toxin from the venom of spider Phoneutria nigriventer blocks calcium channels associated with exocytosis.

The purpose of the present work was to investigate the pharmacological action of a calcium channel-blocking toxin from the venom of the spider Phonetic nigriventer, Tx3-4 on calcium channels coupled to exocytosis of synaptic vesicles. Tx3-4 blocked KCl-induced exocytosis of synaptic vesicles with an IC50 of 1.1 nM. To investigate whether the target of Tx3-4 overlaps with known calcium channels that mediate calcium entry and exocytosis, we used omega-toxins that interact selectively with neuronal calcium channels. The results indicate that the main population of voltage-sensitive calcium channels altered by Tx3-4 is P/Q calcium channels. In conclusion, Tx3-4 is a potent inhibitor of calcium channels involved in the KCl-induced exocytosis of synaptic vesicles in brain cortical synaptosomes.

Protective effect of retinal ischemia by blockers of voltage-dependent calcium channels and intracellular calcium stores.

In the present study, the neuroprotective effect of blockers of voltage-dependent calcium channels (VDCC) and intracellular calcium stores on retinal ischemic damage induced by oxygen deprivation-low glucose insult (ODLG) was investigated. Retinal damage induced by ODLG was dependent on the calcium concentration in the perfusion medium. When incubated in medium containing 2.4 mM CaCl(2), cell death in ischemic retinal slices treated with blockers of VDCC, omega-conotoxin GVIA (1.0 microM), omega-conotoxin MVIIC (100 nM) and nifedipine (1.0 microM), was reduced to 62 +/- 2.3, 46 +/- 4.3 and 47 +/- 3.9%, respectively. In the presence of blockers of intracellular calcium stores, dantrolene (100 microM) and 2-APB (100 microM), the cell death was reduced to 46 +/- 3.2 and 55 +/- 2.9%, respectively. Tetrodotoxin (1.0 microM), reducing the extent of the membrane depolarization reduces the magnitude of calcium influx trough VDCC causing a reduction of the cell death to 55 +/- 4.3. Lactate dehydrogenase content of untreated ischemic retinal slices was reduced by 37% and treatment of ischemic slices with BAPTA-AM (100 microM) or 2-APB (100 microM) abolished the leakage of LDH. Dantrolene (100 microM) and nifedipine (1.0 microM) partially blocked the induced reduction on the LDH content of retinal ischemic slices. Histological analysis of retinal ischemic slices showed 40% reduction of ganglion cells that was prevented by BAPTA-AM or dantrolene. 2-APB partially blocked this reduction whilst nifedipine had no effect, p > 0.95. Conclusion Blockers of VDCC and intracellular calcium-sensitive receptors exert neuroprotective effect on retinal ischemia.

The effect of sevoflurane on intracellular calcium concentration from cholinergic cells.

The mechanism of action of volatile anesthetics is not completely understood. Calcium release from internal stores may alter signaling pathways that influence neurotransmission. Abnormalities of the regulation of intracellular calcium concentration ([Ca2+]i) from patients with malignant hyperthermia is a hallmark of this syndrome indicating the potential of these agents to interact with proteins involved in Ca2+ signaling. In the present study, a cholinergic cell line (SN56) was used to examine whether the release of calcium from intracellular stores occurs in the presence of sevoflurane. Changes in [Ca2+]i were measured using fluo-4, a fluorescent calcium sensitive dye and laser scanning confocal microscopy. Sevoflurane induced an increase on [Ca2+]i from SN56 cells. The sevoflurane-induced increase on [Ca2+]i remained even when the cells were perfused with medium lacking extracellular calcium. However, this effect was abolished by BAPTA-AM, a chelator of intracellular calcium, suggesting the involvement of intracellular Ca2+ stores. Using cyclopiazonic acid, an inhibitor of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase, we investigated whether the depletion of intracellular Ca2+ stores interfered with the effect of sevoflurane. In the presence of this agent, sevoflurane caused a small but not significant rise on [Ca2+]i of the SN56 cells. Dantrolene, an inhibitor of ryanodine-sensitive calcium stores did not modify the sevoflurane increase on [Ca2+]i. Carbachol, a drug that releases Ca2+ from the IP3 pool, abolished the effect of sevoflurane. In addition, xestospongin D, a cell-permeant IP3 receptor antagonist, decreased significantly the sevoflurane increase on [Ca2+]i. Our data suggest that the sevoflurane-induced increase on [Ca2+]i from SN56 cells occurs through the release of calcium from IP3-sensitive calcium stores.

Acetylcholine release induced by the volatile anesthetic sevoflurane in rat brain cortical slices.

1. We have investigated the effect of the volatile anesthetic sevoflurane on acetylcholine (ACh) release from rat brain cortical slices. 2. The release of [3H]-ACh into the incubation fluid was studied after labeling the tissue ACh with [methyl-3H]-choline chloride. 3. We observed that sevoflurane induced an increase on the release of ACh that was dependent on incubation time and anesthetic concentration. The sevoflurane-induced ACh release was not blocked by tetrodotoxin (TTX) and therefore was independent of sodium channels. In addition, the sevoflurane effect was not blocked by ethylene glycol-bis(beta-aminoethyl ether (EGTA) or cadmium (Cd2+), thus independent of extracellular calcium. 4. The sevoflurane-induced ACh release was inhibited by 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetra-acetic acid (BAPTA-AM), suggesting the involvement of intracellular calcium-sensitive stores in the process. Dantrolene, an inhibitor of ryanodine receptors, had no effect but 2-aminoethoxydiphenylborate (2-APB), a membrane-permeable inhibitor of inositol 1,4,5-triphosphate receptor inhibited the sevoflurane-induced release of ACh. 5. It is concluded that sevoflurane-induced release of ACh in brain cortical slices involves the mobilization of calcium from IP3-sensitive calcium stores.