The glyphosate controversy: an update / A controvérsia do glifosato: uma atualização

The demand for food in the world grows year after year due in part to population growth, but also to the improvement of emerging markets. Brazil is one of the largest food producers in the world. In 2017, its cereal, legume and oilseed crops totaled 238.6 million tons, 29.2% more than the year before. Much of the great increase in productivity is due to the incorporation of transgenic seeds, especially cotton, maize and soybean, which possess genes that will increase plant's adaptability to harsh soil and water conditions and, resistance to pests, but also tolerance to herbicides. Virtually, all transgenic soybeans planted in Brazil are resistant to glyphosate, the herbicide initially launched on the market by Monsanto in the 1970s under the trade name Roundup. Due to the existence of several transgenic crops tolerant to glyphosate, such as soy, wheat, corn and canola, this product is the most commercialized herbicide in the world. The use of glyphosate allows the sowing of transgenic crops immediately after application, making the planting and maintenance processes very practical. Soybeans, such as other transgenics, have biological safety already well defined, but the use of glyphosate is still an extremely controversial subject. This review presents some historical aspects of the binomial Roundup Ready soybean/glyphosate and discusses the most recent controversies about the use of glyphosate in Brazil and worldwide.(AU)

A demanda por alimentos no mundo cresce ano após ano devido em parte pelo aumento populacional, mas também pela melhoria dos mercados emergentes. O Brasil é um dos maiores produtores de alimentos do mundo. Em 2017, a safra nacional de cereais, leguminosas e oleaginosas totalizou 238,6 milhões de toneladas, 29,2% superior à obtida em 2016. Boa parte do grande aumento da produtividade se dá pela incorporação de sementes transgênicas, especialmente de algodão, milho e soja, que apresentam genes que conferem maior adaptabilidade da planta ao solo, pragas, condições hídricas, mas também conferem resistência a herbicidas. Praticamente toda a soja transgênica plantada no país é resistente ao glifosato, inicialmente lançado no mercado pela empresa Monsanto nos anos 1970 pelo nome comercial Roundup, que é hoje o herbicida mais utilizado no mundo, principalmente pela disseminação da soja e outros produtos transgênicos como trigo, milho e canola. A utilização do glifosato permite a semeadura de culturas transgênicas imediatamente após a aplicação, tornando o processo de plantio muito prático. A soja, como outros transgênicos, apresenta segurança biológica já definida, mas a utilização do glifosato ainda é um assunto extremamente controverso. Esta revisão apresenta alguns aspectos históricos do binômio soja transgênica Roundup Ready/glifosato e discute as controvérsias mais recentes sobre o uso do glifosato no Brasil e no mundo.(AU)

Identification of membrane proteome of Paracoccidioides lutzii and its regulation by zinc.

AIM: During infection development in the host, Paracoccidioides spp. faces the deprivation of micronutrients, a mechanism called nutritional immunity. This condition induces the remodeling of proteins present in different metabolic pathways. Therefore, we attempted to identify membrane proteins and their regulation by zinc in Paracoccidioides lutzii. MATERIALS & METHODS: Membranes enriched fraction of yeast cells of P. lutzii were isolated, purified and identified by 2D LC-MS/MS detection and database search. RESULTS & CONCLUSION: Zinc deprivation suppressed the expression of membrane proteins such as glycoproteins, those involved in cell wall synthesis and those related to oxidative phosphorylation. This is the first study describing membrane proteins and the effect of zinc deficiency in their regulation in one member of the genus Paracoccidioides.

Characterizing the nuclear proteome of Paracoccidioides spp.

Paracoccidioidomycosis is an endemic disease in Latin America, caused by thermo dimorphic fungi of the genus Paracoccidioides. Although previous proteome analyses of Paracoccidioides spp. have been carried out, the nuclear subproteome of this pathogen has not been described. In this way, we aimed to characterize the nuclear proteome of Paracoccidioides species, in the yeast form. For that, yeast cells were disrupted and submitted to cell fractionation. The purity of the nuclear fraction was confirmed by fluorescence and electron microscopy. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) allowed the identification of 867 proteins. In order to support our enrichment method for nuclear proteins, bioinformatics analysis were applied that allowed the identification of 281 proteins with nuclear localization. The analysis revealed proteins related to DNA maintenance, gene expression, synthesis and processing of messenger and ribosomal RNAs, likewise proteins of nuclear-cytoplasmic traffic. It was also possible to detect some proteins that are poorly expressed, like transcription factors involved in important roles such as resistance to abiotic stress, sporulation, cellular growth and DNA and chromatin maintenance. This is the first descriptive nuclear proteome of Paracoccidioides spp. that can be useful as an important platform base for fungi-specific nuclear processes.

Osmotic stress adaptation of Paracoccidioides lutzii, Pb01, monitored by proteomics.

The ability to respond to stressful conditions is essential for most living organisms. In pathogenic organisms, this response is required for effective transition from a saprophytic lifestyle to the establishment of pathogenic interactions within a susceptible host. Hyperosmotic stress has been used as a model to study signal transduction and seems to cause many cellular adaptations, including the alteration of protein expression and cellular volume as well as size regulation. In this work, we evaluated the proteomic profile of Paracoccidioides lutzii Pb01 yeast cells during osmotic stress induced by potassium chloride. We performed a high accuracy proteomic technique (NanoUPLC-MS(E)) to identify differentially expressed proteins during osmotic shock. The data describe an osmoadaptative response of this fungus when subjected to this treatment. Proteins involved in the synthesis of cell wall components were modulated, which suggested cell wall remodeling. In addition, alterations in the energy metabolism were observed. Furthermore, proteins involved in amino acid metabolism and hydrogen peroxide detoxification were modulated during osmotic stress. Our study suggests that P. lutzii Pb01. presents a vast osmoadaptative response that is composed of different proteins that act together to minimize the effects caused by osmotic stress.

The response of Paracoccidioides spp. to nitrosative stress.

Paracoccidioidomycosis (PCM) is an endemic disease in Latin America caused by species belonging to the genus Paracoccidioides. During infection, immune cells present a variety of defense mechanisms against pathogens. One of these defensive strategies is the production and release of nitric oxide (NO) and S-nitroso thiols (e.g., S-nitrosoglutathione, GSNO), which produce reactive nitrogen species (RNS). This results in damage to DNA and membranes, inhibition of respiration and inactivation of cellular enzymes. In response to nitrosative stress, human pathogenic fungi possess defense mechanisms to prevent the adverse effects of NO, which helps them survive during initial contact with the host immune system. To understand how Paracoccidioides spp. respond to nitrosative stress, we conducted this study to identify genes and proteins that might contribute to this response. The results of proteomic analysis demonstrated that nitrosative stress induced a reduction in the expression of proteins related to the mitochondrial electron transport chain. This hypothesis was supported by the reduced mitochondrial activity observed in the presence of GSNO. Additionally, lipids and branched chain amino acid metabolism enzymes were altered. The role played by enzymes acting in oxidative stress in the RNS response was remarkable. This interface among enzymes acting in both stress responses was confirmed by using a RNA approach to silence the ccp gene in Paracoccidioides. It was observed that mutants with low expression of the ccp gene were more sensitive to nitrosative stress.

Transcriptional and proteomic responses to carbon starvation in Paracoccidioides.

BACKGROUND: The genus Paracoccidioides comprises human thermal dimorphic fungi, which cause paracoccidioidomycosis (PCM), an important mycosis in Latin America. Adaptation to environmental conditions is key to fungal survival during human host infection. The adaptability of carbon metabolism is a vital fitness attribute during pathogenesis. METHODOLOGY/PRINCIPAL FINDINGS: The fungal pathogen Paracoccidioides spp. is exposed to numerous adverse conditions, such as nutrient deprivation, in the human host. In this study, a comprehensive response of Paracoccidioides, Pb01, under carbon starvation was investigated using high-resolution transcriptomic (RNAseq) and proteomic (NanoUPLC-MSE) approaches. A total of 1,063 transcripts and 421 proteins were differentially regulated, providing a global view of metabolic reprogramming during carbon starvation. The main changes were those related to cells shifting to gluconeogenesis and ethanol production, supported by the degradation of amino acids and fatty acids and by the modulation of the glyoxylate and tricarboxylic cycles. This proposed carbon flow hypothesis was supported by gene and protein expression profiles assessed using qRT-PCR and western blot analysis, respectively, as well as using enzymatic, cell dry weight and fungus-macrophage interaction assays. The carbon source provides a survival advantage to Paracoccidioides inside macrophages. CONCLUSIONS/SIGNIFICANCE: For a complete understanding of the physiological processes in an organism, the integration of approaches addressing different levels of regulation is important. To the best of our knowledge, this report presents the first description of the responses of Paracoccidioides spp. to host-like conditions using large-scale expression approaches. The alternative metabolic pathways that could be adopted by the organism during carbon starvation can be important for a better understanding of the fungal adaptation to the host, because systems for detecting and responding to carbon sources play a major role in adaptation and persistence in the host niche.

Centrosome duplication proceeds during mimosine-induced G1 cell cycle arrest.

Centrosome duplication must remain coordinated with cell cycle progression to ensure the formation of a strictly bipolar mitotic spindle, but the mechanisms that regulate this coordination are poorly understood. Previous work has shown that prolonged S-phase is permissive for centrosome duplication, but prolonging either G2 or M-phase cannot support duplication. To examine whether G1 is permissive for centrosome duplication, we release serum-starved G0 cells into mimosine, which delays the cell cycle in G1. We find that in mimosine, centrosome duplication does occur, albeit slowly compared with cells that progress into S-phase; centrosome duplication in mimosine-treated cells also proceeds in the absence of a rise in Cdk2 kinase activity normally associated with the G1/S transition. CHO cells arrested with mimosine can also assemble more than four centrioles (termed "centrosome amplification"), but the extent of centrosome amplification during prolonged G1 is decreased compared to cells that enter S-phase and activate the Cdk2-cyclin complex. Together, our results suggest a model, which predicts that entry into S-phase and the rise in Cdk2 activity associated with this transition are not absolutely required to initiate centrosome duplication, but rather, serve to entrain the centrosome reproduction cycle with cell cycle progression.

Myosin Va is proteolysed in rat cerebellar granule neurons after excitotoxic injury.

Cerebellar granule neurons when exposed to glutamate die through an excitotoxic mechanism induced by overactivation of glutamate receptors. This kind of cell death is mediated by an overload of intracellular calcium involving calpain activation, a Ca2+ -dependent intracellular cysteine protease, among other intracellular responses. On the other hand, class V myosins are proteins that move cargo along actin filaments and one of its members, myosin Va, is involved in vesicles transport. Here we studied the effect of excitotoxicity on myosin Va in cultured cerebellar granule neurons. Western blot analysis of control cultures shows a band corresponding to myosin Va as well as an 80 kDa band corresponding to its proteolytic product by calpain. When cells are exposed to glutamate (500 microM), kainate (100 microM) or NMDA (150 microM) during 3-24 h, the proteolytic processing of myosin Va is markedly increased. This proteolysis is inhibited by leupeptin (100 microM) and calpain inhibitor I (50 microM). These inhibitors also significantly improve the morphological appearance of the neurons possibly through the preservation of the cytoskeleton integrity. Our results suggest that myosin Va is a target for calpain I during an excitotoxic injury and could lead to a new area of research to address the participation of molecular motors in neurotoxicity.

Myosin-Va proteolysis by Ca2+/calpain in depolarized nerve endings from rat brain.

Myosin-Va is a molecular motor that may participate in synaptic vesicle cycling. Calpain cleaves myosin-Va in vitro at methionine 1141 in the tail domain. We show that intracellular proteolysis of myosin-Va occurs in rat cortical synaptosomes depolarized in the presence of calcium, evidenced by the formation of an 80 k polypeptide that co-migrates in SDS-PAGE with the 80 k fragment produced by the in vitro proteolysis of myosin-Va by calpain. Anti-myosin-Va antibody recognized this polypeptide in Western blots and immunoprecipitated it from synaptosome extracts. Calpastatin, a calpain-specific inhibitor, or leupeptin, a general cysteine protease inhibitor, suppressed or blocked formation of the 80 k polypeptide depending on membrane permeability. We conclude that myosin-Va undergoes intracellular proteolysis by endogenous calpain, when synaptosomes are depolarized in the presence of calcium, at the same cleavage site previously identified in vitro, thus, making it a target for calcium signaling during synaptic activation.

Myosins II and V in chromaffin cells: myosin V is a chromaffin vesicle molecular motor involved in secretion.

The presence of myosin II and V in chromaffin cells and their subcellular distribution is described. Myosin II and V distribution in sucrose density gradients showed only a strong correlation between the distribution of myosin V and secretory vesicle markers. Confocal microscopy images demonstrated colocalization of myosin V with dopamine beta-hydroxylase, a chromaffin vesicle marker, whereas myosin II was present mainly in the cell cortex. Cell depolarization induced, in a Ca2+ and time-dependent manner, the dissociation of myosin V from chromaffin vesicles suggesting that this association was not permanent but determined by secretory cycle requirements. Myosin II was also found in the crude granule fraction, however, its distribution was not affected by cell depolarization. Myosin V head antibodies were able to inhibit secretion whereas myosin II antibodies had no inhibitory effect. The pattern of inhibition indicated that these treatments interfered with the transport of vesicles from the reserve to the release-ready compartment, suggesting the involvement of myosin V and not myosin II in this transport process. The results described here suggest that myosin V is a molecular motor involved in chromaffin vesicle secretion. However, these results do not discard an indirect role for myosin II in secretion through its interaction with F-actin networks.

Molecular motors involved in chromaffin cell secretion.

Neurosecretory cells, including chromaffin cells, possess a mesh of filamentous actin underneath the plasma membrane. It has been proposed that filamentous actin network separates the secretory vesicles into two compartments: the reserve pool and the release-ready vesicle pool. Disassembly of chromaffin cell cortical filamentous actin in response to stimulation allows the movement of vesicles from the reserve pool into the release-ready vesicle pool. Electron microscopy of cytoskeletons revealed the presence of polygonal areas almost devoid of actin filaments in stimulated cells. The percentage of stimulated cells showing disrupted cytoskeleton correlates well with the increase in secretion in these cells. Fine filaments also remain in these areas of disassembly, and these reacted with actin antibodies, as demonstrated by immunogold staining. In addition, the movement of vesicles between pools requires Ca(2+) and ATP, a condition for activation of a molecular motor. Confocal microscopy images demonstrated colocalization of myosin Va with dopamine-beta-hydroxylase. Cell depolarization induced the dissociation of myosin Va from chromaffin vesicles. 2,3-Butadione-2-monoxime (BDM), an inhibitor of myosin ATPase, inhibited secretion, suggesting a blockage for chromaffin vesicle transport between the reserve pool and the release-ready vesicle pool. On the other hand, myosin II subcellular distribution was not affected by cell depolarization. Confocal microscopy images show myosin II to be localized in the cell cortex and in some perinuclear structures. Chromaffin vesicles were not stained by myosin II antibody.

Expression of constructs of the neuronal isoform of myosin-Va interferes with the distribution of melanosomes and other vesicles in melanoma cells.

Myosin-Va has been implicated in melanosome translocation, but the exact molecular mechanisms underlying this function are not known. In the dilute, S91 melanoma cells, melanosomes move to the cell periphery but do not accumulate in the tips of dendrites as occurs in wild-type B16 melanocytes; rather, they return and accumulate primarily at the pericentrosomal region in a microtubule-dependent manner. Expression of the full-length neuronal isoform of myosin-Va in S91 cells causes melanosomes to disperse, occupying a cellular area approximately twice that observed in non-transfected cells, suggesting a partial rescue of the dilute phenotype. Overexpression of the full tail domain in S91 cells is not sufficient to induce melanosome dispersion, rather it causes melanosomal clumping. Overexpression of the head and head-neck domains of myosin-Va in B16 cells does not alter the melanosome distribution. However, overexpression of the full tail domain in these cells induces melanosome aggregation and the appearance of tail-associated, aggregated particles or vesicular structures that exhibit variable degrees of staining for melanosomal and Golgi beta-COP markers, as well as colocalization with the endogenous myosin-Va. Altogether, the present data suggest that myosin-Va plays a role in regulating the direction of microtubule-dependent melanosome translocation, in addition to promoting the capture of melanosomes at the cell periphery as suggested by previous studies. These studies also reinforce the notion that myosin-V has a broader function in melanocytes by acting on vesicular targeting or intracellular protein trafficking.

Morphometric alterations in hepatocytes and ultrastructural distribution of liver glycogen in pacu (Piaractus mesopotamicus Holmberg, 1887) during food restriction and refeeding

The morphometric alterations in hepatocytes and the ultrastructural distribution of tissue glycogen in pacu (Piaractus mesootamicus) were studied following food restriction and refeeding. Fish (200-300g) were allocated to control and experimental groups. The experimental group was sampled after 0, 2, 7, 30 and 60 days of food restriction and after 7 and 30 days of refeeding. The control group, which was fed daily, was sampled on the same days. The morphometric results were analyzed by ANOVA in a 2x7 (feeding x days) factorial design and the averages compared by the Tukey test. Transmission electron microscopy showed liver glycogen mobilization during food restriction. The levels of glycogen did not return to normal after up to 30 days of refeeding. There was a decrease in the cytoplasmic area and volume after seven days without food whereas changes in the nuclear area and volume appeared after two days of food restriction. Recovery of the nuclear and cytoplasmic area and volume occurred after 7 days and 30 days of refeeding, respectively. These results indicate tha liver glycogen supplies at least part of the energy requirement during food restriction in juvenile pacu. Thirty days of refeeding were not enough to re-establish the pre-restriction carbohydrate levels, probably because of the extra energy demand associated with the high metabolic rate that occurred during the compensatory process of refeeding at elevated ambient temperatures. However, the recovey seen in the morphometric parameters of the hepatocytes indicated a functional re-adjustment of the liver stimulated by the restored food supply.