Validation of a liquid chromatography ultraviolet method for determination of herbicide diuron and its metabolites in soil samples.

Diuron is one of the most widely herbicide used worldwide, which can undergo degradation producing three primary metabolites: 3,4-dichlorophenylurea, 3-(3,4-dichlorophenyl)-1-methylurea, and 3,4-dichloroaniline. Since the persistence of diuron and its by-products in ecosystems involves risk of toxicity to environment and human health, a reliable quantitative method for simultaneous monitoring of these compounds is required. Hence, a simple method without preconcentration step was validated for quantitation of diuron and its main metabolites by high performance liquid chromatography with ultraviolet detection. Separation was achieved in less than 11 minutes using a C18 column, mobile phase composed of acetonitrile and water (45:55 v/v) at 0.86 mL min-1 and detection at 254 nm. The validated method using solid-liquid extraction followed by an isocratic chromatographic elution proved to be specific, precise and linear (R2 ˃ 0.99), presenting more than 90% of recovery. The method was successfully applied to quantify diuron and their by-products in soil samples collected in a sugarcane cultivation area, focusing on the environmental control.

A protein kinase screen of Neurospora crassa mutant strains reveals that the SNF1 protein kinase promotes glycogen synthase phosphorylation.

Glycogen functions as a carbohydrate reserve in a variety of organisms and its metabolism is highly regulated. The activities of glycogen synthase and glycogen phosphorylase, the rate-limiting enzymes of the synthesis and degradation processes, respectively, are regulated by allosteric modulation and reversible phosphorylation. To identify the protein kinases affecting glycogen metabolism in Neurospora crassa, we performed a screen of 84 serine/threonine kinase knockout strains. We identified multiple kinases that have already been described as controlling glycogen metabolism in different organisms, such as NcSNF1, NcPHO85, NcGSK3, NcPKA, PSK2 homologue and NcATG1. In addition, many hypothetical kinases have been implicated in the control of glycogen metabolism. Two kinases, NcIME-2 and NcNIMA, already functionally characterized but with no functions related to glycogen metabolism regulation, were also identified. Among the kinases identified, it is important to mention the role of NcSNF1. We showed in the present study that this kinase was implicated in glycogen synthase phosphorylation, as demonstrated by the higher levels of glycogen accumulated during growth, along with a higher glycogen synthase (GSN) ±glucose 6-phosphate activity ratio and a lesser set of phosphorylated GSN isoforms in strain Ncsnf1KO, when compared with the wild-type strain. The results led us to conclude that, in N. crassa, this kinase promotes phosphorylation of glycogen synthase either directly or indirectly, which is the opposite of what is described for Saccharomyces cerevisiae. The kinases also play a role in gene expression regulation, in that gdn, the gene encoding the debranching enzyme, was down-regulated by the proteins identified in the screen. Some kinases affected growth and development, suggesting a connection linking glycogen metabolism with cell growth and development.

Ferric iron uptake genes are differentially expressed in the presence of copper sulfides in Acidithiobacillus ferrooxidans strain LR.

Acidithiobacillus ferrooxidans is one of the most widely used microorganisms in bioleaching operations to recover copper from low-grade copper sulfide ores. This work aimed to investigate the relative expression of genes related to the iron uptake system when A. ferrooxidans LR was maintained in contact with chalcopyrite or bornite as the sole energy source. Real-time quantitative PCR analysis revealed that the presence of bornite had no effect on the expression of seven genes related to the siderophore-mediated Fe(III) uptake system, while in the presence of chalcopyrite the expression of the genes was up-regulated. Bioinformatic analysis of the genomic region where these genes were found revealed the existence of three new putative DNA-binding sequences for the ferric iron uptake transcriptional regulator (Fur). Electrophoretic mobility shift assays demonstrated that a purified A. ferrooxidans His-tagged Fur protein was able to bind in vitro to each of these putative Fur boxes, suggesting that Fur regulated the expression of these genes. The expression of fur and two known Fur-regulated genes, mntH and dsrK, was also investigated in the presence of chalcopyrite. While the expression of fur and mntH was up-regulated, the expression of dsrK was down-regulated. The low amount of ferrous iron in the medium was probably responsible for the up-regulation of fur and the genes related to the siderophore-mediated Fe(III) uptake system when A. ferrooxidans LR was kept in the presence of chalcopyrite. A homology model of the A. ferrooxidans Fur was constructed and revealed that the putative DNA-binding surface presents conserved positively charged residues, supporting a previously suggested mode of interaction with DNA. The up-regulation of fur and the siderophore-mediated Fe(III) uptake genes, and the down-regulation of dsrK suggest that in the presence of chalcopyrite Fur acts as a transcription inducer and repressor.

Gene expression modulation by chalcopyrite and bornite in Acidithiobacillus ferrooxidans.

Acidithiobacillus ferrooxidans is a mesophilic, acidophilic, chemolithoautotrophic bacterium that obtains energy from the oxidation of ferrous iron (Fe2+), elemental sulfur and reduced sulfur compounds. The industrial interest in A. ferrooxidans resides in its capacity to oxidize insoluble metal sulfides into soluble metal sulfates, thus allowing the recovery of the desired metals from low-grade sulfide ores. In the present work, RNA arbitrarily primed PCR (RAP-PCR) was performed to identify cDNAs differentially expressed in A. ferrooxidans cells grown in the presence of Fe2+ and cells maintained for 24 h in the presence of the copper sulfides bornite and chalcopyrite. Eighteen cDNAs corresponding to genes with known function were identified, and their relative expression was further characterized by real-time quantitative PCR. Bornite had a mild effect on the expression of the 18 genes analyzed. None of these genes was down-regulated and among the few genes up-regulated, it is worth mentioning lepA and def-2 that are involved in protein synthesis. Chalcopyrite presented the most significant changes. Five genes related to protein processing were down-regulated, and another 5 genes related to the transport system were up-regulated. The up- and down-regulation of these genes in the presence of bornite and chalcopyrite could be due to alterations in the ideal pH, presence of copper ions in solution and nutrient limitation. The results suggest that gene expression modulation might be important for the A. ferrooxidans early response to copper sulfides.

THP-1 monocytes but not macrophages as a potential alternative for CD34+ dendritic cells to identify chemical skin sensitizers.

Early detection of the sensitizing potential of chemicals is an emerging issue for chemical, pharmaceutical and cosmetic industries. In our institute, an in vitro classification model for prediction of chemical-induced skin sensitization based on gene expression signatures in human CD34+ progenitor-derived dendritic cells (DC) has been developed. This primary cell model is able to closely mimic the induction phase of sensitization by Langerhans cells in the skin, but it has drawbacks, such as the availability of cord blood. The aim of this study was to investigate whether human in vitro cultured THP-1 monocytes or macrophages display a similar expression profile for 13 predictive gene markers previously identified in DC and whether they also possess a discriminating capacity towards skin sensitizers and non-sensitizers based on these marker genes. To this end, the cell models were exposed to 5 skin sensitizers (ammonium hexachloroplatinate IV, 1-chloro-2,4-dinitrobenzene, eugenol, para-phenylenediamine, and tetramethylthiuram disulfide) and 5 non-sensitizers (l-glutamic acid, methyl salicylate, sodium dodecyl sulfate, tributyltin chloride, and zinc sulfate) for 6, 10, and 24 h, and mRNA expression of the 13 genes was analyzed using real-time RT-PCR. The transcriptional response of 7 out of 13 genes in THP-1 monocytes was significantly correlated with DC, whereas only 2 out of 13 genes in THP-1 macrophages. After a cross-validation of a discriminant analysis of the gene expression profiles in the THP-1 monocytes, this cell model demonstrated to also have a capacity to distinguish skin sensitizers from non-sensitizers. However, the DC model was superior to the monocyte model for discrimination of (non-)sensitizing chemicals.

Aflatoxins and ochratoxin A in different cocoa clones (Theobroma cacao L.) developed in the southern region of Bahia, Brazil.

Brazil is the sixth largest producer of cocoa beans in the world, after Côte d'Ivoire, Ghana, Indonesia, Nigeria and Cameroon. The southern region of Bahia stands out as the country's largest producer, accounting for approximately 60% of production. Due to damage caused by infestation of the cocoa crop with the fungus Moniliophthora perniciosa, which causes 'witch's broom disease', research in cocoa beans has led to the cloning of species that are resistant to the disease; however, there is little information about the development of other fungal genera in these clones, such as Aspergillus, which do not represent a phytopathogenicity problem but can grow during the pre-processing of cocoa beans and produce mycotoxins. Thus, the aim of this work was to determine the presence of aflatoxin (AF) and ochratoxin A (OTA) in cocoa clones developed in Brazil. Aflatoxin and ochratoxin A contamination were determined in 130 samples from 13 cocoa clones grown in the south of Bahia by ultra-performance liquid chromatography with a fluorescence detector. The method was evaluated for limit of detection (LOD) (0.05-0.90 µg kg-1), limit of quantification (0.10-2.50 µg kg-1) and recovery (RSD) (89.40-95.80%) for AFB1, AFB2, AFG1, AFG2 and OTA. Aflatoxin contamination was detected in 38% of the samples in the range of

Eye darkening as a reliable, easy and inexpensive indicator of stress in fish.

We expand the use of eye darkening (ED) to indicate non-social stress in the fish Nile tilapia Oreochromis niloticus (L.). ED is easily estimated, not requiring any sophisticated equipment, and is non-invasive, facilitating the collection of several measures of stress over time. In the current study, we showed the following: (i) high- and low-ED occur spontaneously, indicating different fish reactions to adjustments to a novel environment; (ii) fish confinement or air exposure clearly increases ED (air exposure is a stronger stressor than confinement), and the time to restore basal values indicates the severity of the impact of the stressor on the fish (this response is not affected by period of the day, e.g., morning or afternoon); and (iii) in adults, females were more responsive (slower recovery) to 2-min air exposure than to 30-min confinement.

[Littré hernia as a cause of bowel obstruction]. / Hérnia de Littré causando obstrução intestinal.

The authors describe a case of a 60-year-old male with a history of a mass and pain at the right inguinal and epigastric areas. He also reported symptoms of bowel obstruction. Physical examination revealed a mass at right inguinal area, which was not reducible or pulsatile. Surgical findings included hernial sac contents with loop of ileum with signs of ischemia and a Meckel's diverticulum. Histopathological examination showed herniation of the Meckel diverticulum -Littré hernia. The article discusses the history and the incidence of this rare form of hernia.

Hérnia de Littré causando obstrução intestinal / Littré hernia bowel obstruction

The authors describe a case of a 60-year-old male with a history of a mass and pain at the right inguinal and epigastric areas. He also reported symptoms of bowel obstruction. Physical examination revealed a mass at right inguinal area, which was not reducible or pulsatile. Surgical findings included hernial sac contents with loop of ileum with signs of ischemia and a Meckel's diverticulum. Histopathological examination showed herniation of the Meckel diverticulum -Littré hernia. The article discusses the history and the incidence of this rare form of hernia.