Hypoxia-induced autophagy drives colorectal cancer initiation and progression by activating the PRKC/PKC-EZR (ezrin) pathway.

In solid tumors, cancer stem cells (CSCs) or tumor-initiating cells (TICs) are often found in hypoxic niches. Nevertheless, the influence of hypoxia on TICs is poorly understood. Using previously established, TIC-enrichedpatient-derived colorectal cancer (CRC) cultures, we show that hypoxia increases the self-renewal capacity of TICs while inducing proliferation arrest in their more differentiated counterpart cultures. Gene expression data revealed macroautophagy/autophagy as one of the major pathways induced by hypoxia in TICs. Interestingly, hypoxia-induced autophagy was found to induce phosphorylation of EZR (ezrin) at Thr567 residue, which could be reversed by knocking down ATG5, BNIP3, BNIP3L, or BECN1. Furthermore, we identified PRKCA/PKCα as a potential kinase involved in hypoxia-induced autophagy-mediated TIC self-renewal. Genetic targeting of autophagy or pharmacological inhibition of PRKC/PKC and EZR resulted in decreased tumor-initiating potential of TICs. In addition, we observed significantly reduced in vivo tumor initiation and growth after a stable knockdown of ATG5. Analysis of human CRC samples showed that p-EZR is often present in TICs located in the hypoxic and autophagic regions of the tumor. Altogether, our results establish the hypoxia-autophagy-PKC-EZR signaling axis as a novel regulatory mechanism of TIC self-renewal and CRC progression. Autophagy inhibition might thus represent a promising therapeutic strategy for cancer patients. ABBREVIATIONS: ATG: autophagy related; BECN1: beclin 1; BNIP3: BCL2 interacting protein 3; BNIP3L: BCL2 interacting protein 3 like; CQ: chloroquine; CSC: cancer stem cells; CRC: colorectal cancer; HIF1A/HIF-1α: hypoxia inducible factor 1 subunit alpha; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PRKC/PKC: protein kinase C; SQSTM1/p62: sequestosome 1; TICs: tumor-initiating cells.

The Lmgpi15 gene, encoding a component of the glycosylphosphatidylinositol anchor biosynthesis pathway, is required for morphogenesis and pathogenicity in Leptosphaeria maculans.

Random insertional mutagenesis was used to investigate pathogenicity determinants in Leptosphaeria maculans. One tagged nonpathogenic mutant, termed m20, was analysed in detail here. The mutant phenotype was investigated by microscopic analyses of infected plant tissues and in vitro growth assays. Complementation and silencing experiments were used to identify the altered gene. Its function was determined by bioinformatics analyses, cell biology experiments and functional studies. The mutant was blocked at the invasive growth phase after an unaffected initial penetration stage, and displayed a reduced growth rate and an aberrant hyphal morphology in vitro. The T-DNA insertion occurred in the intergenic region between two head-to-tail genes, leading to a complex deregulation of their expression. The unique gene accounting for the mutant phenotype was suggested to be the orthologue of the poorly conserved Saccharomyces cerevisiae gpi15, which encodes for one component of the glycosylphosphatidylinositol (GPI) anchor biosynthesis pathway. Consistent with this predicted function, a functional translational fusion with the green fluorescent protein (GFP) was targeted to the endoplasmic reticulum. Moreover, the mutant exhibited an altered cell wall and addition of glucosamine relieved growth defects. It is concluded that the GPI anchor biosynthetic pathway is required for morphogenesis, cell wall integrity and pathogenicity in Leptosphaeria maculans.

Identification of an amino acid determinant of pH regiospecificity in a seed lipoxygenase from Momordica charantia.

Lipoxygenases (LOX) form a heterogeneous family of lipid peroxidizing enzymes, which catalyze specific dioxygenation of polyunsaturated fatty acids. According to their positional specificity of linoleic acid oxygenation plant LOX have been classified into linoleate 9- and linoleate 13-LOX and recent reports identified a critical valine at the active site of 9-LOX. In contrast, more bulky phenylalanine or histidine residues were found at this position in 13-LOX. We have recently cloned a LOX-isoform from Momordica charantia and multiple amino acid alignments indicated the existence of a glutamine (Gln599) at the position were 13-LOX usually carry histidine or phenylalanine residues. Analyzing the pH-dependence of the positional specificity of linoleic acid oxygenation we observed that at pH-values higher than 7.5 this enzyme constitutes a linoleate 13-LOX whereas at lower pH, 9-H(P)ODE was the major reaction product. Site-directed mutagenesis of glutamine 599 to histidine (Gln599His) converted the enzyme to a pure 13-LOX. These data confirm previous observation suggesting that reaction specificity of certain LOX-isoforms is not an absolute enzyme property but may be impacted by reaction conditions such as pH of the reaction mixture. We extended this concept by identifying glutamine 599 as sequence determinant for such pH-dependence of the reaction specificity. Although the biological relevance for this alteration switch remains to be investigated it is of particular interest that it occurs at near physiological conditions in the pH-range between 7 and 8.

Contribuições e principais intervenções da terapia cognitivo comportamental no tratamento do transtorno bipolar / Contributions and Main Interventions of Cognitive-Behavioral Therapy in the Treatment of Bipolar Disorder

Este trabalho tem como objetivo averiguar as contribuições da Terapia Cognitivo- Comportamental no tratamento do Transtorno Bipolar e focar em aspectos específicos das intervenções realizadas no tratamento. O trabalho consiste em uma revisão narrativa sobre este tema. A busca bibliográfica foi realizada através dos sites pubmed e google acadêmico. Os resultados mostraram que a Terapia Cognitivo-Comportamental auxilia na prevenção de recaídas, em episódios de humor mais curtos, menor número de hospitalizações e menor variabilidade dos sintomas maníacos. Dentre as principais técnicas utilizadas, a psicoeducação parece ser a mais utilizada e eficaz no tratamento do Transtorno Bipolar. No entanto, a farmacoterapia ainda é considerada a principal escolha no tratamento.

This study aims to investigate the contributions of Cognitive-Behavioral Therapy in the treatment of Bipolar Disorder and to focus on specific aspects of the interventions performed in the treatment. This papper consists of a narrative review on this theme. The bibliographic search was conducted through the pubmed and google academic sites. The results showed that Cognitive-Behavioral Therapy assists in the prevention of relapses, shorter episodes of mood, fewer hospitalizations and less variability of manic symptoms. Among the main techniques used, psychoeducation seems to be the most used and effective in the treatment of Bipolar Disorder. However, pharmacotherapy is still considered the main treatment choice.

Boilysin and thermolysin in dipeptide synthesis: a comparative study.

Boilysin (BLN) is an engineered, highly thermostable neutral protease from Bacillus stearothermophilus. Its high resistance is based on the stabilization of a surface loop (amino acid residues 55-69) by eight amino acid exchanges, including the introduction of a disulphide bond. In the present study, BLN was compared with the well-known and structurally related thermolysin (from B. thermoproteolyticus) with respect to their dipeptide- synthetic properties. The synthesis of the aspartame precursor N-(benzyloxycarbonyl)-l-aspartyl-l-phenylalanine methyl ester (Z-Asp-Phe-OMe) was used as the model reaction to study its enantioselectivity as well as the influence of neutral salt, temperature and calcium- ion concentration on peptide synthesis. The reactions were performed in homogeneous reaction systems containing DMSO or aliphatic alcohols. Furthermore, the substrate specificity in the synthesis of Z-Asp-X-OMe (X=Phe, d-Phe, Ala, Ile, Leu, Met, Tyr or Val) was examined. The two enzymes showed no difference in time course of reaction by the use of salts as activators or different alcohols as co-solvents. Both enzymes showed a high enantioselectivity towards the amino component in the reaction. They were strongly activated by NaCl, whereas the final product yields were strongly decreased at high NaCl concentrations. Aliphatic alcohols act as an inhibitor, but allow higher product yields compared with purely aqueous medium. Differences between the two enzymes are found at higher temperatures (>/=60 degrees C) in the absence or at low concentrations of Ca(2+) ions; BLN proved superior under these conditions, because its stability is less dependent on Ca(2+) ions. The substrate specificities of BLN and thermolysin at the P(')(1) position follow the same tendencies, with differences in the initial rates for the conversion of Z-Asp with Ile-OMe, Leu-OMe and Val-OMe.