Beta-amyloid peptides enhance the proliferative response of activated CD4CD28 lymphocytes from Alzheimer disease patients and from healthy elderly

Alzheimer's disease (AD) is the most frequent form of dementia among elderly. Despite the vast amount of literature on non-specific immune mechanisms in AD there is still little information about the potential antigen-specific immune response in this pathology. It is known that early stages of AD include β-amyloid (Aβ)- reactive antibodies production and inflammatory response. Despite some evidence gathered proving cellular immune response background in AD pathology, the specific reactions of CD4(+) and CD8(+) cells remain unknown as the previous investigations yielded conflicting results. Here we investigated the CD4(+)CD28(+) population of human peripheral blood T cells and showed that soluble β-amyloids alone were unable to stimulate these cells to proliferate significantly, resulting only in minor, probably antigen-specific, proliferative response. On the other hand, the exposure of in vitro pre-stimulated lymphocytes to soluble Aβ peptides significantly enhanced the proliferative response of these cells which had also lead to increased levels of TNF, IL-10 and IL-6. We also proved that Aβ peptide-enhanced proliferative response of CD4(+)CD28(+) cells is autonomous and independent from disease status while being associated with the initial, ex vivo activation status of the CD4(+) cells. In conclusion, we suggest that the effect of Aβ peptides on the immune system of AD patients does not depend on the specific reactivity to Aβ epitope(s), but is rather a consequence of an unspecific modulation of the cell cycle dynamics and cytokine production by T cells, occurring simultaneously in a huge proportion of Aβ peptide-exposed T lymphocytes and affecting the immune system performance.     

DAPK plays an important role in panobinostat-induced autophagy and commits cells to apoptosis under autophagy deficient conditions

The histone deacetylase inhibitor (HDACi) LBH589 has been verified as an effective anticancer agent. The identification and characterization of new targets for LBH589 action would further enhance our understanding of the molecular mechanisms involved in HDACi therapy. The role of the tumor suppressor death-associated protein kinase (DAPK) in LBH589-induced cytotoxicity has not been investigated to date. Stable DAPK knockdown (shRNA) and DAPK overexpressing (DAPK+++) cell lines were generated from HCT116 wildtype colon cancer cells. LBH589 inhibited cell proliferation, reduced the long-term survival, and up-regulated and activated DAPK in colorectal cancer cells. Moreover, LBH589 significantly suppressed the growth of colon tumor xenografts and in accordance with the in vitro studies, increased DAPK levels were detected immunohistochemically. LBH589 induced a DAPK-dependent autophagy as assessed by punctuate accumulation of LC3-II, the formation of acidic vesicular organelles, and degradation of p62 protein. LBH589-induced autophagy seems to be predominantly caused by DAPK protein interactions than by its kinase activity. Caspase inhibitor zVAD increased autophagosome formation, decreased the cleavage of caspase 3 and PARP but didn’t rescue the cells from LBH589-induced cell death in crystal violet staining suggesting both caspase-dependent as well as caspase-independent apoptosis pathways. Pre-treatment with the autophagy inhibitor Bafilomycin A1 caused caspase 3-mediated apoptosis in a DAPK-dependent manner. Altogether our data suggest that DAPK induces autophagy in response to HDACi-treatment. In autophagy deficient cells, DAPK plays an essential role in committing cells to HDACi-induced apoptosis.     

Genome comparison of barley and maize smut fungi reveals targeted loss of RNA silencing components and species-specific presence of transposable elements

Ustilago hordei is a biotrophic parasite of barley (Hordeum vulgare). After seedling infection, the fungus persists in the plant until head emergence when fungal spores develop and are released from sori formed at kernel positions. The 26.1-Mb U. hordei genome contains 7113 protein encoding genes with high synteny to the smaller genomes of the related, maize-infecting smut fungi Ustilago maydis and Sporisorium reilianum but has a larger repeat content that affected genome evolution at important loci, including mating-type and effector loci. The U. hordei genome encodes components involved in RNA interference and heterochromatin formation, normally involved in genome defense, that are lacking in the U. maydis genome due to clean excision events. These excision events were possibly a result of former presence of repetitive DNA and of an efficient homologous recombination system in U. maydis. We found evidence of repeat-induced point mutations in the genome of U. hordei, indicating that smut fungi use different strategies to counteract the deleterious effects of repetitive DNA. The complement of U. hordei effector genes is comparable to the other two smuts but reveals differences in family expansion and clustering. The availability of the genome sequence will facilitate the identification of genes responsible for virulence and evolution of smut fungi on their respective hosts.     

Implication of Glutathione in the In Vitro Antiplasmodial Mechanism of Action of Ellagic Acid

The search for new antimalarial chemotherapy has become increasingly urgent due to parasite resistance to current drugs. Ellagic acid (EA) is a polyphenol, recently found in various plant products, that has effective antimalarial activity in vitro and in vivo without toxicity. To further understand the antimalarial mechanism of action of EA in vitro, we evaluated the effects of EA, ascorbic acid and N-acetyl-L-cysteine (NAC), alone and/or in combination on the production of reactive oxygen species (ROS) during the trophozoite and schizonte stages of the erythrocytic cycle of P. falciparum. The parasitized erythrocytes were pre-labelled with DCFDA (dichlorofluorescein diacetate). We showed that NAC had no effect on ROS production, contrary to ascorbic acid and EA, which considerably reduced ROS production. Surprisingly, EA reduced the production of the ROS with concentrations (6.6×10⁻⁹ − 6.6×10⁻⁶ M) ten-fold lower than ascorbic acid (113×10⁻⁶ M). Additionally, the in vitro drug sensitivity of EA with antioxidants showed that antiplasmodial activity is independent of the ROS production inside parasites, which was confirmed by the additive activity of EA and desferrioxamine. Finally, EA could act by reducing the glutathione content inside the Plasmodium parasite. This was consolidated by the decrease in the antiplasmodial efficacy of EA in the murine model Plasmodium yoelii- high GSH strain, known for its high glutathione content. Given its low toxicity and now known mechanism of action, EA appears as a promising antiplasmodial compound.     

The Top 10 fungal pathogens in molecular plant pathology

The aim of this review was to survey all fungal pathologists with an association with the journal Molecular Plant Pathology and ask them to nominate which fungal pathogens they would place in a 'Top 10' based on scientific/economic importance. The survey generated 495 votes from the international community, and resulted in the generation of a Top 10 fungal plant pathogen list for Molecular Plant Pathology. The Top 10 list includes, in rank order, (1) Magnaporthe oryzae; (2) Botrytis cinerea; (3) Puccinia spp.; (4) Fusarium graminearum; (5) Fusarium oxysporum; (6) Blumeria graminis; (7) Mycosphaerella graminicola; (8) Colletotrichum spp.; (9) Ustilago maydis; (10) Melampsora lini, with honourable mentions for fungi just missing out on the Top 10, including Phakopsora pachyrhizi and Rhizoctonia solani. This article presents a short resumé of each fungus in the Top 10 list and its importance, with the intent of initiating discussion and debate amongst the plant mycology community, as well as laying down a bench-mark. It will be interesting to see in future years how perceptions change and what fungi will comprise any future Top 10.     

Multiplexed quantification of estrogen receptor and HER2/Neu in tissue and cell lysates by peptide immunoaffinity enrichment mass spectrometry

Access to a wider range of quantitative protein assays would significantly impact the number and use of tissue markers in guiding disease treatment. Quantitative mass spectrometry-based peptide and protein assays, such as immuno-SRM assays, have seen tremendous growth in recent years in application to protein quantification in biological fluids such as plasma or urine. Here, we extend the capability of the technique by demonstrating the application of a multiplexed immuno-SRM assay for quantification of estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) levels in cell line lysates and human surgical specimens. The performance of the assay was characterized using peptide response curves, with linear ranges covering approximately four orders of magnitude and limits of detection in the low fmol/mg lysate range. Reproducibility was acceptable with median coefficients of variation of approximately 10%. We applied the assay to measurements of ER and HER2 in well-characterized cell line lysates with good discernment based on ER/HER2 status. Finally, the proteins were measured in surgically resected breast cancers, and the results showed good correlation with ER/HER2 status determined by clinical assays. This is the first implementation of the peptide-based immuno-SRM assay technology in cell lysates and human surgical specimens.     

Molecular mechanisms of β-lactam resistance in Streptococcus pneumoniae

Alterations in the target enzymes for β-lactam antibiotics, the penicillin-binding proteins (PBPs), have been recognized as a major resistance mechanism in Streptococcus pneumoniae. Mutations in PBPs that confer a reduced affinity to β-lactams have been identified in laboratory mutants and clinical isolates, and document an astounding variability of sites involved in this phenotype. Whereas point mutations are selected in the laboratory, clinical isolates display a mosaic structure of the affected PBP genes, the result of interspecies gene transfer and recombination events. Depending on the selective β-lactam, different combinations of PBP genes and mutations within are involved in conferring resistance, and astoundingly in non-PBP genes as well.     

Penicillin-binding protein 2x of Streptococcus pneumoniae: three new mutational pathways for remodelling an essential enzyme into a resistance determinant

Mutations in the transpeptidase domain of penicillin-binding protein 2x (PBP2x) of Streptococcus pneumoniae that reduce the affinity to beta-lactams are important determinants of resistance to these antibiotics. We have now analyzed in vitro and in vivo properties of PBP2x variants from cefotaxime-resistant laboratory mutants and a clinical isolate. The patterns of two to four resistance-specific mutations present in each of the proteins, all of which are placed between 6.6 and 24 Å around the active site, fall into three categories according to their positions in the three-dimensional structure. The first PBP2x group is characterized by mutations at the end of helix α11 and carries the well-known T550A change and/or one mutation on the surface of the penicillin-binding domain in close contact with the C-terminal domain. All group I proteins display very low acylation efficiencies, ≤ 1700 M^− 1 s^− 1, for cefotaxime. The second class represented by PBP2x of the mutant C505 shows acylation efficiencies below 100 M^− 1 s^− 1 for both cefotaxime and benzylpenicillin and contains the mutation L403F at a critical site close to the active serine. PBP2x of the clinical isolate 669 reveals a third mutational pathway where at least the two mutations Q552E and S389L are important for resistance, and acylation efficiency is reduced for both beta-lactams to around 10,000 M^− 1 s^− 1. In each group, at least one mutation is located in close vicinity to the active site and mediates a resistance phenotype in vivo alone, whereas other mutations might exhibit secondary effects only in context with other alterations.