Fungal communities in Brazilian cassava tubers and food products.

Cassava (Manihot esculenta Crantz) is one of the most widely cultivated foods in the world and is of great socio-economic importance, especially in developing countries. It is predominantly consumed in boiled form, but also is used to produce a number of products, including cassava starch, sour starch, cassava flour and tapioca flour (hydrated cassava starch). Fungal spoilage can occur throughout the production chain, impairing both productivity and quality, as well as posing a potential risk of contamination by mycotoxins. We used multidisciplinary approaches based on phenotypic and molecular data (ITS/BenA/TEF-1a/RPB2 loci) to investigate the mycobiota of 101 samples (including roots, soil and products) collected in the state of São Paulo, Brazil. A total of 20 fungal groups/genera were morphologically characterized, and 37 different species were molecularly identified. The predominant groups in cassava tubers were Fusarium spp., Penicillium spp. and Trichoderma spp. In cassava products, the most frequent groups were Penicillium spp. and Paecilomyces spp. Potentially toxigenic species were also found, including Paecilomyces saturatus, Penicillium citrinum, P. paneum, P. brevicompactum, P. chrysogenum, Fusarium foetens and Fusarium mundagurra. In soil-cultivated cassava samples, the groups found most frequently were Penicillium spp., Cladosporium spp. and Fusarium spp. Some of the species found in cassava tubers and/or product samples were also present in the soil, including F. mundagurra, Neocosmospora solani, P. citrinum and P. brevicompactum. In general, there was a higher occurrence of Penicillium spp., Fusarium spp. and Trichoderma spp., and the predominant species were F. fabacearum and P. citrinum. The mycobiota of Brazilian cassava proved to be extremely diverse, and the occurrence of several species in cassava tubers and/or products are reported herein for the first time. Potentially toxigenic species were found in cassava tubers, cassava products and soil, showing how important it is to constantly monitor these substrates.

Aspergillus section Flavi and aflatoxins in Brazilian cassava (Manihot esculenta Crantz) and products.

Aflatoxins are carcinogenic compounds produced by some species of Aspergillus, especially those belonging to Aspergillus section Flavi. Their occurrence in food may start in the field, in the post-harvest, or during storage due to inadequate handling and storage. Because cassava is a staple food for a high percentage of the Brazilian population, we evaluated the presence of aflatoxin-producing species in cassava tubers, cassava products (cassava flour, cassava starch, sour starch, and tapioca flour), and in soil samples collected from cassava fields. In addition, the levels of aflatoxin contamination in cassava products were quantified. A total of 101 samples were analyzed, and 45 strains of Aspergillus section Flavi were isolated. Among the identified species, Aspergillus flavus, Aspergillus arachidicola, Aspergillus novoparasiticus, and Aspergillus parasiticus were found. The majority of strains (73.3%) tested for their aflatoxin-producing ability in synthetic media was positive. Despite that, cassava and cassava products were essentially free of aflatoxins, and only one sample of cassava flour contained traces of AFB1 (0.35 µg/kg).

Potential of volatile compounds produced by fungi to influence sensory quality of coffee beverage.

Fungi are known producers of a large number of volatile compounds (VCs). Several VCs such as 2,4,6 trichloroanisole (TCA), geosmin and terpenes have been found in coffee beverages, and these compounds can be responsible for off-flavor development. However, few studies have related the fungal contamination of coffee with the sensory characteristics of the beverage. The aim of this research was to investigate the production of VCs by fungi isolated from coffee and their potential as modifiers of the sensory coffee beverage quality. Three species were isolated from coffee from the southwest of São Paulo state and selected for the study: Penicillium brevicompactum, Aspergillus luchuensis (belonging to section Nigri) and Penicillium sp. nov. (related to Penicillium crustosum). VCs produced by the fungal inoculated in raw coffee beans were extracted and tentatively identified by SPME-GC-MS. Different VCs that may interfere in the coffee beverage quality were detected in the raw coffee beans inoculated with these fungal species (mainly A. luchuensis). Oct-1-en-3-ol was detected in the raw coffee inoculated with A. luchuensis. This compound, which is characterized by earthy and moldy/mushroom aroma, can be related to negative characteristics of coffee beverage in sensory analysis. On the other hand, the presence of some fungal species in the coffee, even at a high percentage of infection, did not necessarily result in loss of the sensorial quality of the beverage, since the samples with a high infection of P. brevicompactum showed positive sensory evaluation.

Multifaceted mechanisms for cell survival and drug targeting in chronic myelogenous leukemia.

Treatment outcomes for chronic myelogenous leukemia (CML) have shown major improvements as a result of the development of the tyrosine kinase inhibitors (TKIs) imatinib, nilotinib and dasatinib for the disease-specific molecular target BCR-ABL1 tyrosine kinase (TK), but a cure of CML by BCR-ABL1 TKIs has been rarely achieved. CML cells are protected from cytotoxic insults, including those by TKIs, through various collaborative BCR-ABL1- mediated and -independent mechanisms, as well as cell-intrinsic and -extrinsic molecular mechanisms. These protective mechanisms include overlapping cell signaling pathways for normal hematopoietic proliferation, modulation of molecules associated with the BCL2 family protein-regulated programmed cell death pathway, autophagic cell protection capability, bone marrow environment-mediated cell protective signaling, abnormally upregulated genetic instability and other BCR-ABL1- independent kinase activities. To develop a more effective treatment strategy for a cure by means of total leukemic cell killing, a thorough understanding of how CML cells survive and resist cytotoxic insults is essential. In this article, we review current knowledge about multifaceted BCR-ABL1-related and -unrelated mechanisms for survival and death of CML cells and present suggestions for the development of new therapeutic strategies for complete elimination of residual CML cells during TKI treatment.

Cytogenetic and molecular abnormalities in myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal hematological disorders characterized by ineffective hematopoiesis which causes peripheral cytopenias and a risk of progression to acute myeloid leukemia. Although various forms of chromosomal abnormalities have been detected in approximately 50-60% of patients with de novo MDS and in up to 80% of patients with therapy-related MDS, their molecular significance for pathogenesis and disease progression is not yet fully understood. Recent technical advances in molecular biology have disclosed more accurately details of pathological chromosomal and molecular aberrations in MDS. Such details could not be identified with conventional cytogenetical techniques, including G-banding. In particular, with recent technical advances in comparative genome hybridization or single nucleotide polymorphism array technology, several candidate genes for the pathogenesis of MDS have been identified, which are located in minimally deleted or uniparental disomy segments. Moreover, epigenetic deregulation of gene expression is also likely to be involved in the pathogenesis of MDS. Accordingly, in addition to classical oncogenic abnormalities, such as p53 abnormalities, or NRAS mutation, various molecular abnormalities, such as TET2, RPS14, or c-CBL, have been identified and/or proposed as the novel candidates for molecular basis of the development and progression of MDS. A better understanding of the causative molecular events underlying MDS pathogenesis is essential for the development and establishment of a more effective treatment resulting in a complete cure for MDS. We here review current knowledge regarding the molecular significance of chromosomal and genetic aberrations in MDS and the proposed molecular mechanisms of action of new agents for MDS, such as lenalidomide or azacitidine.

Eµ/miR-125b transgenic mice develop lethal B-cell malignancies.

MicroRNA-125b-1 (miR-125b-1) is a target of a chromosomal translocation t(11;14)(q24;q32) recurrently found in human B-cell precursor acute lymphoblastic leukemia (BCP-ALL). This translocation results in overexpression of miR-125b controlled by immunoglobulin heavy chain gene (IGH) regulatory elements. In addition, we found that six out of twenty-one BCP-ALL patients without t(11;14)(q24;q32) showed overexpression of miR-125b. Interestingly, four out of nine patients with BCR/ABL-positive BCP-ALL and one patient with B-cell lymphoid crisis that had progressed from chronic myelogenous leukemia overexpressed miR-125b. To examine the role of the deregulated expression of miR-125b in the development of B-cell tumor in vivo, we generated transgenic mice mimicking the t(11;14)(q24;q32) (Eµ/miR-125b-TG mice). Eµ/miR-125b-TG mice overexpressed miR-125b driven by IGH enhancer and promoter and developed IgM-negative or IgM-positive lethal B-cell malignancies with clonal proliferation. B cells obtained from the Eµ/miR-125b-TG mice were resistant to apoptosis induced by serum starvation. We identified Trp53inp1, a pro-apoptotic gene induced by cell stress, as a novel target gene of miR-125b in hematopoietic cells in vitro and in vivo. Our results provide direct evidence that miR-125b has important roles in the tumorigenesis of precursor B cells.

AV-65, a novel Wnt/ß-catenin signal inhibitor, successfully suppresses progression of multiple myeloma in a mouse model.

Multiple myeloma (MM) is a malignant neoplasm of plasma cells. Although new molecular targeting agents against MM have been developed based on the better understanding of the underlying pathogenesis, MM still remains an incurable disease. We previously demonstrated that ß-catenin, a downstream effector in the Wnt pathway, is a potential target in MM using RNA interference in an in vivo experimental mouse model. In this study, we have screened a library of more than 100 000 small-molecule chemical compounds for novel Wnt/ß-catenin signaling inhibitors using a high-throughput transcriptional screening technology. We identified AV-65, which diminished ß-catenin protein levels and T-cell factor transcriptional activity. AV-65 then decreased c-myc, cyclin D1 and survivin expression, resulting in the inhibition of MM cell proliferation through the apoptotic pathway. AV-65 treatment prolonged the survival of MM-bearing mice. These findings indicate that this compound represents a novel and attractive therapeutic agent against MM. This study also illustrates the potential of high-throughput transcriptional screening to identify candidates for anticancer drug discovery.

Growth and mycotoxin production by fungi in atmospheres containing 80% carbon dioxide and 20% oxygen.

The effect of atmosphere containing 80% CO(2) and 20% O(2) on growth of Mucor plumbeus, Fusarium oxysporum, Byssochlamys fulva, Byssochlamys nivea, Penicillium commune, Penicillium roqueforti, Aspergillus flavus, Eurotium chevalieri and Xeromyces bisporus was investigated. Production of aflatoxin by A. flavus, patulin by B. nivea, roquefortine C by P. roqueforti, and cyclopiazonic acid by P. commune was also studied. Fungal growth was evaluated by three methods: colony diameter, hyphal length or mycelium dry weight and ergosterol content. Among the nine fungal species examined, two E. chevalieri and X. bisporus, did not grow under these conditions. In this study, fungi differed in their response to modified atmospheres in biomass, ergosterol content, mycotoxin production and morphology. Reductions of 57.8-96.9%, 73.7-99.6% and 91.5-99.9% were obtained in colony diameter, hyphal length and ergosterol content, respectively, under this atmosphere compared to air. Ergosterol content was more affected in most species than other measurements. Patulin, cyclopiazonic acid and roquefortine C were produced in this atmosphere, although levels were very low and aflatoxin was not produced at all. Growth was quite extensive as measured by colony diameters, but hyphal lengths were low and ergosterol production was also affected in all species of this study.

Drug susceptibility and clonality of methicillin-resistant Staphylococcus epidermidis in hospitalized patients with hematological malignancies.

PURPOSE: To evaluate the clonal relatedness and drug susceptibility of Streptococcus epidermidis isolated from hematological patients. METHODS: All S. epidermidis isolated from hematological patients who developed bloodstream infections between June 2005 and December 2007 were included. The clonal relationship was tested by means of pulsed-field gel electrophoresis (PFGE) analysis. RESULTS: Fifteen methicillin-resistant S. epidermidis (MRSE) isolates were examined from patients' blood culture samples. Two subgroups that differed approximately by 40% in their PFGE banding were identified. In clinical practice, two cases were cured with cephalosporin only, thus demonstrating sensitivity of the strains to beta-lactam antibiotics. CONCLUSIONS: Our results represent two significant findings. One is the major capability of MRSE to colonize patients. The other is that some MRSE isolates proved to be sensitive to clindamycin, minocycline, and cephalosporin, so that using antibiotics to which MRSE is sensitive as first-line therapy can avoid the need for vancomycin in clinical settings.

Galectin-9 exhibits anti-myeloma activity through JNK and p38 MAP kinase pathways.

Galectins constitute a family of lectins that specifically exhibit the affinity for beta-galactosides and modulate various biological events. Galectin-9 is a tandem-repeat type galectin with two carbohydrate recognition domains and has recently been shown to have an anti-proliferative effect on cancer cells. We investigated the effect of recombinant protease-resistant galectin-9 (hGal9) on multiple myeloma (MM). In vitro, hGal9 inhibited the cell proliferation of five myeloma cell lines examined, including a bortezomib-resistant subcell line, with IC(50) between 75.1 and 280.0 nM, and this effect was mediated by the induction of apoptosis with the activation of caspase-8, -9, and -3. hGal9-activated Jun NH(2)-terminal kinase (JNK) and p38 MAPK signaling pathways followed by H2AX phosphorylation. Importantly, the inhibition of either JNK or p38 MAPK partly inhibited the anti-proliferative effect of hGal9, indicating the crucial role of these pathways in the anti-MM effect of hGal9. hGal9 also induced cell death in patient-derived myeloma cells, some with poor-risk factors, such as chromosomal deletion of 13q or translocation t(4;14)(p16;q32). Finally, hGal9 potently inhibited the growth of human myeloma cells xenografted in nude mice. These suggest that hGal9 is a new therapeutic target for MM that may overcome resistance to conventional chemotherapy.

Modified ELISPOT assay may predict T-cell hyporesponsiveness to non-inherited maternal antigens.

Clinical reports have suggested the existence of immunological tolerance to noninherited maternal antigens (NIMA) in human leukocyte antigen (HLA) mismatched allogeneic stem cell transplantation (allo-SCT). We studied the T-cell reactivity using IFN-gamma enzyme-linked immunospot (ELISPOT) assay in three HLA fully matched allo-SCT cases and one healthy volunteer family case. In HLA fully matched allo-SCT cases, ELISPOT assay could detect the hyporesponsiveness of T cells from donors to the B cells from recipients. Moreover, ELISPOT assay showed that the T cells from an individual responded to B cell from his mother significantly weakly than those from an unrelated HLA-haploidentical individual. These observations suggest that our IFN-gamma ELISPOT assay-based method may predict the presence of immunological tolerance to NIMA.

Defective expression of polarity protein PAR-3 gene (PARD3) in esophageal squamous cell carcinoma.

The partition-defective 3 (PAR-3) protein is implicated in the formation of tight junctions at epithelial cell-cell contacts. We investigated DNA copy number aberrations in human esophageal squamous cell carcinoma (ESCC) cell lines using a high-density oligonucleotide microarray and found a homozygous deletion of PARD3 (the gene encoding PAR-3). Exogenous expression of PARD3 in ESCC cells lacking this gene enhanced the recruitment of zonula occludens 1 (ZO-1), a marker of tight junctions, to sites of cell-cell contact. Conversely, knockdown of PARD3 in ESCC cells expressing this gene caused a disruption of ZO-1 localization at cell-cell borders. A copy number loss of PARD3 was observed in 15% of primary ESCC cells. Expression of PARD3 was significantly reduced in primary ESCC tumors compared with their nontumorous counterparts, and this reduced expression was associated with positive lymph node metastasis and poor differentiation. Our results suggest that deletion and reduced expression of PARD3 may be a novel mechanism that drives the progression of ESCC.