Comparison between the univariate and multivariate analysis on the partial characterization of the endoglucanase produced in the solid state fermentation by Aspergillus oryzae ATCC 10124

Endoglucanase production by Aspergillus oryzae ATCC 10124 cultivated in rice husks or peanut shells was optimized by experimental design as a function of humidity, time, and temperature. The optimum temperature for the endoglucanase activity was estimated by a univariate analysis (one factor at the time) as 50°C (rice husks) and 60°C (peanut shells), however, by a multivariate analysis (synergism of factors), it was determined a different temperature (56°C) for endoglucanase from peanut shells. For the optimum pH, values determined by univariate and multivariate analysis were 5 and 5.2 (rice husk) and 5 and 7.6 (peanut shells). In addition, the best half-lives were observed at 50°C as 22.8?hr (rice husks) and 7.3?hr (peanut shells), also, 80% of residual activities was obtained between 30 and 50°C for both substrates, and the pH stability was improved at 5–7 (rice hulls) and 6–9 (peanut shells). Both endoglucanases obtained presented different characteristics as a result of the versatility of fungi in different substrates.     

Conservation implications of genetic structure in the narrowest endemic quillwort from the Eastern Amazon

The quillwort Isoëtes cangae is a critically endangered species occurring in a single lake in Serra dos Carajás, Eastern Amazon. Low genetic diversity and small effective population sizes (N _e) are expected for narrow endemic species (NES). Conservation biology studies centered in a single species show some limitations, but they are still useful considering the limited time and resources available for protection of species at risk of extinction. Here, we evaluated the genetic diversity, population structure, N _e, and minimum viable population (MVP) of I. cangae to provide information for effective conservation programs. Our analyses were based on 55 individuals collected from the Amendoim Lake and 35,638 neutral SNPs. Our results indicated a single panmictic population, moderate levels of genetic diversity, and N _e in the order of thousands, contrasting the expected for NES. Negative F_IS values were also found, suggesting that I. cangae is not under risk of inbreeding depression. Our findings imply that I. cangae contains enough genetic diversity to ensure evolutionary potential and that all individuals should be treated as one demographic unit. These results provide essential information to optimize ex situ conservation efforts and genetic diversity monitoring, which are currently applied to guide I. cangae conservation plans.     

Leishmania infantum: Lipophosphoglycan intraspecific variation and interaction with vertebrate and invertebrate hosts

Interspecies variations in lipophosphoglycan (LPG) have been the focus of intense study over the years due its role in specificity during sand fly-Leishmania interaction. This cell surface glycoconjugate is highly polymorphic among species with variations in sugars that branch off the conserved Gal(β1,4)Man(α1)-PO(4) backbone of repeat units. However, the degree of intraspecies polymorphism in LPG of Leishmania infantum (syn. Leishmania chagasi) is not known. In this study, intraspecific variation in the repeat units of LPG was evaluated in 16 strains of L. infantum from Brazil, France, Algeria and Tunisia. The structural polymorphism in the L. infantum LPG repeat units was relatively slight and consisted of three types: type I does not have side chains; type II has one β-glucose residue that branches off the disaccharide-phosphate repeat units and type III has up to three glucose residues (oligo-glucosylated). The significance of these modifications was investigated during in vivo interaction of L. infantum with Lutzomyia longipalpis, and in vitro interaction of the parasites and respective LPGs with murine macrophages. There were no consequential differences in the parasite densities in sand fly midguts infected with Leishmania strains exhibiting type I, II and III LPGs. However, higher nitric oxide production was observed in macrophages exposed to glucosylated type II LPG.     

The effects of hypercaloric diets on glucose homeostasis in the rat: influence of saturated and monounsaturated dietary lipids

Consumption of energy-dense/high-fat diets is strongly and positively associated with overweight and obesity, which are associated with increase in the prevalence of certain chronic diseases. We evaluated the effect of hypercaloric/fat or normocaloric diets on some biochemical parameters in rats. Seventy-two rats were divided into four groups that were fed for 16 weeks with diets: normocaloric [9.12% soy oil, normocaloric soy oil (NSO)], hypercaloric olive oil [43.8% olive oil, hypercaloric olive oil (HOO)], hypercaloric saturated fat [43.8% saturated fat, hypercaloric saturated fat (HSF)] and normocaloric saturated fat [43.8% saturated fat, normocaloric saturated fat (NSF)]. HSF rats consumed more calories daily than the others and gained more retroperitoneal fat, although HSF and HOO rats had higher body weight. In liver, glycogen synthesis and concentration were higher in rats HSF and NSF. In plasma, total cholesterol (TC) levels were higher in HSF rats than in the others, and triacylglycerol (TAG) levels were lower in HOO and higher in HSF rats in relation to the others. In liver, TC and TAG were elevated in HSF, NSF and HOO rats. Paraoxonase 1 activity, which is related to high-density lipoprotein cholesterol and has anti-atherogenic role was lower in rats HSF. In HOO rats, glucose tolerance test was altered, but insulin tolerance test was normal. These results suggest that consumption of energy-dense/high-fat diets, both saturated or monounsaturated, causes damaging effects. However, more studies are necessary to understand the mechanisms by which these diets cause the metabolic alterations observed.     

Use of in vivo and in vitro systems to select Leishmania amazonensis expressing green fluorescent protein

Various Leishmania species were engineered with green fluorescent protein (GFP) using episomal vectors that encoded an antibiotic resistance gene, such as aminoglycoside geneticin sulphate (G418). Most reports of GFP-Leishmania have used the flagellated extracellular promastigote, the stage of parasite detected in the midgut of the sandfly vector; fewer studies have been performed with amastigotes, the stage of parasite detected in mammals. In this study, comparisons were made regarding the efficiency for in vitro G418 selection of GFP-Leishmania amazonensis promastigotes and amastigotes and the use of in vivo G418 selection. The GFP-promastigotes retained episomal plasmid for a prolonged period and G418 treatment was necessary and efficient for in vitro selection. In contrast, GFP-amastigotes showed low retention of the episomal plasmid in the absence of G418 selection and low sensitivity to antibiotics in vitro. The use of protocols for G418 selection using infected BALB/c mice also indicated low sensitivity to antibiotics against amastigotes in cutaneous lesions.     

Estrogen receptor alpha (ERS1) SNPs c454-397T>C (PvuII) and c454-351A>G (XbaI) are risk biomarkers for breast cancer development

There are several risk factors related to Breast Cancer (BC) risks and response to chemotherapy with SERMs. Recently some single nucleotide polymorphisms (SNPs) on ESR1 gene have been associated to this disease. However, data are still inconclusive. The present study aimed to investigate the association of SNPs c454-397T>C (also called PvuII) and c454-351A>G (so called XbaI) to incidence of sporadic BC; ERα expression in BC; tamoxifen hormonetherapy (HT-TMX) responsiveness. To do so, a cohort of BC patients was analyzed through retrospective data collection, immunohistochemistry to ERα protein, and genotyping for PvuII and XbaI SNPs by PCR–RFLP, confirmed by sequencing. Significant difference in PvuII alleles frequencies were found BC patients when compared to control samples. Patients with P allele have a 5.14-fold increased BC risk. We found higher P and X alleles frequencies in ERα positive BC and the pp and xx genotypes were observed exclusively in patients with HT-TMX-responsive BC. Taken together, data indicates that P allele as a novel sporadic BC biomarker whereas p and x alleles enhanced chemotherapy responsiveness.     

GALNT11 as a new molecular marker in chronic lymphocytic leukemia

Aberrant mucin O-glycosylation often occurs in different cancers and is characterized by immature expression of simple mucin-type carbohydrates. At present, there are some controversial reports about the Tn antigen (GalNAcα-O-Ser/Thr) expression and there is a great lack of information about the [UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase (GalNAc-Ts)] expression in chronic lymphocytic leukemia (CLL). To gain insight in these issues we evaluated the Tn antigen expression in CLL patient samples using two Tn binding proteins with different fine specificity. We also studied the expression from 14 GalNAc-Ts genes in CLL patients by RT-PCR. Our results have provided additional information about the expression level of the Tn antigen, suggesting that a low density of Tn residues is expressed in CLL cells. We also found that GALNT11 was expressed in CLL cells and normal T cell whereas little or no expression was found in normal B cells. Based on these results, GALNT11 expression was assessed by qPCR in a cohort of 50 CLL patients. We found significant over-expression of GALNT11 in 96% of B–CLL cells when compared to normal B cells. Moreover, we confirmed the expression of this enzyme at the protein level. Finally we found that GALNT11 expression was significantly associated with the mutational status of the immunoglobulin heavy chain variable region (IGHV), [?²(1) = 18.26; P < 0.0001], lipoprotein lipase expression [?²(1) = 13.72; P = 0.0002] and disease prognosis [?²(1) = 15.49; P < 0.0001]. Our evidence suggests that CLL patient samples harbor aberrant O-glycosylation highlighted by Tn antigen expression and that the over-expression of GALNT11 constitutes a new molecular marker for CLL.     

Isocitrate dehydrogenase 1 R132C mutation occurs exclusively in microsatellite stable colorectal cancers with the CpG island methylator phenotype

The CpG Island Methylator Phenotype (CIMP) is fundamental to an important subset of colorectal cancer; however, its cause is unknown. CIMP is associated with microsatellite instability but is also found in BRAF mutant microsatellite stable cancers that are associated with poor prognosis. The isocitrate dehydrogenase 1 (IDH1) gene causes CIMP in glioma due to an activating mutation that produces the 2-hydroxyglutarate oncometabolite. We therefore examined IDH1 alteration as a potential cause of CIMP in colorectal cancer. The IDH1 mutational hotspot was screened in 86 CIMP-positive and 80 CIMP-negative cancers. The entire coding sequence was examined in 81 CIMP-positive colorectal cancers. Forty-seven cancers varying by CIMP-status and IDH1 mutation status were examined using Illumina 450K DNA methylation microarrays. The R132C IDH1 mutation was detected in 4/166 cancers. All IDH1 mutations were in CIMP cancers that were BRAF mutant and microsatellite stable (4/45, 8.9%). Unsupervised hierarchical cluster analysis identified an IDH1 mutation-like methylation signature in approximately half of the CIMP-positive cancers. IDH1 mutation appears to cause CIMP in a small proportion of BRAF mutant, microsatellite stable colorectal cancers. This study provides a precedent that a single gene mutation may cause CIMP in colorectal cancer, and that this will be associated with a specific epigenetic signature and clinicopathological features.