The structures of polysaccharides and glycolipids of Aspergillus fumigatus grown in the presence of human serum

A study was made of polysaccharides and glycosphingolipids isolated from Aspergillus fumigatus grown in media supplemented with human serum from healthy donors. Fractionation of Cetavlon-precipitated polysaccharides on Sephacryl S-400 gave rise to an excluded fraction (Fraction I) with molecular weight of >400 kDa and an included peak (Fraction II) with an average molecular weight of 30–80 kDa. Fraction I comprises about 5% of total polysaccharide and was identified as a glycogen-like molecule. Its structure was deduced from methylation data, treatment with amyloglucosidase, a red-brown coloration produced with an iodine solution and by ¹H and ¹³C-NMR spectroscopy. It was previously suggested that higher amounts of glycogen-like polysaccharide (20%) were present in A. fumigatus grown in serum-free medium. Fraction II was identified as a galactomannan and was the main polysaccharide of A. fumigatus grown in serum-supplemented medium. Its structure was elucidated mainly by ¹³C-NMR spectroscopy combined with partial acetolysis and methylation analysis. The ¹³C-NMR spectrum of the galactomannan showed a much greater complexity in the -d-galf and -d-manp C-1 regions, than was evident for galactomannan from serum-free cultures previously described, reflecting differences in the glycosylation pattern, stimulated in serum-supplemented medium.No differences in A. fumigatus glycosphingolipid could be detected between serum-containing and serum-free growth conditions.Our results demonstrate that the change in polysaccharide structure is a more specific response to the altered growth conditions and not merely a symptom of more general changes.This revised version was published online in October 2005 with corrections to the Cover Date.     

High-efficiency cryopreservation of Araucaria angustifolia (Bertol.) Kuntze embryogenic cultures: ultrastructural characterization and morpho-physiological features

Here we evaluated and characterized the growth dynamics of A. angustifolia embryogenic cultures (EC) submitted to different cryotreatment incubation times through morphological and time-lapse cell tracking analyzes. The EC submitted to cryopreservation protocol were evaluated by regrowth rates, and ultrastructural characterization by transmission electron microscopy (TEM). The results indicated that A. angustifolia EC support all the cryoprotection times evaluated, without cell proliferation inhibition, but with noticeable genotype-dependent response in all tested cell lines. The use of 1M DMSO showed non-inhibitory effects to EC regrowth independent of cell line or cryotreatment incubation time. However, after cryopreservation, Cr01 cell line regrowth was 100 % for all cryotreatments incubation times evaluated (30, 60, 120, 240 min), while Cr02 cell line only showed 100 % regrowth in 240 min of cryotreatment. The 100 % cell regrowth obtained in both cell lines indicates that the proposed protocol can be successful applied to A. angustifolia EC cryopreservation. Cell tracking analysis showed a survival and initial proliferation of embryogenic cells, with the first cell regrowth signs after 30 days in culture. TEM analysis revealed a conspicuous cell wall thickening in embryogenic cells after cryotreatment and after thawing, which may be related to osmotic stress response caused by the cryopreservation process. An increased heterochromatin presence was also observed in cryotreated or after thawing cells, may possibly be acting as a cell defense mechanism, decreasing the DNA vulnerability to cleavage and preserving the cell integrity.

     

Identification of stably expressed reference small non‐coding RNAs for microRNA quantification in high‐grade serous ovarian carcinoma tissues

MicroRNAs (miRNAs) belong to a family of small non‐coding RNAs (sncRNAs) playing important roles in human carcinogenesis. Multiple investigations reported miRNAs aberrantly expressed in several cancers, including high‐grade serous ovarian carcinoma (HGS‐OvCa). Quantitative PCR is widely used in studies investigating miRNA expression and the identification of reliable endogenous controls is crucial for proper data normalization. In this study, we aimed to experimentally identify the most stable reference sncRNAs for normalization of miRNA qPCR expression data in HGS‐OvCa. Eleven putative reference sncRNAs for normalization (U6, SNORD48, miR‐92a‐3p, let‐7a‐5p, SNORD61, SNORD72, SNORD68, miR‐103a‐3p, miR‐423‐3p, miR‐191‐5p, miR‐16‐5p) were analysed on a total of 75 HGS‐OvCa and 30 normal tissues, using a highly specific qPCR. Both the normal tissues considered to initiate HGS‐OvCa malignant transformation, namely ovary and fallopian tube epithelia, were included in our study. Stability of candidate endogenous controls was evaluated using an equivalence test and validated by geNorm and NormFinder algorithms. Combining results from the three different statistical approaches, SNORD48 emerged as stably and equivalently expressed between malignant and normal tissues. Among malignant samples, considering groups based on residual tumour, miR‐191‐5p was identified as the most equivalent sncRNA. On the basis of our results, we support the use of SNORD48 as best reference sncRNA for relative quantification in miRNA expression studies between HGS‐OvCa and normal controls, including the first time both the normal tissues supposed to be HGS‐OvCa progenitors. In addition, we recommend miR‐191‐5p as best reference sncRNA in miRNA expression studies with prognostic intent on HGS‐OvCa tissues.     

CDK1 Is a Synthetic Lethal Target for KRAS Mutant Tumours

Activating KRAS mutations are found in approximately 20% of human cancers but no RAS-directed therapies are currently available. Here we describe a novel, robust, KRAS synthetic lethal interaction with the cyclin dependent kinase, CDK1. This was discovered using parallel siRNA screens in KRAS mutant and wild type colorectal isogenic tumour cells and subsequently validated in a genetically diverse panel of 26 colorectal and pancreatic tumour cell models. This established that the KRAS/CDK1 synthetic lethality applies in tumour cells with either amino acid position 12 (p.G12V, pG12D, p.G12S) or amino acid position 13 (p.G13D) KRAS mutations and can also be replicated in vivo in a xenograft model using a small molecule CDK1 inhibitor. Mechanistically, CDK1 inhibition caused a reduction in the S-phase fraction of KRAS mutant cells, an effect also characterised by modulation of Rb, a master control of the G₁/S checkpoint. Taken together, these observations suggest that the KRAS/CDK1 interaction is a robust synthetic lethal effect worthy of further investigation.     

Population Genetics of Plasmodium vivax in the Peruvian Amazon

Background

Characterizing the parasite dynamics and population structure provides useful information to understand the dynamic of transmission and to better target control interventions. Despite considerable efforts for its control, vivax malaria remains a major health problem in Peru. In this study, we have explored the population genetics of Plasmodium vivax isolates from Iquitos, the main city in the Peruvian Amazon, and 25 neighbouring peri-urban as well as rural villages along the Iquitos-Nauta Road.

Methodology/ Results

From April to December 2008, 292 P. vivax isolates were collected and successfully genotyped using 14 neutral microsatellites. Analysis of the molecular data revealed a similar proportion of monoclonal and polyclonal infections in urban areas, while in rural areas monoclonal infections were predominant (p = 0.002). Multiplicity of infection was higher in urban (MOI = 1.5–2) compared to rural areas (MOI = 1) (p = 0.003). The level of genetic diversity was similar in all areas (He = 0.66–0.76, p = 0.32) though genetic differentiation between areas was substantial (PHI_PT = 0.17, p<0.0001). Principal coordinate analysis showed a marked differentiation between parasites from urban and rural areas. Linkage disequilibrium was detected in all the areas (IAs = 0.08–0.49, for all p<0.0001). Gene flow among the areas was stablished through Bayesian analysis of migration models. Recent bottleneck events were detected in 4 areas and a recent parasite expansion in one of the isolated areas. In total, 87 unique haplotypes grouped in 2 or 3 genetic clusters described a sub-structured parasite population.

Conclusion/Significance

Our study shows a sub-structured parasite population with clonal propagation, with most of its components recently affected by bottleneck events. Iquitos city is the main source of parasite spreading for all the peripheral study areas. The routes of transmission and gene flow and the reduction of the parasite population described are important from the public health perspective as well for the formulation of future control policies.     

Culture of Risk in Vulnerable Communities: The Case of Barranquilla,Colombia, in the Context of Globalized (In)Security

This article addresses both the culture of risk as an analytical and intervention category in vulnerable communities. The main objective is to show that risk analysis and intervention is only viable when the cultural values of the communities that face a particular kind of vulnerability are taken into account. We present the results from a study of a representative sample of vulnerable communities in Barranquilla, Colombia, in which various techniques of data collection were applied to identify the perception of risk and its relationship with the cultural typologies developed by Cultural Theory. We explain the causes and effects of perception and response when faced with risk in different contexts.     

A fungal glycoprotein mitigates passion fruit woodiness disease caused by Cowpea aphid-borne mosaic virus (CABMV) in Passiflora edulis

BioControl - Passion fruit woodiness disease is responsible for severe losses in passion fruit production around the world. The disease is caused by Cowpea aphid-borne mosaic virus (CABMV), an...