Synchrotron x-ray microdiffraction reveals intrinsic structural features of amyloid deposits in situ

Amyloidoses are increasingly recognized as a major public health concern in Western countries. All amyloidoses share common morphological, structural, and tinctorial properties. These consist of staining by specific dyes, a fibrillar aspect in electron microscopy and a typical cross-β folding in x-ray diffraction patterns. Most studies that aim at deciphering the amyloid structure rely on fibers generated in vitro or extracted from tissues using protocols that may modify their intrinsic structure. Therefore, the fine details of the in situ architecture of the deposits remain unknown. Here, we present to our knowledge the first data obtained on ex vivo human renal tissue sections using x-ray microdiffraction. The typical cross-β features from fixed paraffin-embedded samples are similar to those formed in vitro or extracted from tissues. Moreover, the fiber orientation maps obtained across glomerular sections reveal an intrinsic texture that is correlated with the glomerulus morphology. These results are of the highest importance to understanding the formation of amyloid deposits and are thus expected to trigger new incentives for tissue investigation. Moreover, the access to intrinsic structural parameters such as fiber size and orientation using synchrotron x-ray microdiffraction, could provide valuable information concerning in situ mechanisms and deposit formation with potential benefits for diagnostic and therapeutic purposes.     

Mutually exclusive distribution of the sap and eag S-layer genes and the lytB/lytA cell wall hydrolase genes in Bacillus thuringiensis

Recently, two Bacillus thuringiensis strains were reported to synthesize parasporal inclusion bodies made not of the expected crystal (Cry) proteins but rather of the surface layer proteins (SLP) Sap (encoded by sap) and EA1 (encoded by eag), respectively. Whether the presence of the sap and eag genes is restricted to these two B. thuringiensis strains or ubiquitous in B. thuringiensis is unknown. We report here the distribution of the sap and eag genes in B. thuringiensis. Strains in the Bacillus cereus group were added for comparison purposes. We show that sap and eag are either present in tandem in 35% of the B. thuringiensis strains analysed and absent in 65% of the strains. When absent, a different tandem, the lytB/lytA cell wall hydrolase genes, is present. The distribution of the sap and eag S-layer and the lytB/lytA cell wall hydrolase genes is not species-specific in B. thuringiensis, B. cereus and Bacillus weihenstephanensis. Bacillus anthracis and Bacillus mycoides harbor sap and eag but not lytB/lytA. The sap, eag and lytB/lytA genes were absent in Bacillus pseudomycoides. Clearly, the distribution of the sap and eag S-layer and the lytB/lytA cell wall hydrolase genes in B. thuringiensis and in the Bacillus cereus group is mutually exclusive. We also showed that two genes involved in cell wall metabolism, csaA and csaB, are present not only upstream of the sap and eag S-layer genes, but also upstream of the lytB/lytA tandem in strains where sap and eag are absent. Bootstrapped neighbor-joining trees were inferred from the translated amino acid sequences of sap, eag and the tandem lytB/lytA, respectively.     

Evaluation of dental enamel caries assessment using Quantitative Light Induced Fluorescence and Optical Coherence Tomography

An in vitro study of morphological alterations between sound dental structure and artificially induced white spot lesions in human teeth, was performed through the loss of fluorescence by Quantitative Light‐Induced Fluorescence (QLF) and the alterations of the light attenuation coefficient by Optical Coherence Tomography (OCT). To analyze the OCT images using a commercially available system, a special algorithm was applied, whereas the QLF images were analyzed using the software available in the commercial system employed. When analyzing the sound region against white spot lesions region by QLF, a reduction in the fluorescence intensity was observed, whilst an increase of light attenuation by the OCT system occurred. Comparison of the percentage of alteration between optical properties of sound and artificial enamel caries regions showed that OCT processed images through the attenuation of light enhanced the tooth optical alterations more than fluorescence detected by QLF System.

QLF versus OCT imaging of enamel caries: a photonics assessment     

Molecular diagnosis of neonatal diabetes mellitus using next-generation sequencing of the whole exome

Background

Accurate molecular diagnosis of monogenic non-autoimmune neonatal diabetes mellitus (NDM) is critical for patient care, as patients carrying a mutation in KCNJ11 or ABCC8 can be treated by oral sulfonylurea drugs instead of insulin therapy. This diagnosis is currently based on Sanger sequencing of at least 42 PCR fragments from the KCNJ11, ABCC8, and INS genes. Here, we assessed the feasibility of using the next-generation whole exome sequencing (WES) for the NDM molecular diagnosis.

Methodology/Principal Findings

We carried out WES for a patient presenting with permanent NDM, for whom mutations in KCNJ11, ABCC8 and INS and abnormalities in chromosome 6q24 had been previously excluded. A solution hybridization selection was performed to generate WES in 76 bp paired-end reads, by using two channels of the sequencing instrument. WES quality was assessed using a high-resolution oligonucleotide whole-genome genotyping array. From our WES with high-quality reads, we identified a novel non-synonymous mutation in ABCC8 (c.1455G>C/p.Q485H), despite a previous negative sequencing of this gene. This mutation, confirmed by Sanger sequencing, was not present in 348 controls and in the patient''s mother, father and young brother, all of whom are normoglycemic.

Conclusions/Significance

WES identified a novel de novo ABCC8 mutation in a NDM patient. Compared to the current Sanger protocol, WES is a comprehensive, cost-efficient and rapid method to identify mutations in NDM patients. We suggest WES as a near future tool of choice for further molecular diagnosis of NDM cases, negative for chr6q24, KCNJ11 and INS abnormalities.     

Heterozygote excess in a self-incompatible and partially clonal forest tree species -- Prunus avium L

Wild cherry (Prunus avium L.), a partially asexual self-incompatible forest tree, shows heterozygote excess, which is a poorly studied phenomenon. In three natural populations, we found significant heterozygote excess at almost all investigated loci (eight microsatellites and markers for the self-incompatibility locus). We examined four hypotheses to account for this observed heterozygote excess. First, negative F(IS) can result from a lack of selfed progeny in small populations of outcrossing species. A second explanation for negative F(IS) is selection during the life cycle of the most heterozygous individuals. A third explanation is negative assortative mating when reproduction occurs between individuals bearing phenotypes more dissimilar than by chance. The last explanation for negative F(IS) relies on asexual reproduction. Expectations for each hypothesis were tested using empirical data. Patterns of F(IS) differed among loci. Nevertheless, our experimental results did not confirm the small sample size hypothesis. Although one locus is probably under a hitch-hiking effect from the SI locus, we rejected the effect of the self-incompatibility locus for the genome as a whole. Similarly, although one locus showed a clear pattern consistent with the selection of heterozygous individuals, the heterosis effect over the whole genome was rejected. Finally, our results revealed that clonality probably explains significant negative F(IS) in wild cherry populations when considering all individuals. More theoretical effort is needed to develop expectations and hypotheses, and test them in the case of species combining self-incompatibility and partially asexual reproduction.     

Sugarcane (Saccharum X officinarum): A Reference Study for the Regulation of Genetically Modified Cultivars in Brazil

Global interest in sugarcane has increased significantly in recent years due to its economic impact on sustainable energy production. Sugarcane breeding and better agronomic practices have contributed to a huge increase in sugarcane yield in the last 30?years. Additional increases in sugarcane yield are expected to result from the use of biotechnology tools in the near future. Genetically modified (GM) sugarcane that incorporates genes to increase resistance to biotic and abiotic stresses could play a major role in achieving this goal. However, to bring GM sugarcane to the market, it is necessary to follow a regulatory process that will evaluate the environmental and health impacts of this crop. The regulatory review process is usually accomplished through a comparison of the biology and composition of the GM cultivar and a non-GM counterpart. This review intends to provide information on non-GM sugarcane biology, genetics, breeding, agronomic management, processing, products and byproducts, as well as the current technologies used to develop GM sugarcane, with the aim of assisting regulators in the decision-making process regarding the commercial release of GM sugarcane cultivars.     

Antioxidant activity of new ruthenium compounds

Many biological properties have been attributed to ruthenium complexes including anti-tumor activity and the attenuation of reperfusion damage and infarct size. In this work, we characterize the antioxidant activity of trans-[RuCl2(nic)4] where nic is 3-pyridinecarboxylic acid and trans-[RuCl2(i-nic)4] where i-nic is 4-pyridinecarboxylic acid by (i) evaluation of total antioxidant potential (TRAP); (ii) prevention of DNA damage induced by hydrogen peroxide using the alkaline comet assay; and (iii) the prevention of lipid peroxidation and cell death induced by iron in liver slices. Our results suggest that nic has stronger antioxidant potential when compared to the i-nic. Higher doses (above 200 microM) of these compounds gave genotoxic effects, but the antioxidant potential could be obtained with the use lower doses (0.1-10 microM).     

Resistance to abomasal nematodes and individual genetic variability in reindeer

Resistance to parasites is believed to have a widespread influence on demographic and adaptive processes. In systems where parasites impose a fitness cost on their host, heterozygotes may be selected because they are more resistant to parasites than homozygotes. Our objective was to assess the relationships between genomewide individual heterozygosity and abomasal nematode burdens in female Svalbard reindeer (Rangifer tarandus platyrhynchus) after the effects of host age, locality, season, and year had been accounted for. Samples were obtained from 306 female reindeer that were culled and genotyped at nine microsatellite loci. Reindeer in our study populations are mainly parasitized by the gastrointestinal nematodes Ostertagia gruehneri and Marshallagia marshalli. The infection intensity of each parasite differed between subpopulations, and among host age classes, seasons and years. We found no significant relationships between abomasal worm burdens, or lumen and mucosa larvae, of either O. gruehneri or M. marshalli and individual heterozygosity (or mean d(2)) alone or in interactions with host age, locality, and year. Although we analysed one of the largest data set available to date on gastrointestinal nematodes of a wild ruminant, we used a typical data set of nine genetic neutral markers that may have had low power to detect heterozygosity-fitness correlations. We conclude that the proportion of the variance in parasite resistance explained by individual heterozygosity for neutral genetic markers is low in Svalbard reindeer and in vertebrates in general, and we suggest that the candidate-gene approach might be more fruitful for further research on gene-fitness correlations.