Large-scale identification of polymorphic microsatellites using an <Emphasis Type="Italic">in silico</Emphasis> approach

Background  

Simple Sequence Repeat (SSR) or microsatellite markers are valuable for genetic research. Experimental methods to develop SSR markers are laborious, time consuming and expensive. In silico approaches have become a practicable and relatively inexpensive alternative during the last decade, although testing putative SSR markers still is time consuming and expensive. In many species only a relatively small percentage of SSR markers turn out to be polymorphic. This is particularly true for markers derived from expressed sequence tags (ESTs). In EST databases a large redundancy of sequences is present, which may contain information on length-polymorphisms in the SSR they contain, and whether they have been derived from heterozygotes or from different genotypes. Up to now, although a number of programs have been developed to identify SSRs in EST sequences, no software can detect putatively polymorphic SSRs.     

Impact of an 8-Year-Old Transgenic Poplar Plantation on the Ectomycorrhizal Fungal Community

The long-term impact of field-deployed genetically modified trees on soil mutualistic organisms is not well known. This study aimed at evaluating the impact of poplars transformed with a binary vector containing the selectable nptII marker and β-glucuronidase reporter genes on ectomycorrhizal (EM) fungi 8 years after field deployment. We generated 2,229 fungal internal transcribed spacer (ITS) PCR products from 1,150 EM root tips and 1,079 fungal soil clones obtained from the organic and mineral soil horizons within the rhizosphere of three control and three transformed poplars. Fifty EM fungal operational taxonomic units were identified from the 1,706 EM fungal ITS amplicons retrieved. Rarefaction curves from both the root tips and soil clones were close to saturation, indicating that most of the EM species present were recovered. Based on qualitative and/or quantitative α- and β-diversity measurements, statistical analyses did not reveal significant differences between EM fungal communities associated with transformed poplars and the untransformed controls. However, EM communities recovered from the root tips and soil cloning analyses differed significantly from each other. We found no evidence of difference in the EM fungal community structure linked to the long-term presence of the transgenic poplars studied, and we showed that coupling root tip analysis with a soil DNA cloning strategy is a complementary approach to better document EM fungal diversity.The poplar has become a model tree species in genetic engineering as it can easily be transformed and clonally propagated and has a small genome size (7, 77, 80). Tree growth, agronomic traits, and timber quality can be improved through genetic engineering (61), thereby avoiding the long reproductive cycles of conventional breeding (47, 59, 83). However, concerns have arisen about the potential impact of genetically modified (GM) trees on the environment (10). The potential environmental hazards linked to GM trees differ from those associated with transgenic crop plants at both spatial and temporal scales (84) because trees are long-lived perennials, unlike annual crop plants. They display several biotic interactions with soil microbial communities such as bacteria and fungi. Interactions between GM trees and these communities could result in exposure to the expression of new traits over several decades, a period longer than those for GM crop plants.Impact studies of GM plants on nontarget organisms usually focus on the potential risk linked to transgene expression (expected effects) that confers a genetic advantage to the transformed plant rather than on unforeseen (pleiotropic) effects from transgene insertion or the expression of other transgene components such as selection markers or reporter genes. The nptII gene, encoding neomycin phosphotransferase II (EC 2.7.1.95), and the GUS gene, encoding β-glucuronidase (GUS; EC 3.2.1.31), are frequently used for genetic selection of transformed cells and for monitoring transgene presence and expression during transgenic plant lifetime (76). The products of the nptII and GUS genes have been subjected to safety assessment studies and were shown to be nondeleterious to human and animal health (21, 23, 27, 51). Nevertheless, pleiotropic effects in crop plants transformed with the nptII and GUS genes have been observed (2, 15, 17, 43). Pleiotropic effects from GM trees coexpressing such selectable markers have also been recorded. For example, Pasonen et al. (56) showed a significant decrease in the number of root tips colonized by Paxillus involutus associated with a line of chitinase-transformed silver birch in vitro. Similar results have been observed in vivo with P. involutus associated with a line of lignin-modified silver birches (72).Many trees in temperate, boreal, tropical, and subtropical forests establish mutualistic interactions with ectomycorrhizal (EM) fungi (42, 66, 67, 68). EM fungi are a polyphyletic group comprising over 5,000 species (49) that play key roles in biogeochemical soil processes and plant health. They represent one-third of the total microbial biomass in the soil of boreal forests (32). Fine roots colonized by EM fungi, also called EM root tips or ectomycorrhizae, display a fungal mantle from which extends the extraradical mycelium to prospect the soil for nutrient uptake. These two anatomical parts can be sampled for EM fungus molecular identification, but some studies have highlighted dissimilarities between the EM fungal diversity recorded in root tip sampling and that recorded in extraradical mycelium sampling (26, 37, 39).Given the potential cumulative effects caused by the presence and stable constitutive expression of transgenes over years on GM tree fitness and on the environment, impact studies of GM trees require long-term field trials (5, 72, 84). In this study, we investigated the potential long-term impact on the EM fungal community of hybrid poplars transformed with the binary vector containing the selectable nptII marker and GUS reporter genes, field deployed for 8 years. This plantation was part of the first confined field trial of transgenic trees in Canada. Hybrid poplars constitutively expressed the nptII gene for kanamycin resistance driven by the NOS promoter (30). The activity of the NOS promoter has been shown to increase in the lower part of transgenic tobacco plants (4). Such a vertical gradient has also been observed in transgenic hybrid poplars, where the NOS promoter activity was 2.4-fold higher in roots than in leaves (87).As no direct negative impact of nptII or GUS gene expression on fungal organisms has been reported in the literature, we first tested the null hypothesis (H₀) that the EM fungal community recorded from transgenic poplars was similar to that from untransformed poplars. Second, since the EM fungal diversity picture can be influenced by the sampling method, we contrasted the EM fungal community recovered from root tips with that recorded in soil cloning analyses. Internal transcribed spacer (ITS) sequences from the nuclear rRNA were produced from both EM root tips and extraradical mycelia to compare the EM fungal communities associated with three control and three transgenic poplars. EM fungal communities were characterized by measuring the usual qualitative and quantitative EM species diversity within each community (α-diversity) and then estimating the nucleotide diversity between EM communities in relation to EM phylotype relative abundances (quantitative β-diversity).     

An Optimized Pentaplex PCR for Detecting DNA Mismatch Repair-Deficient Colorectal Cancers

Purpose

Microsatellite instability (MSI) is used to screen colorectal cancers (CRC) for Lynch Syndrome, and to predict outcome and response to treatment. The current technique for measuring MSI requires DNA from normal and neoplastic tissues, and fails to identify tumors with specific DNA mismatch repair (MMR) defects. We tested a panel of five quasi-monomorphic mononucleotide repeat markers amplified in a single multiplex PCR reaction (pentaplex PCR) to detect MSI.

Experimental Design

We investigated a cohort of 213 CRC patients, comprised of 114 MMR-deficient and 99 MMR-proficient tumors. Immunohistochemical (IHC) analysis evaluated the expression of MLH1, MSH2, PMS2 and MSH6. MSI status was defined by differences in the quasi-monomorphic variation range (QMVR) from a pool of normal DNA samples, and measuring differences in allele lengths in tumor DNA.

Results

Amplification of 426 normal alleles allowed optimization of the QMVR at each marker, and eliminated the requirement for matched reference DNA to define MSI in each sample. Using ≥2/5 unstable markers as the criteria for MSI resulted in a sensitivity of 95.6% (95% CI = 90.1–98.1%) and a positive predictive value of 100% (95% CI = 96.6%–100%). Detection of MSH6-deficiency was limited using all techniques. Data analysis with a three-marker panel (BAT26, NR21 and NR27) was comparable in sensitivity (97.4%) and positive predictive value (96.5%) to the five marker panel. Both approaches were superior to the standard approach to measuring MSI.

Conclusions

An optimized pentaplex (or triplex) PCR offers a facile, robust, very inexpensive, highly sensitive, and specific assay for the identification of MSI in CRC.     

Furin is the major processing enzyme of the cardiac-specific growth factor bone morphogenetic protein 10

Bone morphogenetic protein 10 (BMP10) is a member of the TGF-β superfamily and plays a critical role in heart development. In the postnatal heart, BMP10 is restricted to the right atrium. The inactive pro-BMP10 (～60 kDa) is processed into active BMP10 (～14 kDa) by an unknown protease. Proteolytic cleavage occurs at the RIRR(316)↓ site (human), suggesting the involvement of proprotein convertase(s) (PCs). In vitro digestion of a 12-mer peptide encompassing the predicted cleavage site with furin, PACE4, PC5/6, and PC7, showed that furin cleaves the best, whereas PC7 is inactive on this peptide. Ex vivo studies in COS-1 cells, a cell line lacking PC5/6, revealed efficient processing of pro-BMP10 by endogenous PCs other than PC5/6. The lack of processing of overexpressed pro-BMP10 in the furin- and PACE4-deficient cell line, CHO-FD11, and in furin-deficient LoVo cells, was restored by stable (CHO-FD11/Fur cells) or transient (LoVo cells) expression of furin. Use of cell-permeable and cell surface inhibitors suggested that endogenous PCs process pro-BMP10 mostly intracellularly, but also at the cell surface. Ex vivo experiments in mouse primary hepatocytes (wild type, PC5/6 knock-out, and furin knock-out) corroborated the above findings that pro-BMP10 is a substrate for endogenous furin. Western blot analyses of heart right atria extracts from wild type and PACE4 knock-out adult mice showed no significant difference in the processing of pro-BMP10, implying no in vivo role of PACE4. Overall, our in vitro, ex vivo, and in vivo data suggest that furin is the major convertase responsible for the generation of BMP10.     

The protein disulfide isomerases PDIA4 and PDIA6 mediate resistance to cisplatin-induced cell death in lung adenocarcinoma

Intrinsic and acquired chemoresistance are frequent causes of cancer eradication failure. Thus, long-term cis-diaminedichloroplatine(II) (CDDP) or cisplatin treatment is known to promote tumor cell resistance to apoptosis induction via multiple mechanisms involving gene expression modulation of oncogenes, tumor suppressors and blockade of pro-apoptotic mitochondrial membrane permeabilization. Here, we demonstrate that CDDP-resistant non-small lung cancer cells undergo profound remodeling of their endoplasmic reticulum (ER) proteome (>80 proteins identified by proteomics) and exhibit a dramatic overexpression of two protein disulfide isomerases, PDIA4 and PDIA6, without any alteration in ER-cytosol Ca²⁺ fluxes. Using pharmacological and genetic inhibition, we show that inactivation of both proteins directly stimulates CDDP-induced cell death by different cellular signaling pathways. PDIA4 inactivation restores a classical mitochondrial apoptosis pathway, while knockdown of PDIA6 favors a non-canonical cell death pathway sharing some necroptosis features. Overexpression of both proteins has also been found in lung adenocarcinoma patients, suggesting a clinical importance of these proteins in chemoresistance.     

Characterization of SPACR, a sialoprotein associated with cones and rods present in the interphotoreceptor matrix of the human retina: immunological and lectin binding analysis

Rod and cone photoreceptors project from the outer retinal surface into a carbohydrate-rich interphotoreceptor matrix (IPM). Unique IPM glycoconjugates are distributed around rods and cones. Wheat germ agglutinin (WGA) strongly decorates the rod matrix domains and weakly decorates the cone matrix domains. This study characterizes the major WGA-binding glycoprotein in the human IPM, which we refer to as SPACR (sialoprotein associated with cones and rods). SPACR, which has a molecular weight of 147 kDa, was isolated and purified from the IPM by lectin affinity chromatography. A polyclonal antibody to SPACR was prepared that colocalizes in tissue preparations with WGA-binding domains in the IPM. Sequential digestion of SPACR with N- and O- glycosidases results in a systematic increase in electrophorectic mobility, indicating the presence of both N- and O-linked glycoconjugates. Complete deglycosylation results in a reduction in the relative molecular mass of SPACR by about 30%. Analysis of lectin binding allowed us to identify some of the structural characteristics of SPACR glycoconjugates. Treatment with neuraminidase exposes Galbeta1- 3GalNAc disaccharide as indicated by positive peanut agglutinin (PNA) staining, accompanied by the loss of WGA staining. Maackia amurensis agglutinins (MAA-1 and MAA-2), specific for sialic acid in alpha2-3 linkage to Gal, bind SPACR, while Sambucus nigra agglutinin (SNA), specific for alpha2-6 linked sialic acid, does not, indicating that the dominant glycoconjugate determinant on SPACR is the O-linked carbohydrate, NeuAcalpha2-3Galbeta1-3GalNAc. The abundance of sialic acid in SPACR suggests that this glycoprotein may contribute substantially to the polyanionic nature of the IPM. The carbohydrate chains present on SPACR could also provide sites for extensive crosslinking and participate in the formation of the ordered IPM lattice that surrounds the elongate photoreceptors projecting from the outer retinal surface.      

Environmental implications of the use of agro‐industrial residues for biorefineries: application of a deterministic model for indirect land‐use changes

Biorefining agro‐industrial biomass residues for bioenergy production represents an opportunity for both sustainable energy supply and greenhouse gas (GHG) emissions mitigation. Yet, is bioenergy the most sustainable use for these residues? To assess the importance of the alternative use of these residues, a consequential life cycle assessment (LCA) of 32 energy‐focused biorefinery scenarios was performed based on eight selected agro‐industrial residues and four conversion pathways (two involving bioethanol and two biogas). To specifically address indirect land‐use changes (iLUC) induced by the competing feed/food sector, a deterministic iLUC model, addressing global impacts, was developed. A dedicated biochemical model was developed to establish detailed mass, energy, and substance balances for each biomass conversion pathway, as input to the LCA. The results demonstrated that, even for residual biomass, environmental savings from fossil fuel displacement can be completely outbalanced by iLUC, depending on the feed value of the biomass residue. This was the case of industrial residues (e.g. whey and beet molasses) in most of the scenarios assessed. Overall, the GHGs from iLUC impacts were quantified to 4.1 t CO₂‐eq.ha^?1_demanded yr^?1 corresponding to 1.2–1.4 t CO₂‐eq. t^?1 dry biomass diverted from feed to energy market. Only, bioenergy from straw and wild grass was shown to perform better than the alternative use, as no competition with the feed sector was involved. Biogas for heat and power production was the best performing pathway, in a short‐term context. Focusing on transport fuels, bioethanol was generally preferable to biomethane considering conventional biogas upgrading technologies. Based on the results, agro‐industrial residues cannot be considered burden‐free simply because they are a residual biomass and careful accounting of alternative utilization is a prerequisite to assess the sustainability of a given use. In this endeavor, the iLUC factors and biochemical model proposed herein can be used as templates and directly applied to any bioenergy consequential study involving demand for arable land.     

Differential expression of sarcoplasmic proteins in four heterogeneous ovine skeletal muscles

Fiber-type distribution is known to vary widely within and between muscles according to differences in muscle functions. 2-DE and MALDI-MS were used to investigate the molecular basis of muscle fiber type-related variability. We compared four lamb skeletal muscles with heterogeneous fiber-type composition that are relatively rich in fast-twitch fiber types, i.e., the semimembranosus, vastus medialis, longissimus dorsi, and tensor fasciae latae (TL). Our results clearly showed that none of the glycolytic metabolism enzymes detected, including TL which was most strongly glycolytic, made intermuscular differentiation possible. Muscle differentiation was based on the differential expression of proteins involved in oxidative metabolism, including not only citric acid cycle enzymes but also other classes of proteins with functions related to oxidative metabolism, oxidative stress, and probably to higher protein turnover. Detected proteins were involved in transport (carbonate dehydratase, myoglobin, fatty acid-binding protein), repair of misfolding damage (heat shock protein (HSP) 60 kDa, HSP-27 kDa, alpha-crystallin beta subunit, DJ1, stress-induced phosphoprotein), detoxification or degradation of impaired proteins (GST-Pi, aldehyde dehydrogenase, peroxiredoxin, ubiquitin), and protein synthesis (tRNA-synthetase). The fractionating method led to the detection of proteins involved in different functions related to oxidative metabolism that have not previously been shown concomitancy.