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Purpose: Most BRCA1/2 mutations are of unknown clinical relevance. An increasing amount of evidence indicates that there can be deleterious effects through the disruption of the splicing process. We have investigated the effect of aberrant splicing of BRCA1/2 on hereditary breast/ovarian cancer (HBOC).Experimental Design: DNA variants were analyzed with splicing prediction programs to select putative splicing mutations. Splicing assays of 57 genetic variants were done by lymphocyte reverse transcription-PCR and/or hybrid minigenes in HeLa and nontumor breast epithelial cells.Results: Twenty-four BRCA1/2 variants of Spanish HBOC patients were bioinformatically preselected. Functional assays showed that 12 variants induced anomalous splicing patterns, 6 of which accounted for 58.5% of BRCA1 families. To further evaluate the defective splicing of BRCA1/2, we analyzed 31 Breast Cancer Information Core Database (BIC) and two artificial variants that were generated by mutagenesis. Sixteen variants induced different degrees of aberrant splicing. Altogether, anomalous splicing was caused by 28 BRCA1/2 variants of all types, indicating that any DNA change can disrupt pre-mRNA processing. We show that a wide range of regulatory elements can be involved, including the canonical and cryptic splice sites, the polypyrimidine tract, and splicing enhancers/silencers. Twenty mutations were predicted to truncate the BRCA proteins and/or to delete essential domains, thus supporting a role in HBOC.Conclusions: An important fraction of DNA variants of BRCA1/2 presents splicing aberrations that may represent a relevant disease-causing mechanism in HBOC. The identification of splicing disruptions by functional assays is a valuable tool to discriminate between benign polymorphisms and pathogenic mutations. Clin Cancer Res; 16(6); 1957–67

Supplementary Data from A High Proportion of DNA Variants of BRCA1 and BRCA2 Is Associated with Aberrant Splicing in Breast/Ovarian Cancer Patients

Background W1282X-CFTR variant (c.3846G>A) is the second most common nonsense cystic fibrosis (CF)-causing mutation in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Even though remarkable breakthroughs have been done towards CF treatment with the approval of four CFTR protein modulators, none of these are approved for patients with nonsense mutations. CRISPR gene editing tools can be of great value to permanently correct the genetic defects caused by these mutations. Methods We compared the capacity of homology-directed repair (HDR) mediated by Cas9 or Cas12a to correct W1282X CFTR mutation in the CFF-16HBEge W1282X CFTR cell line (obtained from CFF), using Cas9/gRNA and Cas12a/gRNA ribonucleoproteins (RNPs) and single strand DNA (ssODN) oligonucleotide donors. Results Cas9 shows higher levels of correction than Cas12a as, by electroporating cells with Cas9 RNPs and ssODN donor, nearly 18% of precise editing was achieved compared to just 8% for Cas12a. Such levels of correction increase the abundance of CFTR mRNA and protein, and partially restore CFTR function in the pool of edited cells to 18% of WT CFTR function. Moreover, homozygous corrected clones produced levels of mRNA, protein, and function comparable to those of cells expressing WT CFTR. Conclusion Altogether, this work demonstrates the potential of gene editing as a therapeutic strategy for CF directly correcting the root cause of the disease.

Hybrid transparent contacts based on combinations of a transparent conductive oxide and a few graphene monolayers were developed in order to evaluate their optical and electrical performance with the main aim to use them as front contacts in optoelectronic devices. The assessment of the most suitable strategies for their fabrication was performed by testing different protocols addressing such issues as the protection of the device structure underneath, the limitation of sample temperature during the graphene-monolayer transfer process and the determination of the most suitable stacking structure. Suitable metal ohmic electrodes were also evaluated. Among a number of options tested, the metal contact based on Ti + Ag showed the highest reproducibility and the lowest contact resistivity. Finally, with the objective of extracting the current generated from optoelectronic devices to the output pins of an external package, focusing on a near future commercial application, the electrical properties of the connections made with an ultrasonic bonding machine (sonic welding) between the optimized Ti + Ag metal contacts and Al or Au micro-wires were also evaluated. All these results have an enormous potential as hybrid electrodes based on graphene to be used in novel designs of a future generation of optoelectronic devices, such as solar cells.

The ability to reprogramme any adult cell back into a pluripotent stem cell state coupled with the capability to precisely edit its genomic sequence has given scientists almost unlimited potential to model any genetic disease and holds great promise for regenerative medicine. The discovery of CRISPR/Cas9 coupled with continued development and modification of this system has made it possible to manipulate gene expression at both a genetic and epigenetic level exposing the true power of stem cells. The applications of this technology to model any genetic disease, screen new drugs, generate organs for donation and develop cell therapies appear limitless. In this review, we will provide a SWOT analysis of CRISPR/Cas9 and discuss the most recent developments in induced pluripotent stem cell gene editing and the impact being made in the clinic.

A feasible alternative for agricultural or forestry waste management is the operation of a distributed network of sustainable Medium Combustion Plants (MCPs). However, one of the main factors that hinder its development is the propensity to operational problems derived from corrosion, slagging and fouling characteristics of both bottom and fly ashes. Therefore, a cost-effective approach for these multi-product MCP could be based on predictive tools for an optimal formulation of a fuel blend. This work focuses on the assessment of the ability of these methods to provide guidance for preventing ash-related operational problems and to provide fuel-blending rules. The more widespread tools pertain to two types: compositional classification based on chemical analysis of laboratory ashes, and thermodynamic prediction of the most likely species and phases. Both criterion numbers and compositional maps are ranking methods based on the chemical analysis at a given ashing temperature. Thermodynamic equilibrium modeling is not constrained by any difference in the physical conditions of the MCP compared to those in the laboratory. Both kind of prediction tools have been validated in an MCP firing olive tree pruning residues as well as its typical blends in order to mimic a plausible pattern of fuels along a full year operating campaign. An intensive experimental campaign encompasses plant monitoring and off-line analysis of the ashes along the process line. Interpretation of compositional plots has revealed to be potentially sensitive to ashing temperature. Here are presented examples showing how this variable could lead to either insignificant differences or to a substantial disparity in the a priori fuel diagnosis. Some inconsistencies have been observed between the predictions based on criterion numbers, even for the same fuel and for ranking rules specifically formulated for biomasses. Moreover, it does not match consistently with the information obtained from phase diagrams. Therefore, their use should be limited to the case of a well-established selection of a fuel index for a well-defined fuel provided empirical evidence of an enough good description of the ash behavior, which is not the most frequent case. Thermodynamic equilibrium calculations allow a more precise prediction of the main species in the condensed phase, without the constraint of the ashing temperature. Elemental closure of main ash-forming elements with the chemical analysis of the process ashes presents small differences, and their proximity localization on the phase diagrams denote similar prediction between predicted and process ashes.

Differential Mobility Analyzers (DMA) have been studied for decades to classify a wide range of particle sizes. Its correct behavior is determined by the resolution, influenced by Brownian diffusion losses, distortions in the electric and flow field. The design configuration, especially inlets and outlets, can be a reason of these effects, affecting the DMA reliability. One of these configurations is the tangential inlet for polydisperse aerosol, used in the Vienna-type DMA. The use of a tangential inlet reduces the residence time and favored the aerosol flow distribution in the classification chamber. However, in this configuration swirl effects of non-uniform particle behaviors had previously been observed. In this work, the 3D-Computational Fluid-Dynamics has been used to analyze the distortions expected of this DMA in previous studies. The proposed methodology had allowed determining its correct operation and thus, to develop new designs optimizing their operation. The procedure includes the simulation by Ansys Workbench v15.0 and the method to determine an adequate behavior, including the effect on the particle distribution and penetration towards the classification chamber, and the effect on the transfer function. The calculation results have allowed the design of alternative geometries of the aerosol inlet keeping the tangentiality. Using the new geometry, the flow distribution has improved removing the flow disturbances, the particle penetration has increased until 15% and the particle distribution has deviated from the ideal one only in a 2% in some cases. The best behavior has been observed especially at high flow rates and lower particle sizes.

The construction of Hybrid minigenes provides a robust and simple strategy to study the effects of disease-causing mutations on mRNA splicing when biological material from patient cells is not available. Hybrid minigenes can be used as splicing reporter plasmids allow RNA expression and heterologous splicing reactions between synthetic splicing signals in the vector and endogenous splicing signals in a cloned genomic DNA fragment that contains one or more introns and exons. Minigene-based assay has been used extensively to test the effect of mutations in the splicing of a target sequence. They can also be used to test the ability of CRISPR/Cas9 and one or more associated gRNAs to target specific sequences in the minigene, and determine the effect of these editing events on splicing. As an example, it is shown that CRISPR/Cas9-based, targeted excision of short intronic sequences containing mutations which create cryptic splice signals, can restore normal splicing in a CFTR Hybrid minigene.

This work demonstrates the feasibility of a robust and effective control of the emissions of particulate matter (PM) and heavy metals (HMs) from olive tree pruning combustion, as well as from its blends with two customary local components (a composted fraction of sewage sludge and a fraction of municipal solid waste with a high plastic content), in an approximated proportion of 50/50 (% w.t) to mimic the seasonal supply variability. The study was carried out during a 5 weeks experimental campaign in a 1MWth fluidized bed combustor. The combustor was equipped with a hybrid filtration system, consisting of an electrostatic precipitator and a bag filter module serially connected, for an enhanced control of the PM emissions when dealing with a realistic suite of fuel blends. Raw aerosol showed high concentrations in both PM and HMs, substantially dependent on the blend. However, the hybrid filter (HF) showed a good performance irrespective of these variable loads, leading to emission concentrations below 7 mg...

Mutation screening of the breast cancer genes BRCA1 and BRCA2 identifies a large fraction of variants of uncertain clinical significance (VUS) whose functional and clinical interpretations pose a challenge for genomic medicine. Likewise, an increasing amount of evidence indicates that genetic variants can have deleterious effects on pre-mRNA splicing. Our goal was to investigate the impact on splicing of a set of reported variants of BRCA2 exons 17 and 18 to assess their role in hereditary breast cancer and to identify critical regulatory elements that may constitute hotspots for spliceogenic variants. A splicing reporter minigene with BRCA2 exons 14 to-20 (MGBR2_ex14-20) was constructed in the pSAD vector. Fifty-two candidate variants were selected with splicing prediction programs, introduced in MGBR2_ex14-20 by site-directed mutagenesis and assayed in triplicate in MCF-7 cells. Wild type MGBR2_ex14-20 produced a stable transcript of the expected size (1,802 nucleotides) and structure (V1-[BRCA2_exons_14–20]–V2). Functional mapping by microdeletions revealed essential sequences for exon recognition on the 3’ end of exon 17 (c.7944-7973) and the 5’ end of exon 18 (c.7979-7988, c.7999-8013). Thirty out of the 52 selected variants induced anomalous splicing in minigene assays with >16 different aberrant transcripts, where exon skipping was the most common event. A wide range of splicing motifs were affected including the canonical splice sites (15 variants), novel alternative sites (3 variants), the polypyrimidine tract (3 variants) and enhancers/silencers (9 variants). According to the guidelines of the American College of Medical Genetics and Genomics (ACMG), 20 variants could be classified as pathogenic (c.7806-2A>G, c.7806-1G>A, c.7806-1G>T, c.7806-1_7806-2dup, c.7976+1G>A, c.7977-3_7978del, c.7977-2A>T, c.7977-1G>T, c.7977-1G>C, c.8009C>A, c.8331+1G>T and c.8331+2T>C) or likely pathogenic (c.7806-9T>G, c.7976G>C, c.7976G>A, c.7977-7C>G, c.7985C>G, c.8023A>G, c.8035G>T and c.8331G>A), accounting for 30.8% of all pathogenic/likely pathogenic variants of exons 17–18 at the BRCA Share database. The remaining 8 variants (c.7975A>G, c.7977-6T>G, c.7988A>T, c.7992T>A, c.8007A>G, c.8009C>T, c.8009C>G, and c.8072C>T) induced partial splicing anomalies with important ratios of the full-length transcript (≥70%), so that they remained classified as VUS. Aberrant splicing is therefore especially prevalent in BRCA2 exons 17 and 18 due to the presence of active ESEs involved in exon recognition. Splicing functional assays with minigenes are a valuable strategy for the initial characterization of the splicing outcomes and the subsequent clinical interpretation of variants of any disease-gene, although these results should be checked, whenever possible, against patient RNA., EAV's lab was supported by grants from the Spanish Ministry of Economy and Competitivity, Plan Nacional de I+D+I 2013-2016, ISCIII (Fis: PI13/01749) co-funded by FEDER from Regional Development European Funds (European Union), and grant CSI090U14 from the Consejería de Educación (ORDEN EDU/122/2014), Junta de Castilla y León. EFB was supported by a predoctoral fellowship from the University of Valladolid and Banco de Santander (2015-2019).

New architectures of transparent conductive electrodes (TCEs) incorporating graphene monolayers in different configurations have been explored with the aim to improve the performance of silicon-heterojunction (SHJ) cell front transparent contacts. In SHJ technology, front electrodes play an important additional role as anti-reflectance (AR) coatings. In this work, different transparent-conductive-oxide (TCO) thin films have been combined with graphene monolayers in different configurations, yielding advanced transparent electrodes specifically designed to minimize surface reflection over a wide range of wavelengths and angles of incidence and to improve electrical performance. A preliminary analysis reveals a strong dependence of the optoelectronic properties of the TCEs on (i) the order in which the different thin films are deposited or the graphene is transferred and (ii) the specific TCO material used. The results shows a clear electrical improvement when three graphene monolayers are placed on top on 80-nm-thick ITO thin film. This optimum TCE presents sheet resistances as low as 55 Ω/sq and an average conductance as high as 13.12 mS. In addition, the spectral reflectance of this TCE also shows an important reduction in its weighted reflectance value of 2&ndash, 3%. Hence, the work undergone so far clearly suggests the possibility to noticeably improve transparent electrodes with this approach and therefore to further enhance silicon-heterojunction cell performance. These results achieved so far clearly open the possibility to noticeably improve TCEs and therefore to further enhance SHJ contact-technology performance.

Recent work regarding the Layer by Layer (LbL) engineering of poly(lactide-co-glycolide) nanoparticles (PLGA NPs) is reviewed here. The LbL engineering of PLGA NPs is applied as a means of generating advanced drug delivery devices with tailored recognition, protection, cargo and release properties. LbL in combination with covalent chemistry is used to attach PEG and folic acid to control cell uptake and direct it towards cancer cells. LbL coatings composed of chitosan and alginate show low protein interactions and can be used as an alternative to Pegylation. The assembly on top of LbL coatings of lipid layers composed of variable percentages of 1,2-dioleoyl-sn-glycero-3-choline (DOPC) and 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) increases NP uptake and directs the NPs towards the endoplasmic reticulum. The antibody anti-TNF-α is encapsulated forming a complex with alginate that is assembled LbL on top of PLGA NPs. The antibody is released in cell culture following first order kinetics. The release kinetics of encapsulated molecules inside PLGA NPs are studied when the PLGA NPs are coated via LbL with different polyelectrolytes. The intracellular release of encapsulated Doxorubicin is studied in the HepG2 cell line by means of Fluorescence Lifetime Imaging.

A strategy of encapsulation of the antiTNF-α antibody on top of poly(lactide-co-glycolide) nanoparticles (PLGA NPs) is presented on the basis of the complexation of antiTNF-α with alginate (Alg) and subsequent assembly layer by layer with poly(L-lysine) (PLL). The assembly of the antiTNF-α/Alg complex with PLL and its stability in PBS and lysozymes are monitored on a planar support using a quartz crystal microbalance with dissipation. The assembly of the antiTNF-α/Alg complex on PLGA NPs is followed by zeta potential measurements. AntiTNF-α release from the PLGA NPs is measured in PBS at 37 and 60 °C and in the HepG2 cell line following NP uptake, using the Q-ADA kit detection kit. The release follows first-order kinetics with an initial burst. Intracellular release of antiTNF-α is confirmed by confocal Raman microscopy.

Cystic fibrosis (CF) is a chronic and progressive autosomal recessive disorder of secretory epithelial cells, which causes obstructions in the lung airways and pancreatic ducts of 70,000 people worldwide (for recent review see Cutting Nat Rev Genet 16(1):45–56, 2015). The finding that mutations in the CFTR gene cause CF (Kerem et al. Science 245(4922):1073–1080, 1989; Riordan et al. Science 245(4922):1066–1073, 1989; Rommens et al. Science 245(4922):1059–1065, 1989), was hailed as the very happy middle of a story whose end is a cure for a fatal disease (Koshland Science 245(4922):1029, 1989). However, despite two licensed drugs (Ramsey et al. N Engl J Med 365(18):1663–1672, 2011; Wainwright et al. N Engl J Med 373(3):220–231, 2015), and a formal demonstration that repeated administration of CFTR cDNA to patients is safe and effects a modest but significant stabilisation of disease (Alton et al. Lancet Respir Med 3(9):684–691, 2015), we are still a long way from a cure, with many patients taking over 100 tablets per day, and a mean age at death of 28 years. The aim of this review is to discuss the impact on the study of CF of gene-editing techniques as they have developed over the last 30 years, up to and including the possibility of editing as a therapeutic approach.

The impact of metal oxide nanoparticles (NPs) on the immune system has been studied in vitro using human peripheral blood lymphocytes (PBLs). Metal oxide NPs (ZnO, CeO 2 , TiO 2 and Al 2 O 3 ) induced changes in the expression levels of adhesion molecules and the C-X-C chemokine receptor type 4 (CXCR4) in these cells. Proliferation studies were carried out with CFSE in response to PHA, finding an increase in T-cell proliferation upon cell exposure to TiO 2 and Al 2 O 3 NPs. For ZnO NPs, a decrease in the chemotactic response to SDF-1α was observed. No changes were found in basophil activation and leukocyte oxidative burst after phagocytosis. Despite the absence of cytotoxicity, metal oxide NPs are not inert; they alter the expression levels of adhesion molecules and chemokine receptors, key actors in the immune response, and affect important cell functions such as T-cell proliferative response to mitogens and chemotaxis. From the Clinical Editor This study demonstrates the immune-modulating effects of four different metal nanoparticles in a human peripheral blood lymphocyte model system. These effects were clearly present even though these nanoparticles did not display cytotocity in ex vivo experiments.

A strategy of encapsulation of the antiTNF-α antibody on top of poly(lactide-co-glycolide) nanoparticles (PLGA NPs) is presented on the basis of the complexation of antiTNF-α with alginate (Alg) and subsequent assembly layer by layer with poly(L-lysine) (PLL). The assembly of the antiTNF-α/Alg complex with PLL and its stability in PBS and lysozymes are monitored on a planar support using a quartz crystal microbalance with dissipation. The assembly of the antiTNF-α/Alg complex on PLGA NPs is followed by zeta potential measurements. AntiTNF-α release from the PLGA NPs is measured in PBS at 37 and 60 °C and in the HepG2 cell line following NP uptake, using the Q-ADA kit detection kit. The release follows first-order kinetics with an initial burst. Intracellular release of antiTNF-α is confirmed by confocal Raman microscopy.

This is an open access article distributed under the terms of the Creative Commons Attribution License., [Introduction]: The underlying pathogenic mechanism of a large fraction of DNA variants of disease-causing genes is the disruption of the splicing process. We aimed to investigate the effect on splicing of the BRCA2 variants c.8488-1G > A (exon 20) and c.9026_9030del (exon 23), as well as 41 BRCA2 variants reported in the Breast Cancer Information Core (BIC) mutation database. [Methods]: DNA variants were analyzed with the splicing prediction programs NNSPLICE and Human Splicing Finder. Functional analyses of candidate variants were performed by lymphocyte RT-PCR and/or hybrid minigene assays. Forty-one BIC variants of exons 19, 20, 23 and 24 were bioinformatically selected and generated by PCR-mutagenesis of the wild type minigenes. [Results]: Lymphocyte RT-PCR of c.8488-1G > A showed intron 19 retention and a 12-nucleotide deletion in exon 20, whereas c.9026_9030del did not show any splicing anomaly. Minigene analysis of c.8488-1G > A displayed the aforementioned aberrant isoforms but also exon 20 skipping. We further evaluated the splicing outcomes of 41 variants of four BRCA2 exons by minigene analysis. Eighteen variants presented splicing aberrations. Most variants (78.9%) disrupted the natural splice sites, whereas four altered putative enhancers/silencers and had a weak effect. Fluorescent RT-PCR of minigenes accurately detected 14 RNA isoforms generated by cryptic site usage, exon skipping and intron retention events. Fourteen variants showed total splicing disruptions and were predicted to truncate or eliminate essential domains of BRCA2. [Conclusions]: A relevant proportion of BRCA2 variants are correlated with splicing disruptions, indicating that RNA analysis is a valuable tool to assess the pathogenicity of a particular DNA change. The minigene system is a straightforward and robust approach to detect variants with an impact on splicing and contributes to a better knowledge of this gene expression step., This work was supported by grants PI10/2910 of the Instituto de Salud Carlos III (Spanish Ministry of Science and Innovation), and CSI004A10-2 (Consejería de Educación) and BIO39/VA27/10 (Consejería de Sanidad) of the Regional Government of Castilla y León. Alberto Acedo is supported by the European Social Fund and Consejeria de Educación de la Junta de Castilla y León under the P.O. Castilla y León 2007-2013., Support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

Control of particulate matter emissions by means of hybrid filter has been included in the experimental plan of a research project in the field of Mediterranean agro-forestry waste biomass combustion at medium scale. Application of hybrid filters to biomass combustion has not been thoroughly experimented so far. An identification of the most important parameters in particulate matter emissions control by means of a hybrid filter was undertaken. The filter involves two of the most significant technologies in fly ash emission control, electrostatic precipitation and fabric filtration A discussion of these parameters and principles of operation of said technologies is presented, as well as the final selection of parameters to be included in the experimental matrix of the project in regards to emissions control. A novel approach is proposed for testing of filtration velocity influence in fabric filter module without impacting on operation of electrostatic precipitation module.

A simple analytical method using low volumes of solvent for determining selected PAHs and NPAHs in PM samples is presented. The proposed extraction method was compared with pressurized fluid (PFE) and microwave (MC) extraction techniques and intermediate precision associated to analytical measurements were estimated. Extraction by agitation with 8 mL of dichloromethane yielded recoveries above 80% compared to those obtained from PFE extraction. Regarding intermediate precision results, values between 10-20% were reached showing increases of dispersion for compounds with high volatility and low levels of concentration. Within the framework of the INTA/CIEMAT research agreement for the PM characterization in gas turbine exhaust, the method was applied for analysis of aluminum foil substrates and quartz filters with mass loading ranged from 0.02 to 2 mg per sample.

11 páginas, 3 figuras, 2 tablas.-- et al.-- El pdf del artículo es la versión post-print., [Purpose]: Most BRCA1/2 mutations are of unknown clinical relevance. An increasing amount of evidence indicates that there can be deleterious effects through the disruption of the splicing process. We have investigated the effect of aberrant splicing of BRCA1/2 on hereditary breast/ovarian cancer (HBOC). [Experimental Design]: DNA variants were analyzed with splicing prediction programs to select putative splicing mutations. Splicing assays of 57 genetic variants were done by lymphocyte reverse transcription-PCR and/or hybrid minigenes in HeLa and nontumor breast epithelial cells. [Results]: Twenty-four BRCA1/2 variants of Spanish HBOC patients were bioinformatically preselected. Functional assays showed that 12 variants induced anomalous splicing patterns, 6 of which accounted for 58.5% of BRCA1 families. To further evaluate the defective splicing of BRCA1/2, we analyzed 31 Breast Cancer Information Core Database (BIC) and two artificial variants that were generated by mutagenesis. Sixteen variants induced different degrees of aberrant splicing. Altogether, anomalous splicing was caused by 28 BRCA1/2 variants of all types, indicating that any DNA change can disrupt pre-mRNA processing. We show that a wide range of regulatory elements can be involved, including the canonical and cryptic splice sites, the polypyrimidine tract, and splicing enhancers/silencers. Twenty mutations were predicted to truncate the BRCA proteins and/or to delete essential domains, thus supporting a role in HBOC. [Conclusions]: An important fraction of DNA variants of BRCA1/2 presents splicing aberrations that may represent a relevant disease-causing mechanism in HBOC. The identification of splicing disruptions by functional assays is a valuable tool to discriminate between benign polymorphisms and pathogenic mutations., PI061102, (Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación, Spain) and 200820I135 (Consejo Superior de Investigaciones Científicas, Ministerio de Ciencia e Innovación, Spain), the Hereditary Cancer Prevention Programme of the Regional Government of Castilla y León, and a grant from the Associazione Italiana Ricerca sul Cancro, Italy (F. Pagani).

10 páginas, 3 figuras, 3 tablas.-- et al.-- El pdf del artículo es la versión post-print., The distribution of BRCA1 and BRCA2 germ line mutations in breast/ovarian cancer families varies among different populations, which typically present a wide spectrum of unique mutations. Splicing mutation 5272-1G>A of BRCA1 and frameshift mutation 5374delTATG of BRCA2 are highly prevalent mutations in Castilla-León (Spain), accounting for 18.4% and 13.6% of BRCA1 and BRCA2 positive families, respectively. To test the presence of founder effects, 9 Spanish 5272-1G>A and 13 5374delTATG families were genotyped with polymorphic markers linked to BRCA1 or BRCA2. All the 5272-1G>A families shared a common haplotype in eight markers (1.1 Mb region) and the mutation age was estimated in 15 generations (∼380 years). A conserved haplotype associated to 5374delTATG was observed in four markers (0.82 Mb). The mutation occurred approximately 48 generations ago (∼1200 years). Each mutation likely arose from a common ancestor that could be traced to a small area of Castilla-León and expanded to other Spanish regions. They can have a significant impact on the clinical management of asymptomatic carriers as well as on the genetic screening strategy to be followed in populations with Spanish ancestries., This work has been supported by the Regional Government of Castilla y León, and grants PI061102, PI052275, PI050864) (Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación), 200820I135 (Consejo Superior de Investigaciones Científicas, Ministerio de Ciencia e Innovación), INT07/191 (Instituto de Salud Carlos III), PGDIT06BTF910101PR (Xunta de Galicia). EB was supported by The Health Department of The Basque Country Government (BOPV, 17 Jul 2008, p.16983).

5 páginas, 1 figura, 1 tabla.-- et al.-- El pdf del artículo es el manuscrito de autor., The mutation spectrum of BRCA1 and BRCA2 presents a wide range of unique mutations in breast/ovarian cancer patients but recurrent mutations with founder effects have also been described. BRCA2 5344delAATA and 9538delAA are recurrent mutations in Castilla-Leo´n (Spain) representing 10.6% of BRCA2 positive families. By genotyping eleven chromosome 13 markers (4.3 Mb) we demonstrate that each mutation shows core haplotypes of 1.66 and 0.87 Mb, respectively, supporting a common ancestor in Castilla-Leo´n. Furthermore, both mutations are associated with earlier onset of breast cancer (5344delAATA: 37.4 years, P = 0.033; 9538delAA: 39.4 years, P = 0.008). The identification of founder effects improves the genetic screening strategy to be followed and facilitates the clinical management of asymptomatic carriers., This work has been supported by the Regional Government of Castilla y León, and grants PI061102 (Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación) and 200820I135 (Consejo Superior de Investigaciones Científicas, Ministerio de Ciencia e Innovación).

9 páginas, 3 figuras, 1 tabla., [Aims]: Hereditary non-polyposis colon cancer (HNPCC) is an autosomal dominant disorder that is genetically heterogeneous because of underlying mutations in mismatch repair (MMR) genes, primarily MLH1, MSH2 and MSH6. One challenge to correctly diagnose HNPCC is that the large size of the causative genes makes identification of mutations both labour intensive and expensive. [Methods]: Our heteroduplex analysis by capillary array electrophoresis (HA-CAE) method, previously developed to increase the throughput and allow other multi-exon genes to be scanned, has been adapted for MMRgenes. The altered peak patterns were then sequenced. Furthermore, the mutational scanning was completed using the Multiplex Ligation-Dependent Probe Amplification (MLPA) test in all negative HA-CAE cases, and these results were confirmed by RT-PCR. [Results]: We studied 216 individuals belonging to 100 unrelated families that met the Amsterdam I/II criteria for HNPCC. We detected 40 different variants that are classified as follows: 8 (20%) deleterious mutation, 8 (20%) unknown pathogenic significance variants and 24 (60%) coding and intronic sequence variants. Pathogenic mutations were detected in 12% of the families and about 42% of these had a deletion variant. Unknown pathogenic significance variants (UVs) affected 13% of the families. We also found 12.5% of novel polymorphisms in the rest of the variants. [Concluding]: In short, using a combined method that includes HA-CAE, MLPA and RT-PCR, it is possible to detect the entire mutational spectrum of MMRgenes. Twenty percent of the mutations found in the three genes have not been reported before. Relatives at risk will be offered predictive molecular analysis with potential exclusion of non-carriers of mutations., This work has been supported by the Regional Government of Castilla and León (Spain).

Carbon Nanotubes (CNTs) have been functionalized with a layer of poly(sulfopropyl methacrylate) (PSPM) synthesized from silane initiators attached to the CNT walls. The PSPM brush was coated with a layer of poly(allyl amine hydrochloride) (PAH) and on top of the PAH layer lipid vesicles composed of 75% 1,2-dioleoyl-sn-glycero-3-phosphocholine and 25% 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] were assembled. The surface modification of the CNTs and lipid assembly were followed by Transmission Electron Microscopy (TEM) and ζ-potential measurements. TEM and Confocal Raman Microscopy (CRM) were used to study the uptake and localization of the surface modified CNTs in HepG2 cells. PSPM modified CNTs were present in the cytoplasm. Proliferation studies based on the MTT assay were used to assess toxicity of the CNTs for the different modifications. Cell proliferation decreases for oxidized CNTs and polymer coated CNTs, having the lowest value for PSPM coated CNTs. The lipid coating on top of the polymers significantly reduces the toxic effect of CNTs.