Genetic variants at the chromosomal region 2q21.3 underlying inhibitor development in patients with severe haemophilia A.

INTRODUCTION: Inhibitor development affects about 30% of patients with severe haemophilia A (HA) and results from different environmental and genetic risk factors. Previously, we identified the missense variant rs3754689 in the LCT gene linked with this predisposition. Since rs3754689 variant is benign and is located in a conserved haplotype region, we hypothesized that the association signal captured by this variant is located in coinherited, neighbouring genes. AIM: To identify novel genetic risk factors associated with inhibitor development in coding regions of R3HDM1, UBXN4, CXCR4, MCM6, DARS and miR128-1 genes. METHODS: Targeted sequencing was performed in 246 severe HA patients (72 with and 174 without inhibitor): 181 previously and 65 newly enrolled. RESULTS: Forty-one common and 152 rare variants passed the quality control. Logistic regression analysis of common variants identified rs3754689 and four additional variants (.011 < P < .047; FDR ranging .2-.38). Logistic regression analysis performed only in the 220 Italian patients showed similar results (.004 < P < .05; FDR ranging .12-.22). Three of these variants (rs3213892 and rs3816155 in the LCT intron 13 and rs961360 in the R3HDM1 intron10-exon11 junction) may affect the expression of UBXN4 and R3HDM1, respectively. Rare variants did not show association with inhibitor development. Identified variants were not replicated in the multi-ethnic SIPPET cohort of 230 severe HA patients. CONCLUSION: Due to the limited sample size that may be responsible of the high FDR values, we could not confirm with certainty the analysed association. Further evaluation of the expression levels of analysed genes will confirm or not their role in inhibitor development.

Selective transcriptional regulation by Myc in cellular growth control and lymphomagenesis.

The c-myc proto-oncogene product, Myc, is a transcription factor that binds thousands of genomic loci. Recent work suggested that rather than up- and downregulating selected groups of genes, Myc targets all active promoters and enhancers in the genome (a phenomenon termed 'invasion') and acts as a general amplifier of transcription. However, the available data did not readily discriminate between direct and indirect effects of Myc on RNA biogenesis. We addressed this issue with genome-wide chromatin immunoprecipitation and RNA expression profiles during B-cell lymphomagenesis in mice, in cultured B cells and fibroblasts. Consistent with long-standing observations, we detected general increases in total RNA or messenger RNA copies per cell (hereby termed 'amplification') when comparing actively proliferating cells with control quiescent cells: this was true whether cells were stimulated by mitogens (requiring endogenous Myc for a proliferative response) or by deregulated, oncogenic Myc activity. RNA amplification and promoter/enhancer invasion by Myc were separable phenomena that could occur without one another. Moreover, whether or not associated with RNA amplification, Myc drove the differential expression of distinct subsets of target genes. Hence, although having the potential to interact with all active or poised regulatory elements in the genome, Myc does not directly act as a global transcriptional amplifier. Instead, our results indicate that Myc activates and represses transcription of discrete gene sets, leading to changes in cellular state that can in turn feed back on global RNA production and turnover.

Degradation dynamics of microRNAs revealed by a novel pulse-chase approach.

The regulation of miRNAs is critical to the definition of cell identity and behavior in normal physiology and disease. To date, the dynamics of miRNA degradation and the mechanisms involved in remain largely obscure, in particular, in higher organisms. Here, we developed a pulse-chase approach based on metabolic RNA labeling to calculate miRNA decay rates at genome-wide scale in mammalian cells. Our analysis revealed heterogeneous miRNA half-lives, with many species behaving as stable molecules (T1/2> 24 h), while others, including passenger miRNAs and a number (25/129) of guide miRNAs, are quickly turned over (T1/2= 4-14 h). Decay rates were coupled with other features, including genomic organization, transcription rates, structural heterogeneity (isomiRs), and target abundance, measured through quantitative experimental approaches. This comprehensive analysis highlighted functional mechanisms that mediate miRNA degradation, as well as the importance of decay dynamics in the regulation of the miRNA pool under both steady-state conditions and during cell transitions.

Complications of whole-exome sequencing for causal gene discovery in primary platelet secretion defects.

Primary platelet secretion defects constitute a heterogeneous group of functional defects characterized by reduced platelet granule secretion upon stimulation by different agonists. The clinical and laboratory heterogeneity of primary platelet secretion defects warrants a tailored approach. We performed a pilot study in order to develop DNA sequence analysis pipelines for gene discovery and to create a list of candidate causal genes for platelet secretion defects. Whole-exome sequencing analysis of 14 unrelated Italian patients with primary secretion defects and 16 controls was performed on Illumina HiSeq. Variant prioritization was carried out using two filtering approaches: identification of rare, potentially damaging variants in platelet candidate genes or by selecting singletons. To corroborate the results, exome sequencing was applied in a family in which platelet secretion defects and a bleeding diathesis were present. Platelet candidate gene analysis revealed gene defects in 10/14 patients, which included ADRA2A, ARHGAP1, DIAPH1, EXOC1, FCGR2A, ITPR1, LTBP1, PTPN7, PTPN12, PRKACG, PRKCD, RAP1GAP, STXBP5L, and VWF The analysis of singletons identified additional gene defects in PLG and PHACTR2 in two other patients. The family analysis confirmed a missense variant p.D1144N in the STXBP5L gene and p.P83H in the KCNMB3 gene as potentially causal. In summary, exome sequencing revealed potential causal variants in 12 of 14 patients with primary platelet secretion defects, highlighting the limitations of the genomic approaches for causal gene identification in this heterogeneous clinical and laboratory phenotype.

Whole-exome sequencing to identify genetic risk variants underlying inhibitor development in severe hemophilia A patients.

The development of neutralizing antibodies (inhibitors) against coagulation factor VIII (FVIII) is the most problematic and costly complication of FVIII replacement therapy that affects up to 30% of previously untreated patients with severe hemophilia A. The development of inhibitors is a multifactorial complication involving environmental and genetic factors. Among the latter, F8 gene mutations, ethnicity, family history of inhibitors, and polymorphisms affecting genes involved in the immune response have been previously investigated. To identify novel genetic elements underling the risk of inhibitor development in patients with severe hemophilia A, we applied whole-exome sequencing (WES) and data analysis in a selected group of 26 Italian patients with (n = 17) and without (n = 9) inhibitors. WES revealed several rare, damaging variants in immunoregulatory genes as novel candidate mutations. A case-control association analysis using Cochran-Armitage and Fisher's exact statistical tests identified 1364 statistically significant variants. Hierarchical clustering of these genetic variants showed 2 distinct patterns of homozygous variants with a protective or harmful role in inhibitor development. When looking solely at coding variants, a total of 28 nonsynonymous variants were identified and replicated in 53 inhibitor-positive and 174 inhibitor-negative Italian severe hemophilia A patients using a TaqMan genotyping assay. The genotyping results revealed 10 variants showing estimated odds ratios in the same direction as in the discovery phase and confirmed the association of the rs3754689 missense variant (OR 0.58; 95% CI 0.36-0.94; P = .028) in a highly conserved haplotype region surrounding the LCT locus on chromosome 2q21 with inhibitor development.

Carbon fiber epoxy composites for both strengthening and health monitoring of structures.

This paper presents a study of the electrical and mechanical behavior of several continuous carbon fibers epoxy composites for both strengthening and monitoring of structures. In these composites, the arrangement of fibers was deliberately diversified to test and understand the ability of the composites for self-sensing low strains. Composites with different arrangements of fibers and textile weaves, mainly unidirectional continuous carbon reinforced composites, were tested at the dynamometer. A two-probe method was considered to measure the relative electrical resistance of these composites during loading. The measured relative electrical resistance includes volume and contact electrical resistances. For all tested specimens, it increases with an increase in tensile strain, at low strain values. This is explained by the improved alignment of fibers and resulting reduction of the number of possible contacts between fibers during loading, increasing as a consequence the contact electrical resistance of the composite. Laboratory tests on strengthening of structural elements were also performed, making hand-made composites by the "wet process", which is commonly used in civil engineering for the strengthening of all types of structures in-situ. Results show that the woven epoxy composite, used for strengthening of concrete elements is also able to sense low deformations, below 1%. Moreover, results clearly show that this textile sensor also improves the mechanical work of the strengthened structural elements, increasing their bearing capacity. Finally, the set of obtained results supports the concept of a textile fabric capable of both structural upgrade and self-monitoring of structures, especially large structures of difficult access and needing constant, sometimes very expensive, health monitoring.

Effects of Multi-Walled Carbon Nanotube Dosages and Sonication Time on Hydration Heat Evolution in Cementitious Composites.

This study aimed to investigate the heat generated during the hydration process in cementitious composites containing multi-walled carbon nanotubes (MWCNTs). The cumulative heat release and heat flow of these cementitious composites were measured over a period of 168 h using isothermal calorimetry. Three different MWCNT dosages, 0.05 wt%, 0.1 wt%, and 0.2 wt%, along with two different sonication times for the solution, which were 20 min and 60 min, were applied in the experimental program. The results reveal that the incorporation of MWCNTs and the use of a naphthalene-based superplasticizer to disperse the nanotubes generally led to a reduction in heat emission during the early stages of hydration, a lower first peak value in the initial stage of hydration, and a significant delay in the acceleration period compared with the reference sample lacking this superplasticizer. Furthermore, the results demonstrate that both the dosage of multi-walled carbon nanotubes (MWCNTs) and the sonication time have an impact on the heat emission and hydration process since the same amount of superplasticizer was applied to all pastes. An increase in the MWCNT dosage led to a decrease in the rate of hydration heat at the main peak for all pastes. Additionally, longer sonication times resulted in lower values of heat generated, reduced main peak values in the heat rate evolution, and generally extended delays in the occurrence of the main peak.

Electrical Properties of the Carbon Nanotube-Reinforced Geopolymer Studied by Impedance Spectroscopy.

Geopolymers, recognized as an ecological alternative to cement concrete, are gaining more and more interest from researchers and the construction industry. Due to the registrable electrical conductivity, this material also attracts the interest of other fields of science and industry as a potential functional material. The article discusses the used geopolymer material, created on the basis of metakaolin and waste Cathode Ray Tubes (CRT) glass, reinforced with ultra-long in-house carbon nanotubes (CNT), in the context of its use as a smart material for Structural Health Monitoring. Long in-house made carbon nanotubes were added to enhance the electrical conductivity of the geopolymer. The impedance spectroscopy method was applied to investigate the conductive properties of this material. The paper shows the microscopic and mechanical characteristics of the materials and presents the results of promising impedance spectroscopy tests.

Recent Advancements in Carbon Nano-Infused Cementitious Composites.

A rising demand for efficient functional materials brings forth research challenges regarding improvements in existing materials. Carbon infused cementitious composites, regardless of being an important research topic worldwide, still present many questions concerning their functionality and properties. The paper aims to highlight the most important materials used for cementitious composites, their properties, and their uses while also including the most relevant of the latest research in that area.

Influence of the Composition and Curing Time on Mechanical Properties of Fluidized Bed Combustion Fly Ash-Based Geopolymer.

This paper presents novel research on a fluidized bed combustion (FBC) fly ash-based geopolymer as a contribution to the problem of FBC fly ash disposal, and a proposal for a new geopolymer composition-an environmentally friendly material that is possible to use in construction. Geopolymer samples of various composition (containing FBC fly ash as the main raw material, metakaolin and CRT glass as additional components, and sodium silicate and sodium hydroxide as activators) were subjected to flexural and compressive strength tests. An investigation on the effect of the demolding time was carried out on one selected mixture. The test showed that both the composition and the demolding time have a decisive influence on the basic mechanical properties. A mixture containing FBC fly ash to metakaolin in a mass ratio of 3:1, removed from the mold after 14 days, was found to be the best in terms of the mechanical parameters expected from a material that could be used in construction, e.g., for the production of precast elements. According to the results obtained, FBC fly ash is a promising and environmentally friendly raw material for the production of geopolymer, with good mechanical properties and low density. Moreover, a high compressive strength can be obtained by curing the geopolymer at ambient temperature.

Characteristics of Metakaolin-Based Geopolymer with Cathode Ray Tube Glass.

Geopolymers can be treated as an environmentally friendly alternative for concrete and enables utilization of various wastes. This paper focuses on the possibility of application of discarded cathode ray tube (CRT) glass inside a metakaolin-based geopolymer in the form of an aggregate, providing an ecological method of recycling of this hazardous material. The main goal of this paper was to develop an optimal composition of a new geopolymer and to describe its behavior under varying curing conditions. A geopolymer made of different mixtures was subjected to flexural and compressive strength tests. The density, mass loss, temperature changes, and metals leaching were determined as well. The results demonstrated that neither the content of CRT glass nor the curing regime has a significant influence on the mechanical behavior. However, the strength of the geopolymer containing 50% CRT glass by mass increased with time in contrast to a geopolymer with a higher CRT glass content. The development of temperature inside the mixture was dependent on the amount of metakaolin. The concentration of toxic metals in an aqueous extract decreased considerably after the encapsulation of CRT glass inside the geopolymer. The presented results indicate that discarded CRT glass can be considered an aggregate for a metakaolin-based geopolymer. The new material shows high strength and makes the CRT glass safe for the environment.

Electrochemically Exfoliated Graphene for High-Durability Cement Composites.

The development of radically new types of corrosion-resistant cement composites is nowadays compulsory in view of the continuous increase of concrete consumption combined with the intrinsically defective nature of concrete. Among various additives being employed in the concrete technology, carbon nanomaterials have emerged as extremely powerful components capable of remarkably enhancing nano- and microstructures as well as properties of cement-based composites. In this study, we demonstrate that cement mortar incorporating electrochemically exfoliated graphene (EEG) exhibits significantly improved fluid transport properties. The addition of 0.05 wt % of EEG to ordinary Portland cement mortar results in the reduction of initial and secondary sorptivity values by 21 and 25%, respectively. This leads to the outstanding resistance of EEG-cement composites to highly corrosive environments, namely, chloride and sulfate solutions. These observations, combined with the previously reported remarkable enhancement of the tensile strength of EEG-cement mortars, represent a major step toward the development of highly durable graphene-based cement composites.

High-Performance Graphene-Based Cementitious Composites.

This study reports on the development of a cementitious composite incorporating electrochemically exfoliated graphene (EEG). This hybrid functional material features significantly enhanced microstructure and mechanical properties, as well as unaffected workability; thus, it outperforms previously reported cementitious composites containing graphene derivatives. The manufacturing of the composite relies on a simple and efficient method that enables the uniform dispersion of EEG within cement matrix in the absence of surfactants. Different from graphene oxide, EEG is found to not agglomerate in cement alkaline environment, thereby not affecting the fluidity of cementitious composites. The addition of 0.05 wt% graphene content to ordinary Portland cement results in an increase up to 79%, 8%, and 9% for the tensile strength, compressive strength, and Young's modulus, respectively. Remarkably, it is found that the addition of EEG promotes the hydration reaction of both alite and belite, thus leading to the formation of a large fraction of 3CaO·2SiO2·3H2O (C-S-H) phase. These findings represent a major step forward toward the practical application of nanomaterials in civil engineering.

Development of Self-Sensing Textile Strengthening System Based on High-Strength Carbon Fiber.

The monitoring of structures is one of the most difficult challenges of engineering in the 21st century. As a result of changes in conditions of use, as well as design errors, many building structures require strengthening. This article presents research on the development of an externally strengthening carbon-fiber textile with a self-sensing option, which is an idea is based on the pattern of resistive strain gauges, where thread is presented in the form of zig-zagging parallel lines. The first laboratory tests showed the system's high efficiency in the measurement of strains, but also revealed its sensitivity to environmental conditions. This article also presents studies on the influence of temperature and humidity on the measurement, and to separate the two effects, resistance changes were tested on unloaded concrete and wooden samples. The models were then placed in a climatic chamber, and the daily cycle of temperature and humidity changes was simulated. The research results confirmed preliminary observations of resistivity growths along with temperature. This effect is more visible on concrete samples, presumably due to its greater amount of natural humidity. The strain measurement with carbon fibers is very sensitive to temperature changes, and applications of this method in practice require compensation.

Next-generation DNA sequencing to identify novel genetic risk factors for cerebral vein thrombosis.

BACKGROUND: Cerebral vein thrombosis (CVT) is a rare, life-threatening disease affecting one adult per 100,000 per year. Genetic risk factors are deficiencies of the natural anticoagulant proteins antithrombin, protein C, protein S or single nucleotide polymorphisms such as factor V Leiden and prothrombin 20210A. In 20% of patients, the cause of CVT remains unknown. AIM: To identify novel genetic risk factors for CVT using targeted next-generation DNA sequencing (NGS). METHODS: We investigated 171 CVT patients and 298 healthy controls. Patients were selected using the following criteria: objective diagnosis of CVT, no active cancer. We performed targeted NGS analysis of the protein-coding regions of 734 candidate genes related to hemostasis and inflammation, 150 ancestry informative markers and 28 thrombosis-associated variants. RESULTS: We identified 3723 common and low frequency variants with minor allele frequency (MAF) >1% in 590 genes. Single variant association testing using logistic regression analysis identified rs8176719 insertion/deletion (indel) variant in the ABO gene associated with CVT (age and sex adjusted OR 2.03; 95% CI 1.52-2.73; P = 2.07 × 10-6; Bonferroni P = 0.008). In addition, we identified 8839 rare variants (MAF ≤ 1%) in 723 genes. Gene-based association analysis of these rare variants using a burden test revealed only a tentative association of non-coding variants located in the F8 locus with CVT. CONCLUSION: Targeted NGS identified a common indel variant rs8176719 in the ABO gene. Gene-based tests of association failed to reveal genomic loci with a cumulative burden of rare variants associated with CVT.

The Drosophila Mre11/Rad50 complex is required to prevent both telomeric fusion and chromosome breakage.

The MRN complex consists of the two evolutionarily conserved components Mre11 and Rad50 and the third less-conserved component Nbs1/Xrs2. This complex mediates telomere maintenance in addition to a variety of functions in response to DNA double-strand breaks, including homologous recombination, nonhomologous end joining (NHEJ), and activation of DNA damage checkpoints. Mutations in the Mre11 gene cause the human ataxia-telangiectasia-like disorder (ATDL). Here, we show that null mutations in the Drosophila mre11 and rad50 genes cause both telomeric fusion and chromosome breakage. Moreover, we demonstrate that these mutations are in the same epistasis group required for telomere capping and mitotic chromosome integrity. Using an antibody against Rad50, we show that this protein is uniformly distributed along mitotic chromosomes, and that Rad50 is unstable in the absence of its binding partner Mre11. To define the roles of rad50 and mre11 in telomere protection, mutant chromosome preparations were immunostained for both HP1 and HOAP, two proteins that protect Drosophila telomeres from fusion. Cytological analysis revealed that mutations in rad50 and mre11 drastically reduce accumulation of HOAP and HP1 at telomeres. This suggests that the MRN complex protects Drosophila telomeres by facilitating recruitment of HOAP and HP1 at chromosome ends.

Lig4 and rad54 are required for repair of DNA double-strand breaks induced by P-element excision in Drosophila.

Site-specific double-strand breaks (DSBs) were generated in the white gene located on the X chromosome of Drosophila by excision of the w(hd) P-element. To investigate the role of nonhomologous end joining (NHEJ) and homologous recombination (HR) in the repair of these breaks, the w(hd) P-element was mobilized in flies carrying mutant alleles of either lig4 or rad54. The survival of both lig4- and rad54-deficient males was reduced to 25% in comparison to the wild type, indicating that both NHEJ and HR are involved in the repair P-induced gaps in males. Survival of lig4-deficient females was not affected at all, implying that HR using the homologous chromosome as a template can partially compensate for the impaired NHEJ pathway. In rad54 mutant females survival was reduced to 70% after w(hd) excision. PCR analysis indicated that the undamaged homologous chromosome may compensate for the potential loss of the broken chromosome in rad54 mutant females after excision. Molecular analysis of the repair junctions revealed microhomology (2-8 bp)-dependent DSB repair in most products. In the absence of Lig4, the 8-bp target site duplication is used more frequently for repair. Our data indicate the presence of efficient alternative end-joining mechanisms, which partly depend on the presence of microhomology but do not require Lig4.

Single Nucleotide Variant rs2232710 in the Protein Z-Dependent Protease Inhibitor (ZPI, SERPINA10) Gene Is Not Associated with Deep Vein Thrombosis.

Rare mutations in PROC, PROS1 or SERPINC1 as well as common variants in F5, F2, F11 and SERPINC1 have been identified as risk factors for deep vein thrombosis (DVT). To identify novel genetic risk factors for DVT, we have developed and applied next-generation DNA sequencing (NGS) of the coding area of hemostatic and proinflammatory genes. Using this strategy, we previously identified a single nucleotide variant (SNV) rs6050 in the FGA gene and novel, rare SNVs in the ADAMTS13 gene associated with DVT. To identify novel coding variants in the genetic predisposition to DVT, we applied NGS analysis of the coding area of 186 hemostatic and proinflammatory genes in 94 DVT cases and 98 controls and we identified 18 variants with putative role in DVT. A group of 585 Italian idiopathic DVT patients and 550 healthy controls was used to genotype all the 18 risk-associated variants identified by NGS. Replication study in the Italian population identified the rs2232710 variant in the protein Z-dependent protease inhibitor (ZPI) gene to be associated with an increased risk of DVT (OR 2.74; 95% CI 1.33-5.65; P = 0.0045; Bonferroni P = 0.081). However, the rs2232710 SNV showed no association with DVT in two Dutch replication cohorts the LETS study (454 patients and 451 controls) and the MEGA study (3799 patients and 4399 controls), indicating that the rs2232710 variant is not a risk factor for DVT.

Disruption of Drosophila Rad50 causes pupal lethality, the accumulation of DNA double-strand breaks and the induction of apoptosis in third instar larvae.

The Rad50/Mre11/Nbs1 protein complex has a crucial role in DNA metabolism, in particular in double-strand break (DSB) repair through homologous recombination (HR). To elucidate the role of the Rad50 protein complex in DSB repair in a multicellular eukaryote, we generated a Rad50 deficient Drosophila strain by P-element mediated mutagenesis. Disruption of Rad50 causes retarded development and pupal lethality. To investigate the mechanism of pupal death, brains and wing imaginal discs from third instar larvae were studied in more detail. Wing imaginal discs from Rad50 mutant larvae displayed a 3.5-fold increase in the induction of spontaneous apoptotic cells in comparison to their heterozygous siblings. This finding correlates with increased levels of phosphorylated histone H2Av, indicating an accumulation of DSBs in Rad50 mutant larvae. A 45-fold increase in the frequency of anaphase bridges was detected in the brains of Rad50 deficient larvae, consistent with a role for Rad50 in telomere maintenance and/or replication of DNA. The induction of DSBs and defects in chromosome segregation are in agreement with a role of Drosophila Rad50 in repairing the DSBs that arise during replication.

The Drosophila melanogaster DNA Ligase IV gene plays a crucial role in the repair of radiation-induced DNA double-strand breaks and acts synergistically with Rad54.

DNA Ligase IV has a crucial role in double-strand break (DSB) repair through nonhomologous end joining (NHEJ). Most notably, its inactivation leads to embryonic lethality in mammals. To elucidate the role of DNA Ligase IV (Lig4) in DSB repair in a multicellular lower eukaryote, we generated viable Lig4-deficient Drosophila strains by P-element-mediated mutagenesis. Embryos and larvae of mutant lines are hypersensitive to ionizing radiation but hardly so to methyl methanesulfonate (MMS) or the crosslinking agent cis-diamminedichloroplatinum (cisDDP). To determine the relative contribution of NHEJ and homologous recombination (HR) in Drosophila, Lig4; Rad54 double-mutant flies were generated. Survival studies demonstrated that both HR and NHEJ have a major role in DSB repair. The synergistic increase in sensitivity seen in the double mutant, in comparison with both single mutants, indicates that both pathways partially overlap. However, during the very first hours after fertilization NHEJ has a minor role in DSB repair after exposure to ionizing radiation. Throughout the first stages of embryogenesis of the fly, HR is the predominant pathway in DSB repair. At late stages of development NHEJ also becomes less important. The residual survival of double mutants after irradiation strongly suggests the existence of a third pathway for the repair of DSBs in Drosophila.