Evaluating the methylation status of CXCR4 promoter: A cost-effective and sensitive two-step PCR method.

The chemokine receptor CXCR4 plays a key role in the bone marrow microenvironment maintenance and in the hematopoietic stem and progenitor cells migration. In addition, CXCR4 is expressed in a broad spectrum of solid tumors where its methylation state has been recently proposed as a biomarker for cancer prognosis. To evaluate methylation status of CXCR4 promoter we developed a sensitive, accurate, specific and cost-effective two-step PCR method that does not require any specific equipment other than a conventional real-time PCR instrument. The principle of the technique relies on a novel normalization strategy which allows the detection and quantification of small methylation differences among pre-amplified DNA samples deriving from low amount of starting material. In addition, the analysis of melting curve profiles of PCR products provides additional information about the methylation status of CpG sites in between the primers. Finally, the principle of this technique can potentially be adapted for the investigation of the methylation status of any other DNA region.

The mitochondrial lncRNA ASncmtRNA-2 is induced in aging and replicative senescence in Endothelial Cells.

Age-associated cardiovascular diseases are at least partially ascribable to vascular cell senescence. Replicative senescence (RS) and stress-induced premature senescence (SIPS) are provoked respectively by endogenous (telomere erosion) and exogenous (H2O2, UV) stimuli resulting in cell cycle arrest in G1 and G2 phases. In both scenarios, mitochondria-derived ROS are important players in senescence initiation. We aimed to define whether a mtDNA-transcribed long-non-coding-RNA (lncRNA), ASncmtRNA-2, has a role in vascular aging and senescence. Aortas of old mice, characterized by increased senescence, showed an increment in ASncmtRNA-2 expression. In vitro analysis of Endothelial Cells (EC) and Vascular Smooth Muscle Cells (VSMC) established that ASncmtRNA-2 is induced in EC, but not in VSMC, during RS. Surprisingly, ASncmtRNA-2 is not upregulated in two different EC SIPS scenarios, treated with H2O2 and UV. The p16 gene displayed similar ASncmtRNA-2 expression patterns, suggesting a possible co-regulation of the two genes. Interestingly, the expression of two miRNAs, hsa-miR-4485 and hsa-miR-1973, with perfect homology to the double strand region of ASncmtRNA-2 and originating at least in part from a mitochondrial transcript, was induced in RS, opening to the possibility that this lncRNA functions as a non-canonical precursor of these miRNAs. Cell cycle analysis of EC transiently over-expressing ASncmtRNA-2 revealed an accumulation of EC in the G2/M phase, but not in the G1 phase. We propose that ASncmtRNA-2 in EC might be involved in the RS establishment by participating in the cell cycle arrest in G2/M phase, possibly through the production of hsa-miR-4485 and hsa-miR-1973. This article is part of a Special Issue entitled: Mitochondria.

Assessment of MDA efficiency for genotyping using cloned embryo biopsies.

The possibility to genotype embryos prior to implantation would have advantages for increasing the speed of selection of cattle. Reliable genotyping requires more DNA than can be obtained from biopsies of embryos, if they are to remain viable. Multiple displacement amplification (MDA) is a whole genome amplification technique used to increase the amount of DNA from biopsies for analysis. Reduced genome coverage resulting in Allele Drop Out (ADO) at heterozygous loci or missing genotypes are drawbacks of MDA. The present article describes the correlation between the input DNA quantity or embryo biopsy size and MDA success. Missing genotypes and ADO drastically increased when fewer than 30-40 cells or the genomic equivalents were used. However, embryo viability was found to be reduced if biopsied with more than 10 cells. Therefore, in vitro cell culture was investigated as a means to increase the number of cells available and the genotyping reliability.

A flexible power electronic converter system with rapid control prototyping for research and teaching.

A flexible power electronic converter embedding a rapid control prototyping platform suitable to be applied in research test setups and teaching laboratories is proposed and described in this paper. The electronic system is composed of three subsystems, namely, i) three half-bridge power boards, ii) a dc-link capacitor bank with a half-bridge power module for active dc-link control, iii) an interfacing board, called motherboard, to couple the power modules with a control unit, iv) a digital control unit with rapid control prototyping functionalities for controlling power electronic circuits. Power modules integrate sensors with related conditioning circuits, driving circuits for power switches, and protection circuits. Conversion circuits exploit GaN electronic switches for optimal performance. The architecture and implementation of the system are described in detail in this manuscript. Main applications are in the implementation of conversion circuits for supplying arbitrary ac or dc voltages or currents, testing of new control algorithms for power electronic converters, testing of systems of electronic converters in, for example, smart nanogrids or renewable energy applications, training of undergraduate and graduate students.

A tool based on Ligation Detection Reaction-Universal Array (LDR-UA) for the characterization of VTEC by identification of virulence-associated and serogroup-specific genes.

Verocytoxigenic Escherichia coli (VTEC) are zoonotic pathogens whose natural reservoir is represented by ruminants, particularly cattle. Infections are mainly acquired by consumption of undercooked contaminated food of animal origin, contact with infected animals and contaminated environment. VTEC O157 is the most frequently isolated serogroup from cases of human disease, however, other VTEC serogroups, such as O26, O111, O145 and O103, are increasingly reported as causing Hemolytic Uremic Syndrome (HUS) worldwide. The identification of VTEC is troublesome, hindering the development of effective prevention strategies. In fact, VTEC are morphologically indistinguishable from harmless E. coli and their pathogenic potential is not strictly dependent on the serogroup, but relies on the presence of a collection of virulence genes. We developed a diagnostic tool for VTEC based on the Ligation Detection Reaction coupled to Universal Array (LDR-UA) for the simultaneous identification of virulence factors and serogroup-associated genes. The method includes the investigation of 40 sites located in 13 fragments from 12 genes (sodCF1/F2, adfO, terB, ehxA, eae, vtx1, vtx2, ihp1, wzx, wbdI, rfbE, dnaK) and was evaluated by performing a trial on a collection of 67 E. coli strains, both VTEC and VT-negative E. coli, as well as on 25 isolates belonging to other related species. Results of this study showed that the LDR-UA technique was specific in identifying the target microorganism. Moreover, due to its higher throughput, the LDR-UA can be a valid and cheaper alternative to real time PCR-based (rt-PCR) methods for VTEC identification.

Comprehensive analysis of Salmonella sequence polymorphisms and development of a LDR-UA assay for the detection and characterization of selected serotypes.

Salmonella is a major cause of food-borne disease, and Salmonella enterica subspecies I includes the most clinically relevant serotypes. Salmonella serotype determination is important for the disease etiology assessment and contamination source tracking. This task will be facilitated by the disclosure of Salmonella serotype sequence polymorphisms, here annotated in seven genes (sefA, safA, safC, bigA, invA, fimA, and phsB) from 139 S. enterica strains, of which 109 belonging to 44 serotypes of subsp. I. One hundred nineteen polymorphic sites were scored and associated to single serotypes or to serotype groups belonging to S. enterica subsp. I. A diagnostic tool was constructed based on the Ligation Detection Reaction-Universal Array (LDR-UA) for the detection of polymorphic sites uniquely associated to serotypes of primary interest (Salmonella Hadar, Salmonella Infantis, Salmonella Enteritidis, Salmonella Typhimurium, Salmonella Gallinarum, Salmonella Virchow, and Salmonella Paratyphi B). The implementation of promiscuous probes allowed the diagnosis of ten further serotypes that could be associated to a unique hybridization pattern. Finally, the sensitivity and applicability of the tool was tested on target DNA dilutions and with controlled meat contamination, allowing the detection of one Salmonella CFU in 25 g of meat.

Abnormal DNA Methylation Induced by Hyperglycemia Reduces CXCR 4 Gene Expression in CD 34+ Stem Cells.

Background CD 34+ stem/progenitor cells are involved in vascular homeostasis and in neovascularization of ischemic tissues. The number of circulating CD 34+ stem cells is a predictive biomarker of adverse cardiovascular outcomes in diabetic patients. Here, we provide evidence that hyperglycemia can be "memorized" by the stem cells through epigenetic changes that contribute to onset and maintenance of their dysfunction in diabetes mellitus. Methods and Results Cord-blood-derived CD 34+ stem cells exposed to high glucose displayed increased reactive oxygen species production, overexpression of p66shc gene, and downregulation of antioxidant genes catalase and manganese superoxide dismutase when compared with normoglycemic cells. This altered oxidative state was associated with impaired migration ability toward stromal-cell-derived factor 1 alpha and reduced protein and mRNA expression of the C-X-C chemokine receptor type 4 ( CXCR 4) receptor. The methylation analysis by bisulfite Sanger sequencing of the CXCR 4 promoter revealed a significant increase in DNA methylation density in high-glucose CD 34+ stem cells that negatively correlated with mRNA expression (Pearson r=-0.76; P=0.004). Consistently, we found, by chromatin immunoprecipitation assay, a more transcriptionally inactive chromatin conformation and reduced RNA polymerase II engagement on the CXCR 4 promoter. Notably, alteration of CXCR 4 DNA methylation, as well as transcriptional and functional defects, persisted in high-glucose CD 34+ stem cells despite recovery in normoglycemic conditions. Importantly, such an epigenetic modification was thoroughly confirmed in bone marrow CD 34+ stem cells isolated from sternal biopsies of diabetic patients undergoing coronary bypass surgery. Conclusions CD 34+ stem cells "memorize" the hyperglycemic environment in the form of epigenetic modifications that collude to alter CXCR 4 receptor expression and migration.

Conserved redox-dependent DNA binding of ROXY glutaredoxins with TGA transcription factors.

The Arabidopsis thaliana CC-type glutaredoxin (GRX) ROXY1 and the bZIP TGA transcription factor (TF) PERIANTHIA (PAN) interact in the nucleus and together regulate petal development. The CC-type GRXs exist exclusively in land plants, and in contrast to the ubiquitously occurring CPYC and CGFS GRX classes, only the CC-type GRXs expanded strongly during land plant evolution. Phylogenetic analyses show that TGA TFs evolved before the CC-type GRXs in charophycean algae. MpROXY1/2 and MpTGA were isolated from the liverwort Marchantia polymorpha to analyze regulatory ROXY/TGA interactions in a basal land plant. Homologous and heterologous protein interaction studies demonstrate that nuclear ROXY/TGA interactions are conserved since the occurrence of CC-type GRXs in bryophytes and mediated by a conserved ROXY C-terminus. Redox EMSA analyses show a redox-sensitive binding of MpTGA to the cis-regulatory as-1-like element. Furthermore, we demonstrate that MpTGA binds together with MpROXY1/2 to this motif under reducing conditions, whereas this interaction is not observed under oxidizing conditions. Remarkably, heterologous complementation studies reveal a strongly conserved land plant ROXY activity, suggesting an ancestral role for CC-type GRXs in modulating the activities of TGA TFs. Super-resolution microscopy experiments detected a strong colocalization of ROXY1 with the active form of the RNA polymerase II in the nucleus. Together, these data shed new light on the function of ROXYs and TGA TFs and the evolution of redox-sensitive transcription regulation processes, which likely contributed to adapt land plants to novel terrestrial habitats.

Metaboloepigenetics: the Emerging Network in Stem Cell Homeostasis Regulation.

Epigenetics harbours all regulatory information that, beyond nucleotide sequences, allows cells to "make decisions" throughout their lifetime in response to the external environment. The information can be transitory or relatively stable, and is even transmittable either to daughter cells or to the next generations through the germ line. Recent discoveries shed light on numerous connections between metabolites and epigenetic chromatin-modifying enzymes, providing a link between the metabolic state of the cell and epigenetics, and ultimately between metabolism, gene expression and cell fate. In this review, we discuss the possible connections between metabolism and epigenetic regulation of stem cell differentiation and self-renewal. Moreover, we describe pertinent literature that could explain how altered metabolic state and nutrition can contribute to disease development through epigenetic modifications. A special section is dedicated to the emerging link between the circadian clock, metabolic transcriptional regulation by epigenetic mechanisms and their implication in stem cell homeostasis.

Methylation profiling by bisulfite sequencing analysis of the mtDNA Non-Coding Region in replicative and senescent Endothelial Cells.

The regulation and function of Mitochondrial DNA (mtDNA) cytosine methylation (5 mC) are largely unexplored. Mitochondria, Endothelial Cell (EC) senescence, and cardiovascular dysfunction are closely related. We extensively investigated the mtDNA Non-Coding Region (NCR) methylation pattern and its variations in EC replicative senescence. We observed previously undescribed 5 mC clusters and a biased distribution of 5 mC among DNA sites and throughout the NCR. The methylation pattern in senescent EC showed non-random variations, including the hypo-methylation of mtDNA replication regulatory sites. Additional experiments opened to a possible role for 5 mC in D-loop formation, rather than in mitochondrial gene expression.

The mitochondrial genome in aging and senescence.

Aging is characterized by a progressive decline in organism functions due to the impairment of all organs. The deterioration of both proliferative tissues in liver, skin and the vascular system, as well as of largely post-mitotic organs, such as the heart and brain could be attributed at least in part to cell senescence. In this review we examine the role of mitochondrial dysfunction and mtDNA mutations in cell aging and senescence. Specifically, we address how p53 and telomerase reverse transcriptase (TERT) activity switch their roles from cytoprotective to detrimental and also examine the role of microRNAs in cell aging. The proposed role of Reactive Oxygen Species (ROS), both as mutating agents and as signalling molecules, underlying these processes is also described.

New labelling technology for molecular probes applied to the ligation detection reaction-universal array system.

The ligation detection reaction (LDR) associated with universal arrays (UA) uses a fluorescently labelled probe (DP) and a Zip Code-extended probe to detect single nucleotide polymorphisms in DNA target sequences. When used for genotyping, the LDR-UA technique uses two DPs, each specific to an allele and labelled with a different fluorophore. The fluorescent signals are processed to calculate the genotype. The uneven decay of fluorophores due to ageing and freezing/thawing cycles and the consequent unequal fluoresce level can lead to erroneous genotype calls. To circumvent this problem, an indirect labelling strategy was developed based on the substitution of the fluorophore with allele-specific 22 bp universal labelling sequences (ULS). Labelling is achieved with fluorescently labelled oligos complementary to the ULS (cULS). The strategy improved the uniformity in probe labelling, and generated results comparable to those using direct-labelled probes, as shown by genotyping 22 polymorphic sites in 70 samples with both strategies. This method can be easily implemented in the routine screening with LDR-UA or other techniques. Moreover, the approach results in a significant cost reduction over traditional direct labelling, and offers the possibility to interchange fluorophores and to increase the fluorescent signal by using multiple-labelled cULS.

Potentials and limitations of molecular diagnostic methods in food safety.

Molecular methods allow the detection of pathogen nucleic acids (DNA and RNA) and, therefore, the detection of contamination in food is carried out with high selectivity and rapidity. In the last 2 decades molecular methods have accompanied traditional diagnostic methods in routine pathogen detection, and might replace them in the upcoming future. In this review the implementation in diagnostics of four of the most used molecular techniques (PCR, NASBA, microarray, LDR) are described and compared, highlighting advantages and limitations of each of them. Drawbacks of molecular methods with regard to traditional ones and the difficulties encountered in pathogen detection from food or clinical specimen are also discussed. Moreover, criteria for the choice of the target sequence for a secure detection and classification of pathogens and possible developments in molecular diagnostics are also proposed.

Nuclear activity of ROXY1, a glutaredoxin interacting with TGA factors, is required for petal development in Arabidopsis thaliana.

Glutaredoxins (GRXs) have thus far been associated mainly with redox-regulated processes participating in stress responses. However, ROXY1, encoding a GRX, has recently been shown to regulate petal primorida initiation and further petal morphogenesis in Arabidopsis thaliana. ROXY1 belongs to a land plant-specific class of GRXs that has a CC-type active site motif, which deviates from ubiquitously occurring CPYC and CGFS GRXs. Expression studies of yellow fluorescent protein-ROXY1 fusion genes driven by the cauliflower mosaic virus 35S promoter reveal a nucleocytoplasmic distribution of ROXY1. We demonstrate that nuclear localization of ROXY1 is indispensable and thus crucial for its activity in flower development. Yeast two-hybrid screens identified TGA transcription factors as interacting proteins, which was confirmed by bimolecular fluorescence complementation experiments showing their nuclear interaction in planta. Overlapping expression patterns of ROXY1 and TGA genes during flower development demonstrate that ROXY1/TGA protein interactions can occur in vivo and support their biological relevance in petal development. Deletion analysis of ROXY1 demonstrates the importance of the C terminus for its functionality and for mediating ROXY1/TGA protein interactions. Phenotypic analysis of the roxy1-2 pan double mutant and an engineered chimeric repressor mutant from PERIANTHIA (PAN), a floral TGA gene, supports a dual role of ROXY1 in petal development. Together, our results show that the ROXY1 protein functions in the nucleus, likely by modifying PAN posttranslationally and thereby regulating its activity in petal primordia initiation. Additionally, ROXY1 affects later petal morphogenesis, probably by modulating other TGA factors that might act redundantly during differentiation of second whorl organs.

The pollen-specific DEFH125 promoter from Antirrhinum is bound in vivo by the MADS-box proteins DEFICIENS and GLOBOSA.

The Antirrhinum DEFH125 MADS-box protein is expressed in maturing pollen and thus likely participates in the regulation of pollen development. Here, we describe the characterization of a 2.5 kbp promoter fragment conferring pollen-specific GUS expression in Antirrhinum, as well as in the distantly related species Arabidopsis. Taking advantage of the higher sensitivity of the diphtheria toxin A-chain (DTA) reporter gene assay, onset of DEFH125 promoter activity could be defined to start at the late unicellular microspore stage. Stamen development in Antirrhinum is governed by the class B MADS-box genes DEFICIENS (DEF) and GLOBOSA (GLO). The respective proteins form a heterodimer and are expressed throughout stamens, except for microspores. Complementary expression patterns of DEFH125 and DEF/GLO during later stamen development tempted us to investigate whether the DEF/GLO heterodimer might bind the DEFH125 promoter and could thus be involved in repressing the DEFH125 expression. The ChIP technique was applied to investigate protein/DNA interactions occurring in vivo. We report the identification of a 200 bp DEFH125 promoter fragment that is in vivo bound by DEF and GLO proteins. This fragment contains a CArG-box motif, known to mediate DNA binding of MADS-box proteins. Implications for a likely function of DEF and GLO in the transcriptional control of DEFH125 are discussed.