Persistence of immersed blood and hair DNA: A preliminary study based on casework.

In some cases, evidence is collected from rivers, canals, lakes or sink pipes. To determine the utility of analyzing these samples and for cases in which DNA was recovered from submerged bulletproof vest parts, we evaluated the time necessary to degrade the blood and, subsequently, DNA on bulletproof vests. In a second experiment, also based on cases, blood was diluted in water from a kitchen sink pipe and incubated at room temperature for different times. Subsequently, DNA quality was assessed. In a parallel experiment, hair roots were incubated in spring water for different time periods. This study demonstrates that after one week of immersion of the bulletproof vest parts in a canal only one sample from more than 100 samples gave a partial genetic profile. No genetic profile were obtained for the 99 other samples. After one month immersion and despite the finding that blood remained detectable on bulletproof vest parts, no genetic profile was obtained for all samples using the classical STR approach. For longer immersion times, no genetic profiles were obtained. In sink pipe water, an incubation time of 72 h (h) was necessary before significant blood degradation occurred. Nevertheless, high inter-sample variability was observed. This high variability may be explained by the variability of water composition coming from nine different sink pipes. For hair root cells incubated in water, we observed that more than 90% of the DNA was degraded after 72 h.

Comparison of performance of genetics 4N6 FLOQSwabs™ with or without surfactant to rayon swabs.

The collection of traces is the first step in the process of forensic genetics analysis. Currently, several different techniques are used (eg. gauze). Nevertheless, swabbing appears to be the most common of these. In a second step, the sampling devices should allow the use of preliminary tests in combination with an immunological confirmatory test (e.g. Hexagon Obti or Hemdirect). Our previous study shows that sampling with Genetics 4N6FLOQswabs™ coated with surfactant reduces by a factor of at least 100 the detection threshold of blood using two immunological tests. The aim of this work was to compare the ability to recover blood trace and the compatibility with immunological confirmatory test of various Genetics 4N6FLOQswabs™ nylon flocked swabs with or without surfactant. The results obtain in this study show that Genetics 4N6FLOQswabs™ not coated with surfactant and Human DNA free FLOQswabs™ were suitable for the used in combination with immunological blood detection tests. Nevertheless, the Genetics 4N6FLOQswabs™ not surfactant coated give a better blood trace recovery.

Nylon flocked swab severely reduces Hexagon Obti sensibility.

Hexagon Obti immunological blood test and flocked swab are widely used in forensic laboratories. Nevertheless, up to now, no compatibility tests have been published between sampling with the ethylene oxide treated flocked swab and the Hexagon Obti blood detection strip. In this study, we investigated this compatibility. Our work shows that sampling with ethylene oxide treated flocked swab reduces by a factor of at least 100 the detection threshold of blood using the Hexagon Obti immunological test.

Down-regulation and decreased activity of cyclin-dependent kinase 2 in H2O2-induced premature senescence.

Premature senescence of human diploid fibroblasts (HDFs) induced by exposure to H2O2 at subcytotoxic concentration is characterized by many biomarkers of normal senescence such as irreversible growth arrest. Cyclin-dependent kinase inhibitor (CdKI) p21(Waf-1) is overexpressed in H2O2- and tert-butylhydroperoxide-induced premature senescence, likely explaining in part the hypophosphorylation of the retinoblastoma protein. p21(Waf-1) is known to inhibit the kinase activity of the cyclin-dependent kinase (CdK) 4 and 6 cyclin complexes. In this work, we investigated whether the kinase activity of the CdK4 and 6 cyclin complexes can be modulated by CdKI p16(Ink-4a), by changes in the protein level of CdKs and cyclins, or by changes in kinase activity of these CdKs not directly involving CdKIs. RNase protection assay, semi-quantitative RT-PCR, Western blot and kinase assay showed that the mRNA level, protein and kinase activity of CdK2 are decreased at 72h after H2O2 stress. These results suggest that the hypophosphorylation of the retinoblastoma protein is mediated in part by a decrease of the kinase activity of CdK2 not directly involving CdKIs. This CdK2-mediated effect should be considered in addition to the inhibition of cyclin D-CdK4 and 6 complexes by CdKI p21(Waf-1).

From the Hayflick mosaic to the mosaics of ageing. Role of stress-induced premature senescence in human ageing.

The Hayflick limit-senescence of proliferative cell types-is a fundamental feature of proliferative cells in vitro. Various human proliferative cell types exposed in vitro to many types of subcytotoxic stresses undergo stress-induced premature senescence (SIPS) (also called stress-induced premature senescence-like phenotype, according to the definition of senescence). The known mechanisms of appearance the main features of SIPS are reviewed: senescent-like morphology, growth arrest, senescence-related changes in gene expression, telomere shortening. Long before telomere-shortening induces senescence, other factors such as culture conditions or lack of 'feeder cells' can trigger either SIPS or prolonged reversible G(0) phase of the cell cycle. In vivo, 'proliferative' cell types of aged individuals are likely to compose a mosaic made of cells irreversibly growth arrested or not. The higher level of stress to which these cells have been exposed throughout their life span, the higher proportion of the cells of this mosaic will be in SIPS rather than in telomere-shortening dependent senescence. All cell types undergoing SIPS in vivo, most notably the ones in stressful conditions, are likely to participate in the tissular changes observed along ageing. For instance, human diploid fibroblasts (HDFs) exposed in vivo and in vitro to pro-inflammatory cytokines display biomarkers of senescence and might participate in the degradation of the extracellular matrix observed in ageing.

Signal transduction in H2O2-induced senescence-like phenotype in human diploid fibroblasts.

A stress-induced senescence-like phenotype is induced by exposure of human diploid fibroblasts to subcytotoxic H2O2 stress. Previous studies showed that TGF-beta1 is responsible for the induction of several biomarkers of replicative senescence within 72 h after stress: senescence-like morphology, senescence-associated beta-galactosidase activity, and an increase in the mRNA steady state level of four senescence-associated genes. Other studies showed that the retinoblastoma protein is responsible for the appearance of these biomarkers in the same conditions. Here we show that sustained p38(MAPK) phosphorylation is responsible for both H2O2-induced overexpression of TGF-beta 1 and subsequent TGF-beta 1-induced appearance of these biomarkers. p38(MAPK) phosphorylation is shown to be necessary for a self-sustained TGF-beta 1 overexpression after H2O2 stress through the activation of ATF-2 transcription factor, thereby creating a regulatory loop between sustained p38(MAPK) activation and sustained TGF-beta 1 overexpression after stress. p38(MAPK) activation is also shown to be responsible in part for the growth arrest observed in stress-induced senescence-like phenotype. At 48 h after stress, ATF-2 starts to interact with hypophosphorylated Rb, which allows the biomarkers of stress-induced senescence-like phenotype to appear. This report gives an overall explanation of how a senescence-like phenotype is established after subcytotoxic H2O2 stress.

Approach of evolutionary theories of ageing, stress, senescence-like phenotypes, calorie restriction and hormesis from the view point of far-from-equilibrium thermodynamics.

B. L. Strehler wrote that "Any system that is not in thermodynamic equilibrium will approach that state at a rate that is a function of absolute temperature and the energy barriers to the rearrangements of components". Far-from-equilibrium thermodynamics allows a global systemic description of the cellular behaviour. This approach transcends the genetic and stochastic considerations on ageing as well as some evolutionary questions about ageing. The fundamental difference between the processes of development and ageing could reflect the intrinsic differences existing between biological systems where an increase in specific entropy production (SEP) is, respectively, still possible or not. The increase of the potential of SEP which probably occurred with evolution might explain in part why life span could increase. However, this SEP-driven increase in life span was possible only in those species which did not take advantage of their increased potential of SEP to ameliorate their reproductive capacity at the expense of possible increases in repair capacity. The criteria of stability of far-from-equilibrium open systems and the theory of attractors also help to sort the possible types of cellular stress responses: normal ageing, hormesis, stress-induced premature senescence, apoptosis or necrosis.

An alternative internal splicing site defines new Ikaros isoforms in Pleurodeles waltl.

The Ikaros gene encodes a family of transcription factors which plays a crucial role in hematopoiesis. To improve our knowledge about the immune system of Pleurodeles waltl, we sequenced the cDNA coding for the Ik-1 isoform of that salamander and analyzed its tissue expression by semi-quantitative RT-PCR. Ikaros transcripts are abundant in the thymus and the spleen, thereby confirming that these organs are, respectively, the primary and secondary lymphoid tissues of Pleurodeles. Analysis of the isoforms produced by this animal revealed two isoforms characteristic of amphibians in which an alternative internal splicing site deletes the 3' half of exon 3 which interestingly comprises the first Zn finger of Ikaros. Ikaros transcripts were found at the earliest stages of development of Pleurodeles indicating that Ikaros has a function at the very early lymphopoietic stages. Moreover, the presence of Ikaros transcripts in spermatozoa suggests that this protein could have another and yet unknown function.

Stress-induced premature senescence or stress-induced senescence-like phenotype: one in vivo reality, two possible definitions?

No consensus exists so far on the definition of cellular senescence. The narrowest definition of senescence is irreversible growth arrest triggered by telomere shortening counting cell generations (definition 1). Other authors gave an enlarged functional definition encompassing any kind of irreversible arrest of proliferative cell types induced by damaging agents or cell cycle deregulations after overexpression of proto-oncogenes (definition 2). As stress increases, the proportion of cells in "stress-induced premature senescence-like phenotype" according to definition 1 or "stress-induced premature senescence," according to definition 2, should increase when a culture reaches growth arrest, and the proportion of cells that reached telomere-dependent replicative senescence due to the end-replication problem should decrease. Stress-induced premature senescence-like phenotype and telomere-dependent replicatively senescent cells share basic similarities such as irreversible growth arrest and resistance to apoptosis, which may appear through different pathways. Irreversible growth arrest after exposure to oxidative stress and generation of DNA damage could be as efficient in avoiding immortalisation as "telomere-dependent" replicative senescence. Probabilities are higher that the senescent cells (according to definition 2) appearing in vivo are in stress-induced premature senescence rather than in telomere-dependent replicative senescence. Examples are given suggesting these cells affect in vivo tissue (patho)physiology and aging.

Efficiency of a novel forensic room-temperature DNA storage medium.

The success of forensic genetics has led to considerable numbers of DNA samples that must be stored. Thus, the ability to preserve the integrity of forensic samples is essential. The possibility of retesting these samples after many years should be guaranteed. DNA storage typically requires the use of freezers. Recently, a new method that enables DNA to be stored at room temperature was developed. This technology is based on the principles of anhydrobiosis and thus permits room-temperature storage of DNA. This study evaluates the ability of this technology to preserve DNA samples mimicking true mixture casework samples for long periods of time. Mixed human DNA from 2 or 3 persons and at low concentrations was dried and stored for a period ranging from 6 months to 2 years in the presence of a desiccant. The quality of the stored DNA was evaluated based on quantitative peak height results from Short Tandem Repeat (STR) genotyping and the number of observed alleles. Furthermore, we determined whether this matrix has a potential inhibitory or enhancing effect on the PCR genotyping reactions. In our previous work, we demonstrated the considerable potential of this new technology. The present study complements our previous work. Our results show that after 2 years of aging at room temperature, there is a decrease in the number of observed alleles and in the peak height of these alleles.

Inositol 1,4,5-trisphosphate 3-kinase B (Itpkb) controls survival, proliferation and cytokine production in mouse peripheral T cells.

Mice genetically-deficient for the B isoform of the inositol 1,4,5-trisphosphate 3-kinase (or Itpkb) have a severe defect in thymocytes differentiation and thus lack peripheral T cells. In order to study the functional role of Itpkb in peripheral T cells, we constructed a new mouse where a transgene encoding mouse Itpkb is specifically and transiently expressed in thymocytes of Itpkb(-)(/)(-) mice. This allows a partial rescue of mature thymocyte/T cell differentiation and thus the functional characterization of peripheral T cells lacking Itpkb. We show here that Itpkb(-)(/)(-) CD4(+) and CD8(+) peripheral T cells present important functional alterations. Indeed, an increased activated/memory phenotype as well as a decreased proliferative capacity and survival were detected in these T cells. These Itpkb-deficient peripheral T cells have also an increased capacity to secrete cytokines upon stimulation. Together, our present results define the important role of Itpkb in peripheral mature T cell fate and function in mouse, suggesting a potential role for Itpkb in autoimmunity.

Evaluation of novel forensic DNA storage methodologies.

An issue in forensic sciences is the secure storage of extracted DNA. Most of the time, DNA is frozen at -20°C or -80°C. Recently, new room temperature DNA storage technologies have been developed based on anhydrobiosis. Two products use this technology: Qiasafe (Qiagen) and Gentegra (Genvault). In this study we focused on the recent Gentegra product and initiated a comparison versus -20°C and Qiasafe storage. We compared the quantity and quality of DNA stored using anhydrobiosis technology against DNA stored at -20°C, by performing STR profiling after short term storage. Furthermore, we studied the quantity and integrity of DNA after long term storage. Our results prove the high potential of this technology but it seems to be extraction dependent. Phenol/chloroform extracted DNA could be stored using the Gentegra matrix for more than 6 months without any obvious degradation. However, DNA extracted using magnetic beads could not be safely stored over the same period. Adaptations are therefore required to store this kind of samples.

Stress-induced premature senescence: from biomarkers to likeliness of in vivo occurrence.

The similarities between the biomarkers of stress-induced premature senescence and replicative senescence are reviewed. The possibility of existence of 'molecular scars', i.e. long-term changes observed after subcytotoxic stress and not observed in replicative senescence, is considered. Lastly, the likeliness of existence of stress-induced premature senescence in vivo is discussed. The possible effects on normal and pathological tissue ageing are predicted.