A Blue Spectral Shift of the Hemoglobin Soret Band Correlates with the Age (Time Since Deposition) of Dried Bloodstains

The ability to determine the time since deposition of a bloodstain found at a crime scene could prove invaluable to law enforcement investigators, defining the time frame in which the individual depositing the evidence was present. Although various methods of accomplishing this have been proposed, none has gained widespread use due to poor time resolution and weak age correlation. We have developed a method for the estimation of the time since deposition (TSD) of dried bloodstains using UV-VIS spectrophotometric analysis of hemoglobin (Hb) that is based upon its characteristic oxidation chemistry. A detailed study of the Hb Soret band (λmax = 412 nm) in aged bloodstains revealed a blue shift (shift to shorter wavelength) as the age of the stain increases. The extent of this shift permits, for the first time, a distinction to be made between bloodstains that were deposited minutes, hours, days and weeks prior to recovery and analysis. The extent of the blue shift was found to be a function of ambient relative humidity and temperature. The method is extremely sensitive, requiring as little as a 1 µl dried bloodstain for analysis. We demonstrate that it might be possible to perform TSD measurements at the crime scene using a portable low-sample-volume spectrophotometer

Inhibition of Effector Function but Not T Cell Activation and Increase in FoxP3 Expression in T Cells Differentiated in the Presence of PP14

Background: T-helper polarization of naïve T cells is determined by a complex mechanism that involves many factors, eventually leading to activation of Th1, Th2, or Th17 responses or alternatively the generation of regulatory T cells. Placental Protein 14 (PP14) is a 28 kDa glycoprotein highly secreted in early pregnancy that is able to desensitize T cell receptor (TCR) signaling and modulate T cell activation. Methodology/Principal Findings: Prolonged antigen-specific stimulation of T cells in the presence of PP14 resulted in an impaired secretion of IFN-c, IL-5 and IL-17 upon restimulation, although the cells proliferated and expressed activation markers. Furthermore, the generation of regulatory CD4 + CD25 high Foxp3 + T cells was induced in the presence of PP14, in both antigen-specific as well as polyclonal stimulation. In accordance with previous reports, we found that the induction of FoxP3 expression by PP14 is accompanied by down regulation of the PI3K-mTOR signaling pathway. Conclusions/Significance: These data suggest that PP14 arrests T cells in a unique activated state that is not accompanied with the acquisition of effector function, together with promoting the generation of regulatory T cells. Taken together, our results may elucidate the role of PP14 in supporting immune tolerance in pregnancy by reducing T cell effector function

Changes in Dry State Hemoglobin over Time Do Not Increase the Potential for Oxidative DNA Damage in Dried Blood

BACKGROUND: Hemoglobin (Hb) is the iron-containing oxygen transport protein present in the red blood cells of vertebrates. Ancient DNA and forensic scientists are particularly interested in Hb reactions in the dry state because both regularly encounter aged, dried bloodstains. The DNA in such stains may be oxidatively damaged and, in theory, may be deteriorated by the presence of Hb. To understand the nature of the oxidative systems potentially available to degrade DNA in the presence of dried Hb, we need to determine what molecular species Hb forms over time. These species will determine what type of iron (i.e. Fe(2+)/Fe(3+)/Fe(4+)) is available to participate in further chemical reactions. The availability of "free" iron will affect the ability of the system to undergo Fenton-type reactions which generate the highly reactive hydroxyl radical (OH*). The OH* can directly damage DNA. METHODOLOGY/PRINCIPAL FINDINGS: Oxygenated Hb (oxyHb) converts over time to oxidized Hb (metHb), but this happens more quickly in the dry state than in the hydrated state, as shown by monitoring stabilized oxyHb. In addition, dry state oxyHb converts into at least one other unknown species other than metHb. Although "free" iron was detectable as both Fe(2+) and Fe(3+) in dry and hydrated oxyHb and metHb, the amount of ions detected did not increase over time. There was no evidence that Hb becomes more prone to generating OH* as it ages in either the hydrated or dry states. CONCLUSIONS: The Hb molecule in the dried state undergoes oxidative changes and releases reactive Fe(II) cations. These changes, however, do not appear to increase the ability of Hb to act as a more aggressive Fenton reagent over time. Nevertheless, the presence of Hb in the vicinity of DNA in dried bloodstains creates the opportunity for OH*-induced oxidative damage to the deoxyribose sugar and the DNA nucleobases

Gene Expression Profile of Neuronal Progenitor Cells Derived from hESCs: Activation of Chromosome 11p15.5 and Comparison to Human Dopaminergic Neurons

BACKGROUND: We initiated differentiation of human embryonic stem cells (hESCs) into dopamine neurons, obtained a purified population of neuronal precursor cells by cell sorting, and determined patterns of gene transcription. METHODOLOGY: Dopaminergic differentiation of hESCs was initiated by culturing hESCs with a feeder layer of PA6 cells. Differentiating cells were then sorted to obtain a pure population of PSA-NCAM-expressing neuronal precursors, which were then analyzed for gene expression using Massive Parallel Signature Sequencing (MPSS). Individual genes as well as regions of the genome which were activated were determined. PRINCIPAL FINDINGS: A number of genes known to be involved in the specification of dopaminergic neurons, including MSX1, CDKN1C, Pitx1 and Pitx2, as well as several novel genes not previously associated with dopaminergic differentiation, were expressed. Notably, we found that a specific region of the genome located on chromosome 11p15.5 was highly activated. This region contains several genes which have previously been associated with the function of dopaminergic neurons, including the gene for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, IGF2, and CDKN1C, which cooperates with Nurr1 in directing the differentiation of dopaminergic neurons. Other genes in this region not previously recognized as being involved in the functions of dopaminergic neurons were also activated, including H19, TSSC4, and HBG2. IGF2 and CDKN1C were also found to be highly expressed in mature human TH-positive dopamine neurons isolated from human brain samples by laser capture. CONCLUSIONS: The present data suggest that the H19-IGF2 imprinting region on chromosome 11p15.5 is involved in the process through which undifferentiated cells are specified to become neuronal precursors and/or dopaminergic neurons