Difficulties of sex determination from forensic bone degraded DNA: A comparison of three methods.

International audienceSex determination is of paramount importance in forensic anthropology. Numerous anthropological methods have been described, including visual assessments and various measurements of bones. Nevertheless, whatever the method used, the percentage of correct classification of a single bone usually varies between 80% and 95%, due to significant intra- and inter-population variations, and sometimes variations coming from secular trends. DNA is increasingly used in a forensic context. But forensic DNA extraction from bone raises several issues, because the samples are very often badly altered and/or in very small quantity. Nuclear DNA is difficult to get from degraded samples, according to low copy number, at least in comparison with mitochondrial DNA. In a forensic context (as in a paeleoanthropological context) DNA sex determination is usually complicated by the weak amount of DNA, the degraded nature of nucleic acids, the presence of enzymatic inhibitors in DNA extracts, the possible faint amplification of Y band and the risk of contamination during either excavation or manipulation of samples. The aim of this work was to compare three methods of DNA sex determination from bones: procedure #1 using a single PCR amplification, procedure #2 using a double PCR amplification, and procedure #3 adding bleaching for decontamination of the bone, instead of simply rubbing the bone. These processes were applied to samples of bones (49 samples coming from 39 individuals) that were in various states of post mortem alteration. The main results are the following. (i) No DNA could be extracted from three skulls (parietal bones, mastoid process), the compact bone of one rib, and the diaphysis of one femur; (ii) there was a contamination in three skulls; and (iii) the Y band did not appear in two male cases, with one of the three procedures (male tibia, procedure #2) and with procedures #2 and #3 (male femur). This study emphasises the main issue while working with altered bones: the impossibility to extract DNA in some cases, and, worth of all, the contamination of the sample or the faint amplification of Y band which leads to a wrong sex answer. Multiple and significant precautions have to be taken to avoid such difficulties

Interleukin-7 partially rescues B-lymphopoiesis in osteopetrotic oc/oc mice through the engagement of B220+ CD11b+ progenitors.

OBJECTIVE: We recently identified in the mouse bone marrow a B-lymphoid/myeloid B220+ CD11b+ progenitor population. This population is accumulated in the osteopetrotic oc/oc mouse, which suggests that it could be controlled by bone marrow factors whose expression varies in this pathologic bone environment. Among the possible factors, interleukin (IL)-7 is involved in the control of B lymphopoiesis and osteoclastogenesis. Therefore, we hypothesized that IL-7 could regulate the accumulation of the B220+ CD11b+ population in oc/oc mice. METHODS: B220+ CD11b+ cells sorted from oc/oc mice were treated with IL-7 and their phenotype was analyzed by flow cytometry and real-time reverse transcriptase polymerase chain reaction (RT-PCR). In vivo, IL-7 was injected in oc/oc mice, and B220+ CD11b+ and B cells, as well as B-cell proliferation and apoptosis, were analyzed by flow cytometry. The expression of B lymphopoiesis and myelopoiesis markers was analyzed by real-time RT-PCR. RESULTS: In vitro, IL-7 induced the differentiation of B220+ CD11b+ cells into B lymphocytes through the induction of Pax5 and the inhibition of myeloid markers. In vivo, IL-7 injections in oc/oc mice induced a decrease of the B220+ CD11b+ population and the partial restoration of B-cell population, which was reduced in oc/oc mice. In parallel, upon IL-7 injections, Pax5 expression was induced in B220+ cells and B-cell apoptosis was reduced. CONCLUSIONS: Our results demonstrate that IL-7 injection can partially rescue B lymphopoiesis in oc/oc mice through the engagement of the B220+ CD11b+ population in the B-lymphoid pathway. Therefore, IL-7 delivery could represent a new therapeutic perspective to circumvent the lymphopenia observed in infantile malignant osteopetrosis patients

The Effect of Parent Marital Status on the Happiness and Aspirations of College Students

The purpose of this study is to examine an effect of marriage or divorce and expressed parental personalities may have on the children\u27s personal view of themselves and their aspirations. Research by Lu (Lu, 1997) shows that characteristics of age and gender had indirect effects of happiness through social support, Thus we hypothesize the following: First, children of parents who stayed married will have a higher sense of happiness, through extensive social support opportunities. Secondly, we believe children of married parents will have a higher focus on attending college and more prestigious career choices. Finally, children of parents who stayed married will have a higher score on a happiness scale, perhaps because of more extensive social support opportunities. Research will be conducted by distributing a survey to participants. The survey is created through Qualtrics, and consists of 50 questions asking about demographics, parent\u27s marital statuses, aspirations, and their views of both parents. One aspect explored in our study focuses on the participant\u27s happiness, which was done by implementing questions from the Subjective Happiness scale (SHS) (Lyubomirsky, 1999), as well as their current aspirations, and whether they perceive themselves as following closely to a certain parental figure, or following their own distinctive path. Data collection is ongoing, and currently approximately 200 participants have completed the survey. Our study may reveal that people with married parents are perceived happier and have higher goals set for themselves

Establishment and characterization of new osteoclast progenitor cell lines derived from osteopetrotic and wild type mice.

Malignant infantile osteopetrosis is a rare and lethal disease characterized by the absence of bone resorption due to inactive osteoclasts (OCLs). Among the murine models of osteopetrosis, the Tcirg1oc/oc mouse is the most resembling to the human pathology. In the majority of patients as in Tcirg1oc/oc mouse, the gene involved is the Tcirg1 gene, encoding the a3 subunit of the vacuolar proton pump. However, to date, no osteoclastic cell lines from osteopetrotic mice are available to facilitate the study of either OCL differentiation in osteopetrosis or the factors involved in the control of Tcirg1 gene expression. Heterozygotes Tcirg1+/oc mice were crossed with p53+/- mice to obtain homozygotes p53-/-Tcirg1oc/oc and p53-/-Tcirg1+/+ animals. The p53-/-Tcirg1oc/oc mice display the same bone and hematological phenotype as the original Tcirg1oc/oc mice. From the bone marrow of these mice, we have derived cell lines named POC-MGoc/oc and POC-MG+/+. These cell lines express standard osteoclastogenic markers and differentiate into OCLs in the presence of RANK-L and M-CSF. Furthermore, both cell lines can be transduced by a lentiviral vector with a high efficiency and without alteration of their OCL differentiation potential. Therefore, these cell lines provide valuable new tools to study the differentiation and function of osteoclasts in normal and resorption defective conditions

Cost effectiveness of osseointegration

Individuals with limb amputation fitted with conventional socket-suspended prostheses often experience socket-related discomfort leading to a significant decrease in quality of life. Bone-anchored prostheses are increasingly acknowledged as viable alternative method of attachment of artificial limb. In this case, the prosthesis is attached directly to the residual skeleton through a percutaneous fixation. To date, a few osseointegration fixations are commercially available. Several devices are at different stages of development particularly in Europe and the US. [1-15] Clearly, surgical procedures are currently blooming worldwide. Indeed, Australia and Queensland, in particular, have one of the fastest growing populations. Previous studies involving either screw-type implants or press-fit fixations for bone-anchorage have focused on biomechanics aspects as well as the clinical benefits and safety of the procedure. [16-25] In principle, bone-anchored prostheses should eliminate lifetime expenses associated with sockets and, consequently, potentially alleviate the financial burden of amputation for governmental organizations. Sadly, publications focusing on cost-effectiveness are sparse. In fact, only one study published by Haggstrom et al (2012), reported that “despite significantly fewer visits for prosthetic service the annual mean costs for osseointegrated prostheses were comparable with socket-suspended prostheses”.[26] Consequently, governmental organizations such as Queensland Artificial Limb Services (QALS) are facing a number of challenges while adjusting financial assistance schemes that should be fair and equitable to their clients fitted with bone-anchored prostheses. Clearly, more scientific evidence extracted from governmental databases is needed to further consolidate the analyses of financial burden associated with both methods of attachment (i.e., conventional sockets prostheses, bone-anchored prostheses). The purposes of the presentation will be: 1. To outline methodological avenues to assess the cost-effectiveness of bone-anchored prostheses compared to conventional sockets prostheses, 2. To highlight the potential obstacles and limitations in cost-effectiveness analyses of bone-anchored prostheses, 3. To present preliminary results of a cost-comparison analysis focusing on the comparison of the costs expressed in dollars over QALS funding cycles for both methods of attachment

Novel mutations in the TCIRG1 gene encoding the a3 subunit of the vacuolar proton pump in patients affected by infantile malignant osteopetrosis

International audienceFifty percent of the infantile malignant osteopetrosis (IMO) cases reported in the literature present mutations in the TCIRG1 gene encoding the 116-kDa osteoclast specific subunit of the vacuolar proton ATPase (ATP6I). In this study, we identified four novel mutations in a series of six IMO patients. All of these mutations correspond to single nucleotide changes and affect splice acceptor or donor sites, resulting in aberrant transcription products. We report also a missense mutation, G405R, previously described in several Costa Rican patients. This independent finding suggests that the highly conserved residue at amino acid 405 plays a critical role in the a3 subunit function. Finally, the results of this study were used to provide a prenatal diagnosis to one of the families

Monocytes differentiation upon treatment with a peptide corresponding to the C-terminus of activated T cell-expressed Tirc7 protein

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Human Primary Osteocyte Differentiation in a Three-Dimensional Culture System.

International audienceAbstract Introduction: Investigations on primary osteocytes, which compose over 90-95% of bone cells, embedded throughout the mineralized matrix, is a major challenge due to their difficult accessibility and the very rare models available in vitro. We engineered a three-dimensional (3D) culture method of primary human osteoblast differentiation into osteocytes. These 3D-differentiated osteocytes were compared with 2D-cultured cells and with human microdissected cortical osteocytes obtained from bone cryosections. Materials and Methods: Human primary osteoblasts were seeded either within the interspace of calibrated biphasic calcium phosphate particles or on plastic culture dishes and cultured for four weeks in the absence of differentiation factors. Osteocyte differentiation was assessed by histological and immunohistological analysis after paraffin embedding of culture after various times as well as by quantitative RT-PCR analysis of a panel of osteoblast and osteocyte markers after nucleic acid extraction. Results: Histological analysis revealed, after only one week, the presence of an osteoid matrix including many lacunae in which the cells were individually embedded, exhibiting characteristics of osteocyte-like cells. Real time PCR expression of a set of bone-related genes confirmed their osteocyte phenotype. Comparison with plastic-cultured cells and mature osteocytes microdissected from human cortical bone allowed to assess their maturation stage as osteoid-osteocytes. Conclusions: This model of primary osteocyte differentiation is a new tool to gain insights into the biology of osteocytes. It should be a suitable method to study the osteoblast-osteocyte differentiation pathway, the osteocyte interaction with the other bone cells and orchestration of bone remodeling transmitted by mechanical loading and shear stress. It should be used in important cancer research areas such as the crosstalk of osteocytes with tumor cells in bone metastasis since it has been recently shown that gene expression in osteocytes is strongly affected by cancer cells of different origin. It could be also a very efficient tool for drug testing and bone tissue engineering applications