mtDNA Heteroplasmy in Hair Shafts versus Buccal Swabs for Forensic Applications

Mitochondrial DNA (mtDNA) analysis has several forensic applications such as criminal investigations, identification of human remains, and missing person investigations. It is also the only type of DNA that is available from certain sample types such as hair shafts. The presence of mtDNA heteroplasmy within and between tissue types can lead to mtDNA sequence differences when comparing samples originating from the same individual. Studies on mtDNA heteroplasmy are increasingly being carried out for their implications in forensic interpretation of mtDNA sequences. Specifically, mtDNA in buccal swabs compared to hair samples from one individual may show differences in sequence due to heteroplasmy, and casework samples compared to reference swabs may exhibit differences that must be correctly interpreted to prevent faulty conclusions made by investigators and scientists alike. Establishing expected rates of heteroplasmy in mtDNA extracted from hair shaft samples and comparison to mtDNA extracted from buccal swab samples will lead to increased confidence in mtDNA interpretation. The goals of this study were to (1) successfully sequence the entire mtDNA control region from buccal swab samples from 5 volunteers using Sanger sequencing, (2) amplify smaller (\u3c300bp) sections of overlapping regions of the mtDNA control region from 15-20 hair shafts collected from three areas of the scalp using three methods of DNA extraction, and (3) evaluate mtDNA sequences from hair shafts and buccal swabs to identify heteroplasmy within and between samples. Overall, only 20% of the extracted hair samples resulted in half of Hypervariable Region 1 (HV1) being successfully sequenced from either the 5´ or 3´ end. Two out of five participants showed length heteroplasmy in the poly-cytosine region beginning at position 303 within the HV2 region. Point heteroplasmy was observed in one participant at one position in the buccal swab (nucleotide position 16093) as well as at two different positions in a hair sample (nucleotide positions 16258 and 16288) that did not show heteroplasmy in the buccal swab. The heteroplasmy seen in the buccal swab could not be compared to the hair sample as position 16093 did not fall within the successfully sequenced region in the hair. Although only a small subset of hair shafts were successfully sequenced, this study has succeeded in showing that mtDNA heteroplasmy seen in a hair shaft may not be present in buccal swab mtDNA. Further research into rates of heteroplasmy in hair shafts vs. buccal swabs is paramount to bettering the interpretative abilities of forensic scientists working with mtDNA and preventing false exclusions

The infuence of the host microbiome on 3,4- methylenedioxymethamphetamine (MDMA)-induced hyperthermia and vice versa

Hyperthermia induced by 3,4-methylenedioxymethamphetamine (MDMA) can be life-threatening. Here, we investigate the role of the gut microbiome and TGR5 bile acid receptors in MDMA-mediated hyperthermia. Fourteen days prior to treatment with MDMA, male Sprague-Dawley rats were provided water or water treated with antibiotics. Animals that had received antibiotics displayed a reduction in gut bacteria and an attenuated hyperthermic response to MDMA. MDMA treated animals showed increased uncoupling protein 1 (UCP1)and TGR5 expression levels in brown adipose tissue and skeletal muscle while increased expression of UCP3 was observed only in skeletal muscle. Antibiotics prior to MDMA administration significantly blunted these increases in gene expression. Furthermore, inhibition of the TGR5 receptor with triamterene or of deiodinase II downstream of the TGR5 receptor with iopanoic acid also resulted in the attenuation of MDMA-induced hyperthermia. MDMA-treatment enriched the relative proportion of a Proteus mirabilis strain in the ceca of animals not pre-treated with antibiotics. These findings suggest a contributing role for the gut microbiota in MDMA-mediated hyperthermia and that MDMA treatment can trigger a rapid remodeling of the composition of the gut microbiome

Pregnancy Weight Gain in Twin Gestations and Maternal and Child Health Outcomes at 5 Years.

Current guidelines for maternal weight gain in twin pregnancy were established in the absence of evidence on its longer-term consequences for maternal and child health. We evaluated the association between weight gain in twin pregnancies and the risk of excess maternal postpartum weight increase, childhood obesity, and child cognitive ability

Diagnostic value of immunostaining in cultured skin fibroblasts from patients with oxidative phosphorylation defects

In the last decades, a large variety of oxidative phosphorylation (OXPHOS) defects have been reported, expressed as an increasing variety of clinical phenotypes. With the expanding number of genes and proteins involved, new screening techniques leading to more effective diagnostic routes are in ever-increasing demand. Cultured skin fibroblasts from a cohort of patients with various OXPHOS defects, previously recognized by enzyme activity studies and blue native PAGE, were investigated with an immunocytochemical technique. Cytospins of cultured fibroblasts were air dried, fixed, and stained with antibodies specifically directed against subunits of each OXPHOS complex. Control cells stained homogeneously and strongly. In fibroblasts from five out of seven patients with a severe deficiency of one of the OXPHOS complexes, a homogeneous reduction of cytoimmunoreactivity of the affected complex was observed. In five out of seven fibroblast strains harboring a mitochondrial tRNA mutation, a mosaic pattern of staining was observed for both complexes I and IV, reflecting the heteroplasmic nature of the defect. The proportion of deficient fibroblasts varied considerably between cell strains from different subjects. The method described offers a convenient and rapid approach to first-line screening of OXPHOS defects. In association with routine assays of enzyme activity, the technique is helpful in orienting molecular investigation further

Transformation of normal human cells in the absence of telomerase activation

Our knowledge of the transformation process has emerged largely from studies of primary rodent cells and animal models. However, numerous attempts to transform human cells using oncogene combinations that are effective in rodents have proven unsuccessful. These findings strongly argue for the study of homologous experimental systems. Here we report that the combined expression of adenovirus E1A, Ha-RasV12, and MDM2 is sufficient to convert a normal human cell into a cancer cell. Notably, transformation did not require telomerase activation. Therefore, we provide evidence that activation of telomere maintenance strategies is not an obligate characteristic of tumorigenic human cells. Copyright © 2002 Cell Press