RESUMO
Bite mark injuries often feature in violent crimes. Conventional morphometric methods for the forensic analysis of bite marks involve elements of subjective interpretation that threaten the credibility of this field. Human DNA recovered from bite marks has the highest evidentiary value, however recovery can be compromised by salivary components. This study assessed the feasibility of matching bacterial DNA sequences amplified from experimental bite marks to those obtained from the teeth responsible, with the aim of evaluating the capability of three genomic regions of streptococcal DNA to discriminate between participant samples. Bite mark and teeth swabs were collected from 16 participants. Bacterial DNA was extracted to provide the template for PCR primers specific for streptococcal 16S ribosomal RNA (16S rRNA) gene, 16S-23S intergenic spacer (ITS) and RNA polymerase beta subunit (rpoB). High throughput sequencing (GS FLX 454), followed by stringent quality filtering, generated reads from bite marks for comparison to those generated from teeth samples. For all three regions, the greatest overlaps of identical reads were between bite mark samples and the corresponding teeth samples. The average proportions of reads identical between bite mark and corresponding teeth samples were 0.31, 0.41 and 0.31, and for non-corresponding samples were 0.11, 0.20 and 0.016, for 16S rRNA, ITS and rpoB, respectively. The probabilities of correctly distinguishing matching and non-matching teeth samples were 0.92 for ITS, 0.99 for 16S rRNA and 1.0 for rpoB. These findings strongly support the tenet that bacterial DNA amplified from bite marks and teeth can provide corroborating information in the identification of assailants.
Assuntos
Proteínas de Bactérias/genética , Mordeduras Humanas/microbiologia , DNA Bacteriano/genética , DNA Espaçador Ribossômico/genética , RNA Ribossômico 16S/genética , Streptococcus/isolamento & purificação , Dente/microbiologia , Adulto , Impressões Digitais de DNA , Primers do DNA/química , Primers do DNA/genética , RNA Polimerases Dirigidas por DNA , Humanos , Reação em Cadeia da Polimerase , Infecções Estreptocócicas/diagnóstico , Infecções Estreptocócicas/genética , Infecções Estreptocócicas/microbiologia , Streptococcus/classificação , Streptococcus/genéticaRESUMO
The adiponutrin/PNPLA3 gene is highly expressed in adipose tissue and liver. Its expression is down-regulated by fasting and rapidly induced by refeeding a high carbohydrate diet. We aimed to determine whether the promoter region of adiponutrin is regulated by glucose and insulin. Endogenous adiponutrin mRNA was increased in mouse 3T3-L1 and human SGBS adipocytes and in human HepG2 cells cultured in 25 mM glucose compared to absence of glucose. A 3100 bp 5'-upstream region of the human adiponutrin gene was cloned into a luciferase reporter plasmid and used in transient transfection studies. Promoter activity was up-regulated by 25 mM glucose, 4.7-fold in HepG2 cells and 2-fold in CHO cells. The effect was shown in CHO cells to be concentration dependent and to depend on glucose metabolism as a non-metabolisable analogue was without effect. In CHO cells constitutively expressing human insulin receptor (CHO-IR), there was a concentration dependent increase of promoter activity by insulin in the presence of glucose. Cotransfection with an expression plasmid for upstream stimulatory factor 2 (USF2), increased promoter activity 1.6-fold in CHO-IR cells. The combined effect of insulin and USF2 (2.3-fold) was greater than the individual effects. Cotransfection of carbohydrate-response element binding protein did not elicit any induction of promoter activity. These results point to potential mechanisms for the observed in vivo nutritional regulation of adiponutrin expression and its up-regulation in fatty liver and by obesity.
