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1.
Leg Med (Tokyo) ; 45: 101715, 2020 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-32413723

RESUMO

The identification of vaginal fluid from casework samples of sexual assaults provides important probative evidence of vaginal intercourse. The aim of this study was to establish a more specific procedure for identifying vaginal fluids for forensic purposes. Vaginal fluid marker candidates have been evaluated quantitatively and five of these markers (ESR1, SERPINB13, KLK13, CYP2B7P1, MUC4) have been amplified simultaneously by a multiplex reverse transcription-polymerase chain reaction (RT-PCR) procedure. Each amplicon has been separated and quantified automatically using chip electrophoresis. Subsequently, in the present study, detectability and cross-reactivity of the developed multiplex procedure were assessed in detail using various forensically relevant body fluids. Then, a cutoff value for the positive detection of vaginal fluids was set for each marker by Youden index. The ability of the multiplex RT-PCR assay to distinguish between vaginal and other body fluids was evaluated statistically using a likelihood ratio (LR) that was estimated using a Bayesian estimation approach to consider the infrequency of detection. A high LR was obtained when all five markers showed positive results (LR = 4.33 × 109; 95% credible interval, 3.95 × 107 -2.87 × 1012). The developed procedure was validated using vaginal fluid samples under various conditions. High LRs were found for aged vaginal fluid stains, although each amplicon peak was low. It was also able to identify vaginal stains mixed with other body fluids. In conclusion, the multiplex RT-PCR-based procedure followed by the statistical evaluation using LR could be a powerful tool for the objective identification of vaginal fluids.

2.
Leg Med (Tokyo) ; 46: 101713, 2020 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-32442862

RESUMO

An evaluation of a Rapid DNA system was performed using buccal swab samples and mock Disaster Victim Identification (DVI) samples collected postmortem. The allelic ladder success rate was 90% and samples analyzed simultaneously with this allelic ladder were used for further analysis. Sample success rate of the Rapid DNA system for buccal swab samples, and blood and muscle DVI samples were calculated. Success rates of buccal swab samples were 100% and 75% using cassettes preloaded with all reagents suitable for high- and low-DNA content samples, respectively. Success rates of fresh DVI samples were 80% to 100%. Success rates of putrefied DVI samples varied widely between 0% and 20% and 50% to 80% depending on cassette and sample types. Conventional DNA analysis was performed for comparison with the results of the Rapid DNA system. DNA quantity and degradation of human DNA were measured using quantitative polymerase chain reaction. DVI samples that yielded more than 1 ng/µL of DNA when extracted with conventional protocols were suitable for analysis using cassettes for both high- and low-DNA content samples. DVI samples with less than 0.1 ng/µL of DNA were suitable only for analysis using cassettes for low-DNA content samples. All alleles called and exported by the Expert system software implemented in the Rapid DNA system were concordant with allele calls made by conventional capillary electrophoresis DNA analysis.

3.
Forensic Sci Int ; 306: 110077, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31821940

RESUMO

Forensic samples are commonly influenced by various environmental factors, including ultraviolet (UV) irradiation; thus, forensic applications of DNA repair (e.g., PreCR™, Restorase®) have been investigated, focusing on short tandem repeat typing. However, current DNA-based examinations are used for both human and body fluid identification. This study thus aims to clarify the efficacy of a DNA repair approach for Streptococcus salivarius DNA-based identification of saliva from UV-damaged samples. Artificial UV-damaged genomic DNA of S. salivarius, drop saliva stains, and buccal swabs were used to evaluate the effects of DNA repair on S. salivarius DNA detection by using PreCR™ repair reagent. To evaluate forensic applications, we prepared mock forensic samples by exposing them to environmental conditions. Melting curve analysis following real-time PCR was applied for qualitatively detecting S. salivarius DNA with a specific melting peak of 80.5°C±0.4°C (n=10, mean ± 3SD). Single PCR was used for quantitative and qualitative analyses, whereas dual PCR was used for S. salivarius DNA qualitative detection. DNA repair experiments using artificial UV-damaged samples revealed a significant increase of only the quantitative value of genomic DNA samples by DNA repair. Moreover, significant quantitative DNA repair effects were not observed in all mock forensic samples, indicating the limitations of DNA repair for actual cell-derived DNA samples. Whereas, differences of qualitative results (with or without detection) were generated for mock forensic samples; thus, we consider the DNA repair strategy as an additional approach for S. salivarius DNA-based identification of saliva from environmentally damaged evidence.


Assuntos
Reparo do DNA , Saliva/microbiologia , Streptococcus salivarius/genética , Raios Ultravioleta/efeitos adversos , Dano ao DNA , DNA Bacteriano/genética , Humanos , Indicadores e Reagentes , Repetições de Microssatélites , Reação em Cadeia da Polimerase em Tempo Real
4.
Jpn Dent Sci Rev ; 55(1): 121-125, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31660092

RESUMO

Half a century has passed since the department for education and research on forensic odontology was established at dentistry-related universities in Japan in 1964. In order to meet the demands of society, the number of universities with a department of forensic odontology increased up until around 2005. In 2007, the Japanese Society of Forensic Dental Science was established, and then a series of reforms such as establishment of the Study Council on Death Cause Investigation in both the National Police Agency and the Cabinet Office of the Japanese government, cabinet decision of enactment and enforcement of new laws on death cause investigation, publication of an article on the Model Core Curriculum of Dental Education, publication of the results of a fact-finding survey on education and research on forensic odontology conducted by the Ministry of Education, Culture, Sports, Science and Technology, inclusion of questions about forensic odontology in the National Board Dental Examination, and compilation of a database on dental findings by the Ministry of Health, Labor and Welfare, proceeded in succession. We introduced the half century of forensic odontology in Japan in chronological order.

5.
Forensic Sci Int Genet ; 42: 103-112, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31302459

RESUMO

Analyzing degraded evidence is an important challenge in forensic casework. Saliva remaining at a crime scene may deteriorate, due to various factors, making it difficult to identify. This study aims to clarify the efficacy of oral gram-positive and -negative bacterial DNA-based identification of saliva for analyzing highly degraded samples. Saliva samples were subjected to three different degradation treatments (heat denaturation: 40-80 °C in wet conditions; microbial decomposition: 1-5 days in humid soil; and ultraviolet (UV) irradiation: 0.01-1 J/cm2). We compared saliva markers' detectability from the degraded samples-oral gram-positive bacterial DNA (Streptococcus salivarius and Streptococcus oralis), oral gram-negative bacterial DNA (Veillonella atypica and Prevotella maculosa) and salivary α-amylase. Oral bacterial DNA was detected using a melting curve analysis following real-time PCR. The efficacy of short tandem repeats (STR) and mitochondrial DNA (mtDNA) analyses were also compared. All oral bacterial DNA were detected with specific melting peaks from the heat-denatured samples, while neither catalytic nor immunochromatographic tests detected salivary α-amylase from the heat (80 °C) samples. The gram-positive bacterial DNA (S. salivarius and S. oralis) was detected from the microbial degradation (1-5 days) samples. In contrast, the gram-negative bacterial DNA (V. atypica and P. maculosa) and salivary α-amylase were not detected from samples treated for more than two days. UV exposure made bacterial DNA-based saliva identification difficult in a dose-dependent manner; however, UV irradiation did not influence protein-based saliva tests using salivary α-amylase as an indicator. As a result of STR and mtDNA typing, partial or null STR profiles were generated from the severely degraded (microbial (2-5 days) and UV (0.1-1 J/cm2) degradation) samples, but full mtDNA profiles were obtained from all degraded samples. The forensic applicability of bacterial DNA test evaluated, using mock case samples, indicates that the oral gram-positive bacterial DNA was more resistant to degradation than the other markers. We conclude that the oral gram-positive bacterial DNA-based examination could be useful for identifying saliva from severely environmentally-exposed forensic samples as well as mtDNA typing.


Assuntos
DNA Bacteriano/genética , Bactérias Gram-Negativas/genética , Bactérias Gram-Positivas/genética , Saliva/microbiologia , Adulto , Biomarcadores/metabolismo , Impressões Digitais de DNA , DNA Mitocondrial/genética , Exposição Ambiental/efeitos adversos , Feminino , Temperatura Alta/efeitos adversos , Humanos , Masculino , Repetições de Microssatélites , Pessoa de Meia-Idade , RNA Ribossômico 16S/genética , Reação em Cadeia da Polimerase em Tempo Real , Saliva/metabolismo , Raios Ultravioleta/efeitos adversos , Adulto Jovem , alfa-Amilases/metabolismo
6.
Leg Med (Tokyo) ; 38: 25-31, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30925381

RESUMO

Age estimation in adults based on aspartic acid racemization (AAR) provides fewer errors and higher precision than that based on bone morphology for the identification of cadavers. The technique has been established in some labs as a routine method. However, as the essential requisites for the technique, a wide age range of teeth of the same type as the target tooth must be collected for calibration for each examination. We investigated whether dentin standard samples could be prepared by increasing the AAR rate via heat. Powdered dentin was prepared from a maxillary first premolar (13 years) and heated for 0-72 h at 110 °C. The extent of AAR increased significantly with heating time and the correlation was strong (r = 0.913; p < 0.01). Similar results were found for a mandibular canine (24 years, r = 0.948; p < 0.01) and a maxillary third molar (20 years, r = 0.944; p < 0.01). We attempted to estimate the age of four maxillary first premolars of persons aged 25-58 years by using the heated samples (18 years, 12 h to 7 days). The differences between the actual and estimated ages were within ±5 years. The stability of the AAR rates in the powdered dentin during storage at 22-25 °C, 4 °C, and -30 °C was examined after 1 year and no significant changes had occurred. We were able to prepare dentin standard samples and created a calibration curve. This is a pilot study that needs to be validated before it can be used in forensic practice.


Assuntos
Determinação da Idade pelos Dentes/métodos , Ácido Aspártico/química , Dentina/química , Temperatura Alta , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Dente Pré-Molar , Feminino , Humanos , Masculino , Maxila , Pessoa de Meia-Idade , Dente Serotino , Projetos Piloto , Fatores de Tempo , Adulto Jovem
7.
J Forensic Leg Med ; 62: 97-102, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30731391

RESUMO

Atmospheric radiocarbon (14C) levels increased from 1955 to 1963 due to atmospheric nuclear weapon tests, and then decreased. As 14C accumulates in human tooth enamel while the enamel is being formed, 14C can be used to estimate the birth year of unidentified bodies. Measurement results of 14C content in tooth enamel using accelerator mass spectrometry vary depending on the enamel's sample site. To address this problem, a method for equalizing samples using a pulverizer was considered in this study. Regarding the tube and cone used as the pulverizer, (1) a polycarbonate tube and stainless steel cone, (2) a stainless steel tube and cone, and (3) a tungsten carbide tube and cone, were compared. In (1), the modern carbon ratio was approximately half that of the normal ratio of 100 pMC, with which accurate dating was impossible, and in (2), a high background value was obtained for IAEA-C1, which was pulverized using a reusable tube and cone. In (3), the 14C content for IAEA-C1 pulverized using reusable tube and cone, which was washed with quartz sand, was 0.31 ±â€¯0.01 pMC. This result did not show any problems regarding background value. Therefore, the use of tungsten carbide products and washing with quartz sand is recommended for 14C measurement of pulverized teeth.


Assuntos
Determinação da Idade pelos Dentes/métodos , Esmalte Dentário/química , Datação Radiométrica , Radioisótopos de Carbono/análise , Odontologia Legal/métodos , Humanos , Espectrometria de Massas , Pós , Quartzo , Manejo de Espécimes , Compostos de Tungstênio
8.
J Forensic Sci ; 64(3): 873-877, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30339736

RESUMO

The stability of salivary α-amylase is a critical factor in both catalytic and immunological method-based forensic saliva identification. This study aimed to assess the sensitivity of catalytic and immunological tests on degraded saliva samples. Degraded saliva stains were prepared by microbial decomposition using humid soil. Salivary α-amylase activity was catalytically detected both qualitatively and quantitatively using the Phadebas® amylase test. As immunological methods, we conducted qualitative and quantitative tests using the RSID™-saliva test and ELISA, respectively. Salivary α-amylase activity of degraded samples (incubated at 37°C for 12 h) was significantly lower than that of controls in the quantitative tests. All the degraded samples obtained by the humid soil produced negative results in the Phadebas® tests, but showed positive results in the RSID™-saliva test and ELISA. These results suggest that immunological tests are effective for testing degraded saliva samples that have lost their enzymatic activity.


Assuntos
Medicina Legal/métodos , Saliva/enzimologia , alfa-Amilases/análise , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoensaio/métodos , Masculino , Solo/química
9.
Forensic Sci Int ; 288: 297-303, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29800936

RESUMO

Forensic facial approximation is a technique used to estimate the antemortem facial features of unknown skeletal remains. In recent years, many researchers have reported nasal tip predictions with positive results. However, the morphological nasal features of the skull can vary widely, and it is hard to obtain accurate values using facial approximation techniques. We assumed that these variations are due to an over-dependence on the values obtained from a single distance metric factor in an anatomical area. Measurements were acquired using cephalometric radiographic images obtained from 190 Japanese individuals (90 men, aged 18-36 years and 100 women, aged 18-46 years). Soft tissue and skeletal features were traced onto acetate sheets. The orbitale (Or), porion (Po), and the Frankfurt Horizontal Plane (FHP) were plotted in addition to the rhinion (Rhi), anterior nasal spine (ANS), subnasale (Sn), prosthion, and point-A (A). From these, the following were measured: a length from rhinion to prosthion; b length from rhinion to the intersection of a line perpendicular to the anterior nasal spine; c length from the prosthion to the intersection of a line perpendicular to the anterior nasal spine; g the proportion of d/b; and f the proportion of c/b. A calculation was generated from these measurements and from proportions of a-h, and applied to the samples. An R-squared (RSQ) test and standard error (SE) were used to compare the actual and predicted values. The errors observed between the predicted and actual values were not greater than 5mm in any of the samples; 91.3% and 71.2% of predicted Sn had an error lower than 2.5 and 1.5mm respectively, from the actual. Reliable results were obtained using the method in the present study. In addition, in the process of obtaining the measurements, we found reliable proportional differences between the sexes in the piriform and axillary alveolar regions.


Assuntos
Grupo com Ancestrais do Continente Asiático , Restos Mortais , Cefalometria , Face/anatomia & histologia , Crânio/anatomia & histologia , Adolescente , Adulto , Pontos de Referência Anatômicos , Feminino , Antropologia Forense/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Estatísticos , Caracteres Sexuais , Adulto Jovem
10.
Leg Med (Tokyo) ; 33: 36-41, 2018 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-29777949

RESUMO

The source of small amounts of touch DNA, which is transferred from the skin to an object when it is handled or touched, could be an issue in the forensic analysis of criminal cases. Here, we performed an extended evaluation of skin- or sweat-characteristic mRNAs to investigate their usability to infer whether an object has been handled or touched by someone. First, we compared the expression levels of candidate genes between skin swabs and other body fluids by quantitative RT-PCR analysis. Among the analyzed genes, corneodesmosin (CDSN), late cornified envelope 1C (LCE1C), filaggrin (FLG), desmocollin 1, and dermcidin were selected for further analysis on the basis of their specificities and sensitivities. Then, we tried to detect these genes from mock casework samples. As a result, CDSN, LCE1C, and FLG could be good markers because of their detectability. Finally, we determined the correlation between the expression of these genes and DNA yield of skin swabs to assess their adaptability as a screening test for touch DNA samples. However, the detectability of these genes was not correlated with the DNA yield of skin swab samples. In conclusion, gene expression analysis of the skin- or sweat-characteristic mRNAs CDSN, LCE1C, and FLG could be useful for inferring the skin origin of touched contact traces, but the use of the expression levels of these mRNAs for the prediction of DNA yield is problematic. To develop a screening test for touch DNA samples, other markers that have a well-correlated sensitivity with DNA analysis should be investigated.

11.
Sci Justice ; 57(6): 404-408, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29173452

RESUMO

Identifying saliva in samples found at crime scenes is important to clarify the tissue origin of DNA obtained for identification of individuals. Recently, a novel messenger RNA-based approach using two saliva-specific markers, Statherin (STATH) and Histatin 3 (HTN3), has been reported. This method can identify saliva more specifically than conventional amylase-based methods. Here, we performed several evaluations related to applying this method to real-world forensic work. First, we evaluated the effects of exposure to blue light (450nm) or to the reagent on Phadebas paper, which are direct methods used to locate saliva stains, on the stability of the RNA markers. The results demonstrate that exposure to the two direct tests did not affect the stability of the RNA markers. Second, we performed a comparative analysis of RNA-based and amylase-based conventional methods to examine the sensitivity and stability of the markers under various storage conditions. Although there was no difference in the sensitivity of the two methods for detecting 1-day-old saliva stains, a time-course study demonstrated that the RNA saliva markers were less stable than amylase, especially in wet conditions. During this time-course experiment, the stability of human DNA was also investigated. Although DNA was also unstable in wet conditions, it was more stable than the RNA markers in dry conditions. Taking the above results into consideration, we suggest that the RNA method could be introduced to current saliva identification procedures and should be used as a supplementary method to strongly support identification of saliva by the amylase-based method.


Assuntos
Histatinas/genética , RNA Mensageiro/análise , Saliva/química , Proteínas e Peptídeos Salivares/genética , Biomarcadores/análise , Medicina Legal , Histatinas/análise , Humanos , Proteínas e Peptídeos Salivares/análise
12.
Int J Legal Med ; 131(2): 359-364, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27349904

RESUMO

The identification of vomit stains may be helpful for crime scene reconstruction. However, there is no specific and convenient method for identifying vomit stain. Therefore, to establish the procedure for forensic identification of vomit stains, we focused on four gastric mucosa-expressing proteins, pepsinogen I (PGA), pepsinogen II (PGC), gastrin (GAST), and mucin 5AC (MUC5AC). We developed enzyme-linked immunosorbent assay (ELISA) procedures for the detection of these four candidate proteins. The specificity and sensitivity of ELISA detection of these proteins were analyzed, and applicability for the identification of vomit in forensic casework samples was also investigated. We found the sensitivities of ELISA for detection of PGA, PGC, GAST, and MUC5AC from the standard protein (peptide) and from diluted gastric mucosa extract were 10.0-100.0 ng/ml and 1:200-1:1600, respectively. PGA and PGC were successfully detected in stomach contents and gastric mucosa samples; however, these also cross-reacted with some urine and semen samples, respectively, because of low level expression in these fluids. MUC5AC was positive for most gastric mucosa samples; however, it was difficult to detect in stomach contents. ELISA detection of GAST was not suitable for the identification of vomit. All aged samples stored up to 90 days gave positive results for ELISA procedures for PGA, PGC, and MUC5AC. Therefore, ELISA detection of these proteins might be applicable to aged samples. PGA was also detected in all actual vomit samples tested. These results suggest that ELISA for the detection of gastric mucosa-expressing proteins, especially PGA, could be an effective tool for the forensic identification of vomit.


Assuntos
Mucosa Gástrica/metabolismo , Conteúdo Gastrointestinal , Vômito , Adulto , Idoso , Idoso de 80 Anos ou mais , Ensaio de Imunoadsorção Enzimática , Medicina Legal/métodos , Gastrinas/metabolismo , Humanos , Pessoa de Meia-Idade , Mucina-5AC/metabolismo , Pepsinogênio A/metabolismo , Pepsinogênio C/metabolismo , Sensibilidade e Especificidade
13.
Leg Med (Tokyo) ; 22: 49-53, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27591539

RESUMO

The identification of blood samples obtained from crime scenes has been an important step in forensic investigation. Recently, a novel approach using the blood-specific methylated CpG site cg06379435 has been reported. In this study, we developed a real-time polymerase-chain-reaction-based method that can simply and rapidly quantitate the methylation ratio of cg06379435 and its neighboring CpGs and set the threshold ratios for blood identification by analyzing various body fluid samples. Blood identification using the thresholds was successfully performed in the analysis of a small amount (1ng) of DNA from blood and various aged blood samples, including 29-year-old stains. We also demonstrated a test for allele-specific blood identification from a mixed DNA sample by bisulfite sequencing analysis of these CpG sites and their neighboring single nucleotide polymorphism, rs7359943 (A/G), which is of relevance in cases where mixed samples are obtained from crime scenes. The stability of DNA methylation in aged samples and the usefulness of neighboring genetic information shown in this study suggest that DNA-methylation-based body fluid identification will play a major role in future forensic investigations.


Assuntos
Alelos , Líquidos Corporais/química , Metilação de DNA/genética , Genética Forense/métodos , Humanos , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA
14.
Expert Opin Drug Metab Toxicol ; 12(7): 743-52, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27144662

RESUMO

INTRODUCTION: The absorption, distribution, metabolism, excretion and toxicity (ADME(T)) of oxime reactivators have been assessed with respect to their polarity, a fundamental requirement for their specific mechanism of action in the intoxication with organophosphorous compounds. The limitations of the therapeutic outcome have been associated not only with the severity of intoxication and to particularities of the toxicants, but also to the reduced lipophilicity and consequent restricted permeability across biological barriers. AREAS COVERED: This article inventories the plethora of mnemotic rules developed throughout the years for defining chemical spaces where drugs share one or more structural and ADME(T) characteristics. Their applicability to oxime is analyzed, especially in relation to intestinal absorption and brain distribution. Other aspects of oximes for antidotal outcome are also reviewed. EXPERT OPINION: The drugability rules are not applicable to oxime reactivators, because the increase in lipophicity and consequent improved permeability across biological barrier comes together with amplified (neuro)toxicity and reduced reactivating capacity. The available data suggest a high solubility and reduced metabolism, assigning the quaternary oximes to the fourth class of Biopharmaceutical Classification Systems. Reliance upon oral absorption data for designing safe centrally acting oximes can be of potential value, with adequate characterization of uptake-influx transporters interplay.


Assuntos
Antídotos/administração & dosagem , Intoxicação por Organofosfatos/tratamento farmacológico , Oximas/administração & dosagem , Animais , Antídotos/química , Antídotos/farmacocinética , Encéfalo/metabolismo , Reativadores da Colinesterase/administração & dosagem , Reativadores da Colinesterase/química , Reativadores da Colinesterase/farmacocinética , Desenho de Fármacos , Humanos , Oximas/química , Oximas/farmacocinética , Permeabilidade , Solubilidade , Distribuição Tecidual
15.
J Forensic Leg Med ; 38: 75-80, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26724561

RESUMO

Skull-photo superimposition is a technique used to identify the relationship between the skull and a photograph of a target person: and facial reconstruction reproduces antemortem facial features from an unknown human skull, or identifies the facial features of unknown human skeletal remains. These techniques are based on soft tissue thickness and the relationships between soft tissue and the skull, i.e., the position of the ear and external acoustic meatus, pupil and orbit, nose and nasal aperture, and lips and teeth. However, the ear and nose region are relatively difficult to identify because of their structure, as the soft tissues of these regions are lined with cartilage. We attempted to establish a more accurate method to determine the position of the nasal tip from the skull. We measured the height of the maxilla and mid-lower facial region in 55 Japanese men and generated a regression equation from the collected data. We obtained a result that was 2.0±0.99mm (mean±SD) distant from the true nasal tip, when applied to a validation set consisting of another 12 Japanese men.


Assuntos
Cefalometria , Antropologia Forense/métodos , Processamento de Imagem Assistida por Computador , Nariz/anatomia & histologia , Crânio/anatomia & histologia , Adulto , Grupo com Ancestrais do Continente Asiático , Humanos , Japão , Masculino , Fotografação , Projetos Piloto , Análise de Regressão , Adulto Jovem
16.
J Forensic Sci ; 61 Suppl 1: S208-12, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26305854

RESUMO

The detection of semen in forensic investigation is considered important evidence of sexual assault. In this study, we report the development of a real-time polymerase chain reaction-based method for identifying semen that can simply and rapidly analyze the semen-specific unmethylated region of the DACT1 gene. Using two fluorescent probes designed for the methylated or unmethylated status, this method could perform quantitative analysis of the methylation status in this region. Furthermore, this method was used to analyze various body fluid samples, including 29-year-old semen and blood stains. The results showed that this method can detect almost exclusively semen or nonsemen signals even in highly decomposed samples, while a few semen or nonsemen samples showed slight signals of the other fluorescence probe. Although there is still a need for further analysis such as setting thresholds to analyze unknown samples, this method could be a useful supplementary tool for identifying semen, especially in old stains such as those in cold-case investigations.


Assuntos
DNA/análise , Reação em Cadeia da Polimerase em Tempo Real , Sêmen/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Líquidos Corporais , Corantes , Humanos , Metilação , Proteínas Nucleares/genética , Saliva , Fatores de Tempo
17.
Anal Bioanal Chem ; 407(23): 7135-44, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26164306

RESUMO

Vaginal fluid is one of the most common body fluids found at crime scenes. Discriminating vaginal fluid from other body fluids is important in forensic science; however, few potential protein markers have been reported to date. Proteomic methods for identifying protein markers have gained attention, although few reports have applied this technology to forensic protein markers. Therefore, to identify characteristic vaginal proteins, we examined various body fluids (nasal secretions, saliva, urine, semen, vaginal fluids, and sweat) using liquid chromatography/electrospray ionization time-of-flight mass spectrometry and peptide mass fingerprinting. We identified three components (average molecular mass values 17,237 ± 2, 18,063 ± 2, and 15,075 ± 1) detectable only in vaginal samples: two human small proline-rich protein 3 (SPRR3) isoforms and a human fatty acid-binding protein 5 (FABP5) with an acetylated (+42) N-terminal region lacking the initiator methionine residue (-131). Using ELISA, these yielded markedly high average values in vaginal fluids. The mass spectra of these proteins were not detected in infant saliva but were detected in the vaginal fluid throughout the menstrual cycle. The results of forensic analysis (detection limit, mixed body fluid samples, casework samples, and blind samples) suggest that these proteins are potential forensic markers. In conclusion, high SPRR3 and FABP5 expression levels, which may be used as potential markers for vaginal fluid identification in forensic science, were detected in vaginal fluids from healthy adults.


Assuntos
Líquidos Corporais/química , Proteínas Ricas em Prolina do Estrato Córneo/análise , Proteínas de Ligação a Ácido Graxo/análise , Mapeamento de Peptídeos/métodos , Estupro/diagnóstico , Vagina/química , Biomarcadores/análise , Biomarcadores/química , Cromatografia Líquida de Alta Pressão/métodos , Feminino , Medicina Legal/métodos , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Massas em Tandem/métodos
19.
Forensic Sci Int Genet ; 14: 11-7, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25270217

RESUMO

Silica column-based RNA purification procedures have widespread use in mRNA profiling for body fluid identification in forensic samples. Also, automated RNA purification systems employing magnetic bead technology have recently become available. In this preliminary study, to ascertain which RNA purification technology is more suitable for the identification of body fluids by real-time reverse transcription polymerase chain reaction (RT-PCR), comparative analyses of the yield and quality of total RNA were performed between automated purification using an EZ1 Advanced Instrument and manual purification using an RNeasy Mini Kit. The yield and size distribution of total RNA were compared by gene expression analysis of two different sized fragments of the ß-actin gene. In addition, the relative amounts of several target genes were compared between the purification methods, and the integrity of total RNA was determined by chip-based electrophoresis. The results of this study suggest that RNeasy can purify higher-quality RNA as compared with automated purification using EZ1. The sensitivity of the RT-PCR analysis, however, was higher in the EZ1-purified samples, likely due to the relative efficiency of EZ1 in extracting short-length RNA from degraded samples. We also show that the quantification of relative levels of body fluid-specific genes could be influenced by the purification procedure. Our results indicate that although use of high-quality RNA is generally required for reproducible results in gene expression analysis, the forensic relevance of short RNA fragments in highly degraded samples cannot be ruled out. Furthermore, our results suggest that automated purification procedures as well as silica column-based manual purification procedures can be used for mRNA-based body fluid identification in forensic samples.


Assuntos
Automação , Líquidos Corporais/metabolismo , RNA Mensageiro/genética , RNA/isolamento & purificação , Expressão Gênica , Humanos , RNA/química , RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
20.
Talanta ; 122: 172-9, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24720980

RESUMO

The liquid chromatographic behavior observed under bimodal retention conditions (reversed phase and hydrophilic interaction) offers a new basis for the determination of some derived lipophilicity indices. The experiments were carried out on a representative group (30 compounds) of pyridinium oximes, therapeutically tested in acetylcholinesterase reactivation, covering a large range of lipophilic character. The chromatographic behavior was observed on a mixed mode acting stationary phase, resulting from covalent functionalization of high purity spherical silica with long chain alkyl groups terminated by a polar environment created through the vicinal diol substitution at the lasting carbon atoms (Acclaim Mixed Mode HILIC 1 column). Elution was achieved by combining different proportions of 5 mM ammonium formiate solutions in water and acetonitrile. The derived lipophilicity indices were compared with logP values resulting from different computational algorithms. The correlations between experimental and computed data sets are significant. To obtain a better insight on the transition from reversed phase to hydrophilic interaction retention mechanisms, the variation of the thermodynamic parameters determined through the van׳t Hoff approach was also discussed.

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