Validation study of endogenous reference genes for normalization of quantitative real time PCR data in post mortem skin tissue

Gene expression profiling may provide insights into the molecular mechanisms underlying wound skin repair contributing to applications in the field of post mortem forensic wound age estimation. Quantitative real-time PCR (qPCR) is the most sensitive technique for gene expression studies but represents a complex procedure with several requirements for accurate data analysis. One of the major pre-requisites is the selection and validation of appropriate endogenous control genes for normalization in a given set of samples. In this study, the gene expression stabilities of ten endogenous controls ACTß, ß2M, PPIA, GAPDH, HPRT1, PGK1, SDHA, TBP, UBC, and YWHAZ were evaluated in two sets of samples: post mortem human wounded and unwounded skin tissue aimed for subsequent wound age estimation studies. The most stable genes were determined by calculating a gene expression normalization factor for each sample based on the geometric mean of the ten selected genes using the reference gene validation software, geNorm. In uninjured skin, the most stable endogenous controls were YWAZ, with the highest stability, followed by PGK1 and GAPDH. In the wounded samples, GAPDH was the most stable gene followed by PGK1, ACTß and HPRT1. Both groups revealed some differences relative to reference gene expression stabilities, nevertheless, PGK1 and GAPDH could serve as two possible common reference genes for qPCR data normalization as both were identified as stable genes in wounded and unwounded skin tissues. The differences of expression stabilities of the reference genes identified in both groups support the need for accurate normalization of gene-expression levels previous to qPCR studies.     

Development and usage of a NIST standard reference material for real time PCR quantitation of human DNA

National Institute of Standards and Technology SRM 2372 human DNA quantitation standard has been produced to support the need for a human-specific DNA quantitation standard in forensic casework and calibration of new quantitative polymerase chain reaction (qPCR) assays. The conventional DNA concentration has been assigned with one of the U.S. National Reference UV/Visible Spectrophotometers, assuming an absorbance of 1.0 at 260 nm equals 50 ng/μL of double stranded DNA. In addition, an interlaboratory study has been conducted, to verify that the SRM 2372 materials perform well in currently used DNA quantitation assays by the forensic DNA community. Each unit of SRM 2372 consists of three well-characterized DNA extracts. Component A is a single-source human male material derived from blood. Component B is a multiple-source human female material derived from blood. Component C was purchased as a purified unsheared genomic human DNA (Sigma-Aldrich Co., St. Louis, MO) obtained as a lyophilized human genomic extract and has both male and female donors. SRM 2372 is intended to enable the comparison of DNA concentration measurements across time and place. Manufacturers can use SRM 2372 to validate the values assigned to their own reference materials. Individual forensic laboratories can use SRM 2372 to validate DNA quantitation methods and to verify the assigned concentration of in-house or commercial DNA calibration standards.     

基于角膜图像的人体死亡时间推断模型初探

目的尸体角膜随死后时间延长发生的形态学变化是规律性较好的指标,常用来判断死亡时间(postmortem interval,PMI)。本文尝试用机器视觉代替人的肉眼主观判断,收集尸体样本以建立通过人体角膜图像推断PMI的模型。方法收集实际案例建立包含505例人体死后角膜图像的数据库,PMI范围为0.24h(约死后14min)至492h(约死后20.5d),大致分为三类(依次为:0~<6h、6~<20h、20h及以上)或二类(0~<15h、15h及以上);使用由华盛顿大学陈天奇博士提出的Xgboost模型分别进行二分类与三分类分析;使用多种卷积神经网络模型分别进行分类和回归学习,并通过比较最终选择了由微软研究院提出的ResNet模型进行分析。结果Xgboost在三分类时预测准确率依次为71.8%、40.7%、65.7%,二分类时为90%、48.5%。ResNet分类模型中,精准率、召回率在三分类时分别依次为:81%、75%,30%、50%,61%、71%,二分类时为:70%、92%,76%、38%。ResNet回归模型中,比较整个模型的预测结果,0~6h内的预测值与真实值较为接近,均值误差为0.5616,均方误差为0.5873,6h之后开始出现较大误差。结论分类和回归模型都在0~6h之内得到了很好的结果,说明在此时间段内,角膜图像噪声较低,可预测性强。     

The GC–MS detection and characterization of reticuline as a marker of opium use

Reticuline (a precursor of opium alkaloids) was detected and characterised as its trimethylsilyl ethers, acetyl esters and methyl ethers by GC–EIMS and GC–CIMS in opium and the urine of opium users after hydrolysis by acid or β-glucuronidase as coextractive of morphine. Because this compound cannot be detected in heroin and poppy seeds, it is suggested as a differentiating marker between opium and heroin use, opium and poppy seeds use, or opium and “pharmaceutical” codeine use in cases when opiate use has been confirmed by detection of morphine and codeine in the urine. As well as being a constituent of opium, reticuline in the urine of opium users may also result from the metabolic demethylation of the three other benzyltetrahydroisoquinoline opium alkaloids: codamine, laudanosine and laudanine.     

Automated washing of FTA Card punches and PCR setup for reference samples using a LIMS-controlled Sias Xantus automated liquid handler

We have implemented and validated automated methods for washing FTA Card punches containing buccal samples and subsequent PCR setup using a Sias Xantus automated liquid handler. The automated methods were controlled by worklists generated by our LabWare Laboratory Information Management System (LIMS). The protocols were validated according to EN/ISO 17025 for use with STR amplifications kits AmpF?STR^® Identifiler^® and Y-filer^® (Applied Biosystems).     

Usefulness of combining clinical and radiological dental findings for a more accurate noninvasive age estimation

The aim of this study was to establish correlations of clinical and radiological dental findings, alone or in combination, with chronological age in adults. Dental findings and orthopantomograms of 984 patients (aged 20-60 years; 524 females/460 males) were analyzed. DMF-T index and distance (alveolar bone level, ABL) between cemento-enamel junction and alveolar bone margin were recorded. Additionally, clinical and radiological findings at each tooth crown and root were collected according to the actual status of destruction and restoration, and a total score for each dentition (TSD) was calculated. After univariate correlation analysis, correlation coefficients for ABL and TSD were improved by using square root (sqrt). However, the determination accuracy was still not satisfactory; 90% of the residuals were in the range of about +/-10. The present study showed that clinical and radiological dental findings could not be used, not even in combination, for accurate age estimation as a single method, but that they could support other methods.     

Simulating transfer and persistence of a chemical marker powder for Lycopodium clavatum spores

In this research a chemical marker powder, based on Lycopodium clavatum spores, was studied to determine its transfer and persistence on a T-shirt. Such chemical marker powders are used to provide evidence that a person has handled a covertly marked object, such as a drug package. The powder was found to transfer readily between a marked item and the person handling it. The powder was found to persist on a T-shirt for up to 13 h; however, there was only a very small amount of powder remaining at this time. The rate of loss of the L. clavatum spores was found to follow a decay curve. The largest decrease in spores from the T-shirt was seen in the first 2 h after the marked item had been handled.     

A multiplex analysis of RNA expression during injury healing in human dermal injuries for injury age estimation

The determination of the timing of a human dermal injury is important in forensic work as it provides valuable information that helps evaluate the relationship between the injury and the crime, and aids in the reconstruction of a crime. In spite of its importance, currently there are no routinely used methods for the accurate estimation of human dermal injury age. In this study, we developed a RNA based multiplex assay to simultaneously investigate the mRNA expression of chymase 1 (CMA1), collagen type III alpha I (COL3A1), dual specificity phosphate 1 (DUSP1), interleukin 1beta (IL1β), interleukin 2 (IL2), interleukin 6 (IL6), interleukin 7 (IL7), tumor necrosis factor alpha (TNFα) and vascular endothelial growth factor A (VEGFA) in human dermal injuries to identify and evaluate their suitability for human dermal injury age estimation. The mRNA expression patterns observed for CMA1, DUSP1, IL1β, IL7, TNFα and VEGFA suggest they have potential to estimate human dermal injury age, especially during the early stages (days) of injury healing.     

大鼠死后脑组织RNA降解与死亡时间推断的研究

目的探讨SD大鼠死后脑组织中甘油醛-3-磷酸脱氢酶(GAPDH)mRNA、18s rRNA含量的比值与死亡时间(PMI)的关系。方法SD大鼠处死后分别置于15℃、20℃环境中,采用TRIzol试剂提取SD大鼠在死后即刻、1、2、3、5、7d时脑组织中的总RNA,采用RT-PCR方法与凝胶图像分析技术检测18s rRNA和GAPDH mRNA的含量,并将二者的比值(GAPDHmRNA/18s rRNA)与PMI进行统计学回归分析。结果SD大鼠死后脑组织中GAPDH mRNA在2d时降解不明显,但到3、5、7d时降解程度显著增加,且随着温度的升高,降解速度增加;而18S rRNA降解缓慢,直到7d仍无显著性降解变化,且降解速度几乎不受温度的影响。对GAPDHmRNA/18s rRNA值(Y)与PMI(X)进行回归相关分析:20℃时,Y=0.62-0.004X(R=-0.981,P<0.001);15℃时,Y=0.965-0.001X(R=-0.709,P<0.001)。结论SD大鼠死后脑组织中GAPDHmRNA和18s rRNA基因的相对稳定性即GAPDHmRNA/18s rRNA值与PMI存在明显相关性。     

A note on exogenous changes in incentives for and deterrence of corruption

In this note we amend the model elaborated by Acemoglu and Verdier (2000, The American Economic Review, 90(1),194–211), to examine how agents respond to an exogenous change in incentives. In particular, we apply the theoretical framework to the consequences on the corruption level in Italy of a famous sentence of the Italian Corte di Cassazione in plenary session: in this sentence, no. 500 of 1999, a revolutionary interpretation of the law has increased the area of civil liability for both the public administration and bureaucrats. This is one of the few cases in which the judge substantially legislates in a system of civil law, and the modification in incentives towards or away from corruption comes from an authority that is not part of the game.      

mRNA在体液斑鉴定研究中内参基因的选择与应用

利用mRNA分析技术鉴定生物检材中各种体液斑迹的组织属性来源是近年来法医物证学的一个重要进展。终点逆转录PCR和实时荧光定量PCR技术是法医学实验室常用的mRNA检测技术,为保证实验结果的准确性,研究者通常选用组成性表达的管家基因作为内参,对不同样本提取RNA进行质控和标准化,以正确评估目标mRNA在各样本中的表达量。本文聚焦近年来使用mRNA分析技术进行体液斑迹组织属性来源鉴定的研究报道,对这些工作中内参基因的选择与应用效果进行综述。     

A comparison of memory for homicide, non-homicidal violence, and positive life experiences

Defendants commonly claim amnesia for their criminal actions especially in cases involving extreme violence. While some claims are malingered or result from physiological factors, other cases may represent genuine partial or complete amnesia resulting from the psychological distress and/or extreme emotion associated with the perpetration of the crime. Fifty Canadian homicide offenders described their memories of their homicide, a non-homicide violent offense, and their most positive adulthood life experience. Self-reported and objective measures of memories for these events revealed that homicides were recalled with the greatest level of detail and sensory information. Although dissociative tendencies were associated with a self-reported memory loss, objective measures of memory quality did not reflect this perceived impairment, suggesting a failure of meta-memory. Recollections of positive life events were superior to those of non-homicidal violence, possibly due to greater impact and meaning attached to such experiences. Findings suggest that memory for homicide typically is enhanced by the powerful emotion associated with its perpetration.     

The marriage license as a hitting license: A comparison of assaults in dating,cohabiting, and married couples

There are many studies of marital and dating violence. However, methodological differences between these studies make it difficult to determine differences in the nature and extent of physical assault between marital status groups. This paper helps fill that gap by analyzing data from two surveys: a study of 526 dating couples at a large midwestern university, and a study of a national probability sample of 5005 married and 237 cohabiting couples. The results show that cohabiting couples have a higher rate of assault than dating and married couples. These findings persist after controls for age, education, and occupational status are introduced. Violence is also more severe in cohabiting than dating or married couples. A number of factors may account for the more frequent violence in cohabiting relationships. These include social isolation, the issue of autonomy and control, and the investment in the relationship.     

A new method for the recovery and evidential comparison of footwear impressions using 3D structured light scanning

Footwear impressions are one of the most common forms of evidence to be found at a crime scene, and can potentially offer the investigator a wealth of intelligence. Our aim is to highlight a new and improved technique for the recovery of footwear impressions, using three-dimensional structured light scanning. Results from this preliminary study demonstrate that this new approach is non-destructive, safe to use and is fast, reliable and accurate. Further, since this is a digital method, there is also the option of digital comparison between items of footwear and footwear impressions, and an increased ability to share recovered footwear impressions between forensic staff thus speeding up the investigation.     

A comparison of methods used in the UK and Ireland for the extraction and detection of semen on swabs and cloth samples

The recent formation of a United Kingdom and Irish working group, the Body Fluids Forum (BFF), highlighted the need to investigate different working practices prior to any inter-laboratory comparison work and identification of best practice. Various dilutions of semen were seeded onto swabs and cloth samples for each BFF member laboratory to test using their standard techniques. The results showed that the detection of acid phosphatase on swabs is best achieved using direct testing rather than on an extract from the swab. Extraction methods for spermatozoa require a balance to be achieved between using a sufficient volume of water to ensure optimal release and minimal volume to ensure a concentrated extract. PSA tests were investigated and found to be more sensitive than Choline. DNA profiles were obtained from samples in which no spermatozoa had been detected during microscopic examination.     

Mental Health Review Tribunals: A comparison of the content of tribunal reports for discharged and not discharged patients and for the effect of legal classification

Abstract

This study examined the content of reports provided for Mental Health Review Tribunals. A retrospective design was used to compare the tribunal reports of fifty patients. Two groups of twenty five patients were compared, one including patients discharged by tribunals and the other patients not discharged. Social combination theory and valence theory were used to analyse the content of the reports and to assess whether they were associated with tribunal outcome. Opinion statements discussing suitability for discharge were more closely associated with outcome than fact statements. For the statements presenting subjects positively or negatively, fact statements were more closely associated with outcome. The valence of both types of statements was also found to be related to outcome, with more positive values being achieved for discharged subjects. Comparison of reports written for previous tribunals before any of the subjects in the study were discharged indicated that those subjects who went on to be discharged at their next tribunal had the least negative valences. In addition to this, the effect of legal classification on outcome was explored, and indicated a difference in the valence of reports for discharged and not discharged patients, depending on classification of mental illness or psychopathic disorder.     

Face recognition as a function of distance and illumination: A practical tool for use in the courtroom

Abstract

This paper describes a practical courtroom tool that provides insight in face recognition accuracy as a function of distance and illumination. Subjects were shown 3/4 target portraits with an exposition time of 12 seconds, immediately followed by a target-absent or target-present lineup of six full-face portraits. Subjects were asked to identify the target. Seven distances (3 to 40 meter) and nine illumination levels (0.3 to 3000 lux) were used which resulted in a 7?9 matrix, with in the cells a hit score and a false alarm score. From these rough data several other measures were derived, like d-prime, diagnostic value and some idealised scores. The scores represent the upper margins of recognition accuracy with the memory component reduced to a minimum. The results clearly indicate a systematic increase of recognition performance with decreasing distance and increasing illumination. The end result is a practical rule of thumb, the Rule of Fifteen: Even in ideal conditions the desired diagnostic value of 15 is reached at not more than 15 meters, not less than 15 lux.     

A Comparison of Civil and Criminal Orders of Protection as Remedies for Domestic Violence Victims in a Midwestern County

Past research has identified many potential advantages of civil protection orders as a means of addressing domestic violence without invoking an official response of the criminal justice system. Using data from a Midwestern county, this exploratory study provides a comparison of civil protection orders with orders of protection that are filed in conjunction with a criminal battering arrest. We examine the demographic characteristics of the respondents/defendants and petitioners/victims, the nature of the abuse leading up to the filing of the protection order, the reasons for filing, the terms of the order, location of the offense, and violations of orders. Our findings reveal many similarities between types of orders, in terms of order stipulations, past abuse histories of petitioners/victims, and respondents/defendants’ likelihood of reoffending. The findings also reveal an important difference in the petitioners’ and victims’ reasons for filing; whereas petitioners in civil cases were more likely to identify emotional abuse as the factor leading up to issuance of the order, victims in criminal cases were more likely to document physical abuse. We discuss these findings in the context of victim preference.     

A comparison of the use of visible microspectrometry and high performance thin layer chromatography for the discrimination of aqueous inks used in porous tip and roller ball pens

Ink lines made by porous tip and roller ball pens have been examined by high performance thin layer chromatography (HPTLC) and by transmission microspectrophotometry (VM) in the visible region. Better discrimination between apparently similar coloured inks was obtained with HPTLC but VM has value as an additional technique for ink examination.