Determination of the post mortem interval in skeletal remains by the comparative use of different physico-chemical methods: Are they reliable as an alternative to 14C?

The determination of the post-mortem interval (PMI) of skeletal remains is a challenging aspect in the forensic field. Previous studies focused their attention on different macroscopic and morphological aspects but a thorough and complete evaluation of the potential of chemical and physical analyses in this field of research has not been performed. In addition to luminol test and Oxford histology index (OHI) reported in a recent paper, widely spread and accessible methods based on physical aspect and chemical characteristics of skeletal remains have been investigated as potential alternatives to dating by determination of ¹⁴C.The investigation was performed on a total of 24 archeological and forensic bone samples with known PMI, with inductively coupled plasma optical emission spectrometer (ICP-OES), inductively coupled plasma quadruple mass spectrometry (ICP-MS), Fourier transform infrared (FT-IR) spectroscopy, energy dispersive X-ray analysis (EDX), powder X-ray diffraction analysis (XRPD) and scanning electron microscopy (SEM). Finally, the feasibility of such alternative methods was discussed. Some results such as carbonates/phosphates ratio from FT-IR, the amounts of organic and inorganic matter by EDX, crystallite sizes with XRPD, and surface morphology obtained by SEM, showed significant trends along with PMI. Though, from a chemical point of view cut-off values and gold-standard methods still present challenges, and rather different techniques together can provide useful information toward the assessment of the PMI of skeletal remains. It is however clear that in a hypothetical flowchart those methods may be placed practically at the same level and a choice should always consider the evaluation of results by each technique, execution times and a costs/benefits relationship.     

Evaluation of the suchey–brooks method of age estimation in an Australian subpopulation using computed tomography of the pubic symphyseal surface

Despite the prominent use of the Suchey–Brooks (S–B) method of age estimation in forensic anthropological practice, it is subject to intrinsic limitations, with reports of differential interpopulation error rates between geographical locations. This study assessed the accuracy of the S–B method to a contemporary adult population in Queensland, Australia and provides robust age parameters calibrated for our population. Three‐dimensional surface reconstructions were generated from computed tomography scans of the pubic symphysis of male and female Caucasian individuals aged 15–70 years (n = 195) in Amira® and Rapidform®. Error was analyzed on the basis of bias, inaccuracy and percentage correct classification for left and right symphyseal surfaces. Application of transition analysis and Chi‐square statistics demonstrated 63.9 and 69.7% correct age classification associated with the left symphyseal surface of Australian males and females, respectively, using the S–B method. Using Bayesian statistics, probability density distributions for each S–B phase were calculated, providing refined age parameters for our population. Mean inaccuracies of 6.77 (±2.76) and 8.28 (±4.41) years were reported for the left surfaces of males and females, respectively; with positive biases for younger individuals (<55 years) and negative biases in older individuals. Significant sexual dimorphism in the application of the S–B method was observed; and asymmetry in phase classification of the pubic symphysis was a frequent phenomenon. These results recommend that the S–B method should be applied with caution in medico‐legal death investigations of Queensland skeletal remains and warrant further investigation of reliable age estimation techniques. Am J Phys Anthropol, 2013. © 2013 Wiley Periodicals, Inc.     

RECLAIMING THE PATIENT'S VOICE AND SPIRIT IN DYING: AN INSIGHT FROM ISRAEL

In the latter half of the 20th century, Western medicine moved death from the home to the hospital. As a result, the process of dying seems to have lost its spiritual dimension, and become a matter of prolonging material life by means of medical technology. The novel quandaries that arose led in turn to medico‐legal regulation. This paper describes the recent regulation of dying in Israel under its Dying Patient Law, 2005. The Law recognizes advance directives in principle, but limits their effect and form through complex medico‐legal artifices. It reflects a culture that places high value on both scientific medicine and the sanctity of life as such, and illustrates a medical culture that pitches battle against death. At the same time, the Law constructs the will of the individual in a medico‐legal language that is alien to the lay person. The paper suggests an alternative approach to advance care planning that is patient‐centred and addresses the psycho‐social needs of the individual in terms of her relational autonomy. From this perspective, advance care planning becomes an opportunity to extract the patient from the medical context and allow her to speak about her approaching death with close ones in her own terms of reference. To this end, there is a need for facilitation of an intimate encounter where patients can speak about their concerns with their loved ones. The paper also presents a methodological approach of attentive listening, which can be applied across diverse cultures and circumstances.     

Flow system for the automatic screening of the effect of phenolic compounds on the luminol–hydrogen peroxide–peroxidase chemiluminescence system

In this work, an automated flow‐based procedure for the screening of the effect of the different phenolic compounds on the chemiluminescence (CL) luminol–hydrogen peroxide–horseradish peroxidase (HRP) system is presented. This procedure involves the combination of multisyringe flow injection analysis (MFSIA) and sequential injection analysis (SIA) techniques and exploits the ability of the different subgroups of phenols, such as cholorophenols, nitrophenols, methylphenols and polyphenols, to enhance or inhibit the described CL system. The implementation of this reaction in the SIA–MSFIA system enabled favourable and precise conditions to evaluate the effect of phenolic compounds, as it involves an in‐line reaction between the phenolic derivative, hydrogen peroxide and peroxidase and subsequent oxidized HRP intermediates generation prior to the fast reaction with the chemiluminogenic reagent. Several studies were then performed with the aim of establishing the appropriate flow system configuration and reaction conditions. It was shown that phenol and chlorophenols produce an enhanced CL response and nitrophenols, methylphenols and polyphenols are inhibitors within the range of concentrations studied (1–100 mg/L). Based on these studies, the developed method was applied to the determination of total polyphenol and phenol content in wine/grape seeds and water samples, respectively, and the results obtained showed good agreement with those furnished by the corresponding Folin–Ciocalteu and 4‐aminoantipyrine reference methods. The developed approach is further pursued by designing an automated generic tool for performing studies of peroxidase‐catalysed CL reactions of luminol focused on the detection of compounds that will affect the rate of those reactions. Copyright © 2011 John Wiley & Sons, Ltd.     

GdX/UBL4A null mice exhibit mild kyphosis and scoliosis accompanied by dysregulation of osteoblastogenesis and chondrogenesis

GdX, also named ubiquitin‐like protein 4A, is a ubiquitin‐domain protein characterized by a ubiquitin‐like domain that regulates the movement of misfolded proteins from the endoplasmic reticulum membrane to proteasome. However, its function in skeletal biology remains unclear. Here, we report that GdX plays a crucial role in skeletal development as mice lacking GdX exhibit skeletal dysplasias, mild kyphosis, and scoliosis. During embryonic stage, GdX knockout mice display decreased bone mineral density and trabecular bone accompanied by delayed osteogenic formation. GdX knockout mice also have blended spine and small body size. At the molecular level, GdX knockout mice showed perturbed expression of osteogenesis‐related genes and cartilage developmental genes, indicative of altered differentiation of mesenchymal cell lineage. Collectively, our results uncovered GdX as a novel regulator in bone development and a potential candidate gene for skeletal dysplasias.     

Kinetics of luminol sonochemiluminescence quenched by purines

A homogeneous chemiluminescence (CL) reaction was initiated by ultrasound irradiation. Luminol sonochemiluminescence (SCL) reaction kinetics were determined under pseudo‐first‐order conditions, and the reaction followed the model for simple rise–fall kinetics. In addition, SCL quenching reactions induced by purines were also investigated in which the interactions between luminol and purines were analysed using the Stern–Volmer (S‐V) mechanism. The results implied that the high rate constant of luminol CL quenched by purines may be attributed to ground state interactions originating from hydrogen bonding. Copyright © 2012 John Wiley & Sons, Ltd.     

不同死后时间骨骼肌运动终板乙酰胆碱酯酶（AChE）降解变化的研究——酶组织化学定量分析

为进一步了解骨髓肌的死后变化特点和规律及其与死后时间（PMI）的关系。本文选用成年实验大鼠,系列观察了死后0－96小时（0－96hpm）骨骼肌运动终板和肌纤维在不同温度条件下AChE（乙酰胆碱酯酶）,SDH（琥珀酸脱氢酶）,LDH（乳酸脱氢酶）,Ca^ -ATPase（钙激活三磷腺苷酶）的酶组织化学变化,并使用图象处理系统对上述酶反应结果进行了定量分析和比较,结果发现,上述改变与PMI有明显的相关关系,其中运动终板AchE因位于突触间隙的特殊位置以及自身的耐活性质,故反应明确,稳定性高,降解缓慢显示更清晰和持久的降解曲线,可作为藉肌肉组织降解变化用于推断死亡时间的标志酶。     

Bisphosphonates: new therapeutic agents for the treatment of bone tumors

Bisphosphonates (BPs) have been used successfully for many years to reduce the skeletal complications associated with the benign and malignant bone diseases that are characterized by enhanced osteoclastic bone resorption. Until recently, it was thought that the clinical efficacy of BPs in the treatment of cancer patients with bone metastases was purely a result of the inhibition of osteoclast-mediated bone resorption. However, recent studies have demonstrated that BPs inhibit the growth, attachment and invasion of cancer cells in culture and promote their apoptosis. These results suggest that BPs are also anti-cancer agents, raising the possibility that BPs could inhibit cancer-cell colonization in visceral organs. However, results from clinical trials are conflicting, and whether BPs possess anti-cancer effects or not remains controversial.     

Micro‐CT evaluation of murine fetal skeletal development yields greater morphometric precision over traditional clear‐staining methods

Traditional techniques for quantification of murine fetal skeletal development (gross measurements, clear‐staining) are severely limited by specimen processing, soft tissue presence, diffuse staining, and unclear landmarks between which to make measurements. Nondestructive microcomputed tomography (micro‐CT) imaging is a versatile, well‐documented tool traditionally used to generate high‐resolution 3‐D images and quantify microarchitectural parameters of trabecular bone. Although previously described as a tool for phenotyping fetal murine specimens, micro‐CT has not previously been used to directly measure individual fetal skeletal structures. Imaging murine fetal skeletons using micro‐CT enables the researcher to nondestructively quantify fetal skeletal development parameters including limb length, total bone volume, and average bone mineral density, as well as identify skeletal malformations. Micro‐CT measurement of fetal limb lengths correlates well with traditional clear‐staining methods (83.98% agreement), decreases variability in measurements (average standard errors: 6.28% for micro‐CT and 10.82% for clear‐staining), decreases data acquisition time by eliminating the need for tissue processing, and preserves the intact fixed fetus for further analysis. Use of the rigorous micro‐CT technique to generate 3‐D images for digital measurement enables isolation of skeletal structures based on degree of mineralization (local radiodensity), eliminating the complications of blurred stain boundaries and soft tissue inclusion that accompany clear‐staining and gross measurement techniques. Microcomputed tomography provides a facile, accurate, and nondestructive method for determining the developmental state of the fetal skeleton using not only limb lengths and identification of malformations, but total skeletal bone volume and average skeletal mineral density as well. Birth Defects Res (Part B) 2008. © 2008 Wiley‐Liss, Inc.     

The influence of dioxygen on luminol chemiluminescence

Assays of peroxy compounds are commonly performed after chromatographic separation of analysed mixtures. In high‐performance liquid chromatography (HPLC), solvent reservoirs are sparged by helium or inline vacuum‐degassed in order to control the compressibility of the solvents for efficient pumping. In this study, we investigated the influence of degassing the reaction solution on the light output of the hemin‐catalyzed luminol oxidation by various oxidants. We found that, when t‐butyl hydroperoxide, hydrogen peroxide, n‐butyl hydroperoxide, iodosobenzene and iodobenzene diacetate were used as oxidants, the luminol chemiluminescence was lowered by 50–70% compared with an equilibrated and degassed solution. The opposite effect was observed when dibenzoyl peroxide and 3‐chloroperoxybenzoic acid were used as oxidants, as the chemiluminescence increased by approximately 20–30%. The reduced chemiluminescence was explained based on the known role of dioxygen in luminol chemiluminescence. The enhancement of chemiluminescence was rationalized by suggesting an alternative mechanism of luminol oxidation valid for peroxyacids and diacyl peroxides in which the reaction of a peroxyacid anion with the diazaquinone led to light emission with a higher quantum yield than the usual path, which is suppressed by the removal of dioxygen from the reaction solution. Copyright © 2009 John Wiley & Sons, Ltd.     

The dead do not lie: using skeletal remains for rapid assessment of historical small-mammal community baselines

Conservation and restoration efforts are often hindered by a lack of historical baselines that pre-date intense anthropogenic environmental change. In this paper I document that natural accumulations of skeletal remains represent a potential source of high-quality data on the historical composition and structure of small-mammal communities. I do so by assessing the fidelity of modern, decadal and centennial-scale time-averaged samples of skeletal remains (concentrated by raptor predation) to the living small-mammal communities from which they are derived. To test the power of skeletal remains to reveal baseline shifts, I employ the design of a natural experiment, comparing two taphonomically similar Great Basin cave localities in areas where anthropogenic land-use practices have diverged within the last century. I find relative stasis at the undisturbed site, but document rapid restructuring of the small-mammal community at the site subjected to recent disturbance. I independently validate this result using historical trapping records to show that dead remains accurately capture both the magnitude and direction of this baseline shift. Surveys of skeletal remains therefore provide a simple, powerful and rapid alternative approach for gaining insight into the historical structure and dynamics of modern small-mammal communities.     

Brief communication: Paleohistopathological analysis of pathology museum specimens: Can periosteal reaction microstructure explain lesion etiology?

The assertion that the microstructure of periosteal new bone formation can be used to differentiate between disease etiologies (Schultz: Yrbk Phys Anthropol 44 2001 106–147; Schultz: Identification of pathological conditions in human skeletal remains, 2nd ed. London: Academic Press 2003 73–109) was tested in a pilot‐study, using diagnosed bone specimens from St George's Hospital Pathology Museum, London, UK. Embedded bone specimens exhibiting pathological periosteal new bone formation were examined using scanning electron microscopy in back‐scattered electron imaging mode (SEM‐BSE). The results suggest that several histological features (i.e. Grenzstreifen, Polsters, and sinuous lacunae) deemed to be diagnostic of specific pathological conditions are of no specific diagnostic value, as they are encountered in pathological conditions of differing disease etiology. These results tie in with a previous investigation demonstrating a lack of diagnostic qualitative or quantitative characteristics seen in the macroscopic and radiographic appearance of periosteal reactions (Weston: Am J Phys Anthropol 137 2008 48–59). Am J Phys Anthropol, 2009. © 2009 Wiley‐Liss, Inc.     

Comparative mitochondrial and nuclear quantitative PCR of historical marine mammal tissue,bone, baleen,and tooth samples

The use of historical and ancient tissue samples for genetic analysis is increasing, with ever greater numbers of samples proving to contain sufficient mitochondrial and even nuclear DNA for multilocus analysis. DNA yield, however, remains highly variable and largely unpredictable based solely on sample morphology or age. Quantification of DNA from historical and degraded samples can greatly improve efficiency of screening DNA extracts prior to attempting sequencing or genotyping, but requires sequence‐specific quantitative polymerase chain reaction (qPCR) based assays to detect such minute quantities of degraded DNA. We present two qPCR assays for marine mammal DNA quantification, and results from analysis of DNA extracted from preserved soft tissues, bone, baleen, and tooth from several cetacean species. These two assays have been shown to amplify DNA from 26 marine mammal species representing 12 families of pinnipeds and cetaceans. Our results indicate that different tissues retain different ratios of mitochondrial to nuclear DNA, and may be more or less suitable for analysis of nuclear loci. Specifically, historical bone and tooth samples average 60‐fold higher ratio of mitochondrial DNA to nuclear DNA than preserved fresh soft tissue, and the ratio is almost 8000‐fold higher in baleen.     

Luminol‐, isoluminol‐ and lucigenin‐enhanced chemiluminescence of rat blood phagocytes stimulated with different activators

Luminol‐, isoluminol‐ or lucigenin‐enhanced chemiluminescence (CL) was used to measure the production of reactive oxygen species by rat blood leukocytes. Opsonized zymosan (OZ), phorbol‐12‐myristate‐13‐acetate (PMA), calcium ionophore A23187 (Ca‐I) or N‐formyl‐Met‐Leu‐Phe (fMLP) were used as activators. The CL signal of isolated blood leukocytes decreased in rank order of luminol > isoluminol > lucigenin. The kinetic pro?les of luminol‐ and isoluminol‐enhanced CL were similar upon stimulation by each activator tested. The remarkably higher luminol and isoluminol CL responses were obtained after OZ stimulation when compared with other activators. However, when lucigenin was used, the PMA‐ and OZ‐stimulated CL were comparable. The presence of plasma increased OZ‐activated CL because of the enhanced phagocytosis of OZ. This was demonstrated by determining the phagocytosis of the ?uorescent OZ using a ?ow cytometer. In contrast, the presence of plasma decreased PMA‐activated CL, due to the antioxidant properties of plasma as determined by the CL method. As far as whole blood is concerned, only OZ activated luminol‐enhanced CL was reliable. Blood volumes over 5 µL decreased CL activity due to the scavenging ability of erythrocytes. The results suggest that 0.5 µL whole blood is suf?cient for routine luminol‐enhanced CL analysis of whole blood oxidative burst in rats. Copyright © 2004 John Wiley & Sons, Ltd.     

From forest fires to fisheries management: Anthropology,conservation biology,and historical ecology

Human‐environmental relationships have long been of interest to a variety of scientists, including ecologists, biologists, anthropologists, and many others. 1 , 2 In anthropology, this interest was especially prevalent among cultural ecologists of the 1970s and earlier, who tended to explain culture as the result of techno‐environmental constraints. 3 More recently researchers have used historical ecology, an approach that focuses on the long‐term dialectical relationship between humans and their environments, as well as long‐term prehuman ecological datasets. 4 - 7 An important contribution of anthropology to historical ecology is that anthropological datasets dealing with ethnohistory, traditional ecological knowledge, and human skeletal analysis, as well as archeological datasets on faunal and floral remains, artifacts, geochemistry, and stratigraphic analysis, provide a deep time perspective (across decades, centuries, and millennia) on the evolution of ecosystems and the place of people in those larger systems. Historical ecological data also have an applied component that can provide important information on the relative abundances of flora and fauna, changes in biogeography, alternations in food webs, landscape evolution, and much more.     

From mouse to man: redefining the role of insulin-like growth factor-I in the acquisition of bone mass

The insulin-like growth factor system (IGF) has been linked to the process of bone acquisition through epidemiologic analyses of large cohorts and in vitro studies of bone cells. But the exact relationship between expression of IGF-I in bone and skeletal homeostasis or pathologic conditions, such as osteoporosis, remains poorly defined. Recent advances in genomic engineering have resulted in the development of better in vivo models to test the role of IGF-I during development and maintenance of the adult skeleton. It is now established that skeletal expression of IGF-I is critical for differentiative bone cell function. It may also be essential for the full anabolic effects of parathyroid hormone on trabecular bone and for some component of biomineralization. Evidence from conditional mutagenesis studies suggests that serum IGF-I may represent more than a storage depot or permissive factor during the final phase of skeletal acquisition. This work re-examines the original tenets of the "somatomedin hypothesis" in light of these newer mouse models and their remarkable skeletal phenotypes. The implications are far reaching and suggest that newer approaches for manipulating the IGF regulatory system may one day be useful as therapeutic adjuncts for the treatment of osteoporosis.     

Technical note: Morphometric maps of long bone shafts and dental roots for imaging topographic thickness variation

Qualitative and quantitative characterization through functional imaging of mineralized tissues is of potential value in the study of the odontoskeletal remains. This technique, widely developed in the medical field, allows the bi‐dimensional, planar representation of some local morphometric properties, i.e., topographic thickness variation, of a three‐dimensional object, such as a long bone shaft. Nonetheless, the use of morphometric maps is still limited in (paleo)anthropology, and their feasibility has not been adequately tested on fossil specimens. Using high‐resolution microtomographic images, here we apply bi‐dimensional virtual “unrolling” and synthetic thickness mapping techniques to compare cortical bone topographic variation across the shaft in a modern and a fossil human adult femur (the Magdalenian from Chancelade). We also test, for the first time, the possibility to virtually unroll and assess for dentine thickness variation in modern and fossil (the Neanderthal child from Roc de Marsal) human deciduous tooth roots. The analyses demonstrate the feasibility of using two‐dimensional morphometric maps for the synthetic functional imaging and comparative biomechanical interpretation of cortical bone thickness variation in extant and fossil specimens and show the interest of using this technique also for the subtle characterization of root architecture and dentine topography. More specifically, our preliminary results support the use of virtual cartography as a tool for assessing to what extent internal root morphology is capable of responding to loading and directional stresses and strains in a predictable way. Am J Phys Anthropol, 2010. © 2010 Wiley‐Liss, Inc.     

A requirement for Fgfr2 in middle ear development

The skeletal structure of the mammalian middle ear, which is composed of three endochondral ossicles suspended within a membranous air‐filled capsule, plays a critical role in conducting sound. Gene mutations that alter skeletal development in the middle ear result in auditory impairment. Mutations in fibroblast growth factor receptor 2 (FGFR2), an important regulator of endochondral and intramembranous bone formation, cause a spectrum of congenital skeletal disorders featuring conductive hearing loss. Although the middle ear malformations in multiple FGFR2 gain‐of‐function disorders are clinically characterized, those in the FGFR2 loss‐of‐function disorder lacrimo‐auriculo‐dento‐digital (LADD) syndrome are relatively undescribed. To better understand conductive hearing loss in LADD, we examined the middle ear skeleton of mice with conditional loss of Fgfr2. We find that decreased auditory function in Fgfr2 mutant mice correlates with hypoplasia of the auditory bulla and ectopic bone growth at sites of tendon/ligament attachment. We show that ectopic bone associated with the intra‐articular ligaments of the incudomalleal joint is derived from Scx‐expressing cells and preceded by decreased expression of the joint progenitor marker Gdf5. Together, these results identify a role for Fgfr2 in development of the middle ear skeletal tissues and suggest potential causes for conductive hearing loss in LADD syndrome.     

Shotgun microbial profiling of fossil remains

Millions to billions of DNA sequences can now be generated from ancient skeletal remains thanks to the massive throughput of next‐generation sequencing platforms. Except in cases of exceptional endogenous DNA preservation, most of the sequences isolated from fossil material do not originate from the specimen of interest, but instead reflect environmental organisms that colonized the specimen after death. Here, we characterize the microbial diversity recovered from seven c. 200‐ to 13 000‐year‐old horse bones collected from northern Siberia. We use a robust, taxonomy‐based assignment approach to identify the microorganisms present in ancient DNA extracts and quantify their relative abundance. Our results suggest that molecular preservation niches exist within ancient samples that can potentially be used to characterize the environments from which the remains are recovered. In addition, microbial community profiling of the seven specimens revealed site‐specific environmental signatures. These microbial communities appear to comprise mainly organisms that colonized the fossils recently. Our approach significantly extends the amount of useful data that can be recovered from ancient specimens using a shotgun sequencing approach. In future, it may be possible to correlate, for example, the accumulation of postmortem DNA damage with the presence and/or abundance of particular microbes.     

Development of a novel chemiluminescence method for the determination of cefazolin sodium in injectable powder and human urine based on a luminol‐Cu(III) complex reaction in alkaline medium

A novel chemiluminescence (CL) method was developed for the determination of cefazolin sodium based on the CL reaction between the [Cu(HIO₆)₂]^5‐Cu(III) complex and luminol in alkaline solution. Results showed that CL emission of Cu(III) complex–luminol in alkaline medium was significantly different from that in acidic medium. A possible mechanism of the enhanced effect of cefazolin on CL emission of the [Cu(HIO₆)₂]^5‐‐ luminol system was proposed. The effect of the reaction conditions on CL emissions was examined. Under optimized conditions, a good linear relationship was obtained between CL intensity and concentrations of cefazolin sodium in the range of 2.0 x 10^‐8 to 2.0 x 10^‐6 g/mL with a correlation coefficient of R² = 0.9978. The limit of detection was 4.58 x 10^‐9 g/mL. The proposed method was applied for the determination of cefazolin sodium in real samples with recoveries of 82.0‐109% with an RSD of 0.7‐2.1%. The proposed method was successfully used for the determination of cefazolin sodium in injectable powder preparations and human urine with satisfactory results. Copyright © 2012 John Wiley & Sons, Ltd.