Assessment of some problematic factors in facial image identification using a 2D/3D superimposition technique.

The number of criminal cases requiring facial image identification of a suspect has been increasing because a surveillance camera is installed everywhere in the city and furthermore, the intercom with the recording function is installed in the home. In this study, we aimed to analyze the usefulness of a 2D/3D facial image superimposition system for image identification when facial aging, facial expression, and twins are under consideration. As a result, the mean values of the average distances calculated from the 16 anatomical landmarks between the 3D facial images of the 50s groups and the 2D facial images of the 20s, 30s, and 40s groups were 2.6, 2.3, and 2.2mm, respectively (facial aging). The mean values of the average distances calculated from 12 anatomical landmarks between the 3D normal facial images and four emotional expressions were 4.9 (laughter), 2.9 (anger), 2.9 (sadness), and 3.6mm (surprised), respectively (facial expressions). The average distance obtained from 11 anatomical landmarks between the same person in twins was 1.1mm, while the average distance between different person in twins was 2.0mm (twins). Facial image identification using the 2D/3D facial image superimposition system demonstrated adequate statistical power and identified an individual with high accuracy, suggesting its usefulness. However, computer technology concerning video image processing and superimpose progress, there is a need to keep familiar with the morphology and anatomy as its base.

Discriminant functions for sex estimation of modern Japanese skulls.

The purpose of this study is to generate a set of discriminant functions in order to estimate the sex of modern Japanese skulls. To conduct the analysis, the anthropological measurement data of 113 individuals (73 males and 40 females) were collected from recent forensic anthropological test records at the National Research Institute of Police Science, Japan. Birth years of the individuals ranged from 1926 to 1979, and age at death was over 19 years for all individuals. A total of 10 anthropological measurements were used in the discriminant function analysis: maximum cranial length, cranial base length, maximum cranial breadth, maximum frontal breadth, basion-bregmatic height, upper facial breadth, bizygomatic breadth, bicondylar breadth, bigonial breadth, and ramal height. As a result, nine discriminant functions were established. The classification accuracy ranged from 79.0 to 89.9% when the measurements of the 113 individuals were substituted into the established functions, from 77.8 to 88.1% when a leave-one-out cross-validation procedure was applied to the data, and from 86.7 to 93.0% when the measurements of 50 new individuals (25 males and 25 females), unrelated to the establishment of the discriminant functions, were used.

Identification of nasal blood by real-time RT-PCR.

A new approach for the identification of body fluid stains by comparing specific mRNA expression levels has been extensively studied in recent years. Here, we examine whether nasal blood, which is regarded as one of the most difficult types of blood to identify, can be identified by comparing mRNA expression levels of target genes specific to saliva, nasal secretion, and blood. The saliva-specific statherin gene (STATH) was found to be expressed at high levels in not only saliva (dCt value: 1.32±1.39, n=5), but also nasal secretions (dCt value: 0.90±1.14, n=5), while the histatin gene (HTN3) was only expressed at high levels in saliva (dCt value: 1.08±2.35, n=5). We also confirmed that the hemoglobin-beta gene (HBB) showed high expression levels in blood (dCt value: -9.51±0.40, n=5). Four nasal blood stains were found to highly express STATH (dCt value: 5.65±3.98) and HBB (dCt value: -8.79±1.67) but not HTN3, suggesting that the stain samples contained both nasal secretions and blood and can therefore be identified as nasal blood stains. Although menstrual blood showed the same expression pattern as nasal blood, the menstrual blood-specific protein matrix metallopeptidase 7 (MMP7) was not expressed in all nasal blood stain samples. Therefore, its expression levels could be used to discriminate between nasal and menstrual blood. In conclusion, real-time RT-PCR was able to identify nasal blood, although the stability of gene expression in nasal blood stains was low over time, suggesting that this assay may not be effective for older stains. Future work should examine the usefulness of this assay under various environmental conditions.

Expression of statherin mRNA and protein in nasal and vaginal secretions.

Nasal secretion has been regarded as one of the most difficult body fluids to identify and is especially difficult to discriminate from vaginal secretions and saliva. At present, few specific markers are known for nasal secretions. The aim of this study is to find a new approach for the identification of nasal secretions. We examined expression levels of statherin and histatin, peptides which are commonly found in saliva, in nasal and vaginal secretions by real-time RT-PCR and ELISA assays. Statherin mRNA was highly expressed in all nasal samples (dCt value=-1.49±1.10, n=8) and was detected even in 1-day-old 0.1-µL stains. However, the stability of mRNA in nasal stains was significantly (P<0.01) lower than in saliva. Low levels of statherin mRNA were detected in 4 of the 17 vaginal samples (dCt value=11.65-14.72). Histatin mRNA was not detected in any nasal or vaginal samples, although it was highly expressed in all saliva samples. ELISA assays with anti-statherin goat polyclonal antibody showed that statherin peptide was detected in all nasal and saliva samples even after dilution of more than 1000-fold. The statherin peptide was not detected in any vaginal samples, including samples that expressed low levels of statherin mRNA. The amount of statherin peptide in vaginal samples might be less than the limit of detection of this assay. In the present study, statherin was highly expressed in nasal secretions, but histatin was not. These markers may be useful for discriminating nasal secretions from vaginal secretions and saliva. However, the usefulness of these markers in practical forensic case samples has not yet been examined. Therefore, further research is required to establish the utility of these assays for identification of nasal secretions.

Rapid discrimination of Legionella by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Molecular typing is an important tool in the surveillance and investigation of human Legionella infection outbreaks. In this study, two molecular typing methods, pulsed-field gel electrophoresis (PFGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), were used to discriminate 23 Legionella pneumophila strains. The usefulness of MALDI-TOF-MS was demonstrated. The MALDI-TOF-MS fingerprinting with filtered small acid-soluble molecules gave different molecular profiles among strains, and the clustal analysis with MALDI-TOF-MS showed a high discrimination of strains the same as that with PFGE. In addition, MALDI-TOF-MS data could be generated within a few hours after the initial culture, although PFGE analyses took several days to complete. Thus, MALDI-TOF-MS offers a simple and rapid discrimination technique that could aid in the tracking of fast-spreading outbreaks of Legionella.

Facial soft tissue thickness in Japanese children.

Facial reconstruction techniques used in forensic anthropology are based on mean soft tissue thickness measurements. Numerous studies of facial tissue thickness in adults have been published on a range of subjects from different ancestral backgrounds. Data on facial thickness in children derive primarily from Caucasoid, African-American, and Hispanic subjects. There are limited data from the few studies of Japanese children (male: skeletal class I only; female: all skeletal classes). The author has previously reported facial tissue thickness data for Japanese girls and boys with skeletal class I and for all three skeletal classes in Japanese girls. The present study reports facial soft tissue thickness data in Japanese children of all skeletal classes, within age subsets. With parental informed consent, diagnostic lateral cephalometric X-ray images were obtained from 339 Japanese children aged 7-18 years (male: 162; female: 177) who attended the Matsumoto Dental University Department of Orthodontics to undergo orthodontic treatment. Soft tissue and skeletal features were traced onto acetate sheets from the X-ray images, and 10 anthropological landmarks on the midsagittal line were measured. Means, SDs, and ranges were then calculated. Differences between male and female measurements in six age groups were compared using t-tests. Significant differences were observed at some landmarks in each age group. The findings were compared with data from other juvenile populations.

Pilot study of facial soft tissue thickness differences among three skeletal classes in Japanese females.

Facial reconstruction is a technique used in forensic anthropology to estimate the appearance of the antemortem face from unknown human skeletal remains. This requires accurate skull assessment (for variables such as age, sex, and race) and soft tissue thickness data. However, the skull can provide only limited information, and further data are needed to reconstruct the face. The authors herein obtained further information from the skull in order to reconstruct the face more accurately. Skulls can be classified into three facial types on the basis of orthodontic skeletal classes (namely, straight facial profile, type I, convex facial profile, type II, and concave facial profile, type III). This concept was applied to facial tissue measurement and soft tissue depth was compared in each skeletal class in a Japanese female population. Differences of soft tissue depth between skeletal classes were observed, and this information may enable more accurate reconstruction than sex-specific depth alone.

Detection of dermcidin for sweat identification by real-time RT-PCR and ELISA.

We evaluated the performance of real-time RT-PCR and ELISA assays for detection of dermcidin (DCD) in sweat and body-fluid stains. DCD, a small antibiotic peptide secreted into human sweat, was detected by real-time RT-PCR in 7-day-old stains containing as small as 10 microL of sweat, and the assay showed high specificity when testing 7-day-old stains containing 30 microL of other body-fluid. ELISA using anti-human dermcidin mouse monoclonal antibody detected DCD sweat diluted up to approximately 10,000-fold and could specifically detect DCD in 10 microL of body-fluid stains. The performance of the two assays was tested during winter on samples that simulated forensic case samples: an undershirt and a sock worn for 20 h, a handkerchief used to wipe the brow several times within 12h, a cap and a cotton glove worn for 4h, and a white robe worn at intervals for 2 years. The result showed that the former assay detected DCD in all sites of the undershirt examined (armpit, back, and breast), and the latter gave a relatively high OD value in the armpit among the three sites. For the socks, although the latter assay gave very high OD values in both the center and toe of the foot sole, the former could not detect DCD in both of them. These results indicate that highly damp conditions, such as inside a shoe, might promote the degradation of mRNA in samples such as socks. In the other case samples, sweat was adequately detected by both assays. This study is the first demonstration of the use of real-time RT-PCR to sensitively identify sweat among body-fluid stains, and it confirmed that dermcidin was an excellent marker for sweat identification. In addition, the usefulness of ELISA was also verified. Positive sweat identification using these assays is expected to assist forensic practice.

Evaluation of mRNA-based approach for identification of saliva and semen.

Multiplex mRNA profiling by a reverse transcription-polymerase chain reaction (RT-PCR) has been reported in the last few years as a new approach for the identification of body fluids. We have also demonstrated the feasibility of identifying body fluids by using a real-time RT-PCR assay. Statherin (STATH) and histatin (HTN3), the selected genes for saliva, and protamin 2 (PRM2) and semenogelin 1 (SEMG1), those selected for semen, showed high specificity to these body fluids. Thus, the sensitivity and specificity of target genes were examined in body fluid stains. All target genes were detected in 0.1 microL 6-day-old stains, and showed high specificity in 7-day-old 30 microL stains. Furthermore, the stability of HTN3 in saliva stains was examined under various environmental conditions over time. The results showed that the degradation of mRNA in the stains was highly affected by wet conditions, and that light was also an important factor. However, mRNA was detectable in an older saliva stain (6 years old) and in an older semen stain (3.5 years old), both of which had been kept under dry and dark conditions. The stability of mRNA beyond our supposition may play an important role in developing new techniques for body fluid identification.

JC viral DNA chip allows geographical localization of unidentified cadavers for rapid identification.

To narrow down the geographical origins of unidentified cadavers, a rapid and simple method to detect JC virus (JCV) genotypes using a DNA chip was developed. Fifty-four probes on a 3mm silicon DNA chip were designed to distinguish 12 JCV genotypes. The detection limit of the DNA chip was 0.001 pg/microL of template JCV DNA concentration, showing a sensitivity equal to that of the conventional method. The analysis requires only a few hours to yield results. This new DNA chip method helps to narrow the search area for human identification much more quickly and easily than use of the conventional method.

Forensic application of Epstein-Barr virus genotype: correlation between viral genotype and geographical area.

Using 50 forensic blood samples, the latent membrane protein 2A (LMP-2A) gene of Epstein-Barr virus (EBV) DNA was amplified to find a geographic correlation among the EBV genotypes. EBV DNA was detected in nine samples. From a phylogenetic analysis using 18 reported sequences as a reference, six EBV subtypes (Ia, Ib, Ic IIa, IIb, and IIc) were found. Japanese isolates were included in subtypes Ia or IIa. All the Asian reference isolates, except isolate D6, were included in subtype Ia or IIa. Mediterranean, an Alaskan and other African isolates were included in types Ib, Ic, IIb and IIc. The EBV genotype in the LMP-2A gene was thus demonstrated as being correlated with the host's geographical location. Typing in the EBV-associated nuclear antigen 2 gene was not related to that in the LMP-2A gene. Detection of the EBV genotype in the LMP-2A gene may be useful for determining the geographical origins of unidentified cadavers.

Sensitive detection of Bacillus anthracis using a binding protein originating from gamma-phage.

Detection of biological weapons is a primary concern in force protection, treaty verification, and safeguarding civilian populations against domestic terrorism. One great concern is the detection of Bacillus anthracis, the causative agent of anthrax. Therefore, there is a pressing need to develop novel methods for rapid, simple, and precise detection of B. anthracis. Here, we report that the C-terminal region of gamma-phage lysin protein (PlyG) binds specifically to the cell wall of B. anthracis and the recombinant protein corresponding to this region (positions, 156-233), PlyGB, is available as a bioprobe for detection of B. anthracis. Our detection method, based on a membrane direct blot assay using recombinant PlyGB, was more rapid and sensitive than the gamma-phage test and was simpler and more inexpensive than genetic methods such as PCR, or immunological methods using specific antibodies. Furthermore, its specificity was comparable to the gamma-phage test. PlyGB is applicable in conventional methods instead of antibodies and could be a potent tool for detection of B. anthracis.

Facial soft tissue thickness in skeletal type I Japanese children.

Facial reconstruction techniques used in forensic anthropology require knowledge of the facial soft tissue thickness of each race if facial features are to be reconstructed correctly. If this is inaccurate, so also will be the reconstructed face. Knowledge of differences by age and sex are also required. Therefore, when unknown human skeletal remains are found, the forensic anthropologist investigates for race, sex, and age, and for other variables of relevance. Cephalometric X-ray images of living persons can help to provide this information. They give an approximately 10% enlargement from true size and can demonstrate the relationship between soft and hard tissue. In the present study, facial soft tissue thickness in Japanese children was measured at 12 anthropological points using X-ray cephalometry in order to establish a database for facial soft tissue thickness. This study of both boys and girls, aged from 6 to 18 years, follows a previous study of Japanese female children only, and focuses on facial soft tissue thickness in only one skeletal type. Sex differences in thickness of tissue were found from 12 years of age upwards. The study provides more detailed and accurate measurements than past reports of facial soft tissue thickness, and reveals the uniqueness of the Japanese child's facial profile.

Development of species identification tests targeting the 16S ribosomal RNA coding region in mitochondrial DNA.

This report describes the development of a species testing system based on the diversity of nucleotide sequences in mitochondrial DNA (mtDNA) among species. Five species, human, cow, pig, dog, and cat, were considered. The partial nucleotide sequences in 16S ribosomal RNA coding region were chosen as the target for discriminating the species. The sequence diversities of this approximately 400 bp long region ranged from 15.7 to 24.1% among the five species. Sequencing analysis of this target on 50 individuals of each species (53 for dogs) revealed that the nucleotide sequences were well preserved within species. Species-specific PCR for each species was also designed, and satisfactory results with regard to both sensitivity and specificity were obtained. A validation study with DNA extracted from bovine bone exposed to the environment revealed that the PCRs designed in this study worked correctly. From the results obtained, it was shown that this testing system could be a good tool for species identification. One successful case report is also demonstrated.

Facial soft tissue thickness in Japanese female children.

Facial reconstruction techniques used in forensic anthropology are based on soft tissue thickness measurements. Many studies of facial tissue thickness in adults have been published that take racial background into account. However, the only data on facial thickness in children are derived from studies of American, British, and Hispanic children. The authors therefore measured facial tissue thickness in Japanese children, with the aim of providing data for producing accurate facial likenesses and to evaluate matching of skull-photo superimposition images. Cephalometric X-ray images give an approximately 10% enlargement from true size and can demonstrate the relationship between soft and hard tissue. Facial soft tissue thickness was measured at 12 anthropological points using X-ray cephalometry.

A new retrieval system for a database of 3D facial images.

A new retrieval system for a 3D facial image database was designed and its reliability was experimentally examined. This system has two steps, firstly to automatically adjust the orientation of all 3D facial images in a database to that of the 2D facial image of a target person, and then to identify the facial image of the target person from the adjusted 3D facial images in the database using a graph-matching method. From the experimental study [M. Yoshino, K. Imaizumi, T. Tanijiri, J.G. Clement, Automatic adjustment of facial orientation in 3D face image database, Jpn. J. Sci. Tech. Iden. 8 (2003) 41-47], it is concluded that the software developed for the first step will be applicable to the automatic adjustment of facial orientation in the 3D facial image database. In 28 out of 110 sets (25.5%), the 3D image of the target person was chosen as the best match (from a database of 132 3D facial images) according to the similarity of the facial image characteristics based on the graph matching. The 3D facial image of the target person was ranked in the top of 10 of the database in 75 out of 110 sets (68.2%). These results suggest that this system is inadequate for the identification level, but may be feasible for screening method in a small database. It will be necessary to further pursue the possibility of realization of a facial image retrieval system for a large database such as suspects' facial images in future.

DNA typing of bone specimens--the potential use of the profiler test as a tool for bone identification.

Twenty-six bone DNA identification cases are described. The postmortem periods of the studied remains ranged from three days to over 30 years, and the locations where the remains were found varied resulting in a variety of postmortem conditions. Nuclear DNA typing using an AmpFLSTR Profiler kit and mitochondrial DNA (mtDNA) typing of hypervariable regions 1 and 2 (HV1 and HV2) in a control region were performed both with decalcified and non-treated bone powder samples. Decalcification was shown to improve the success of DNA typing. The nucleotide sequences of the HV1 and HV2 regions were successfully determined in all cases examined. Nuclear DNA typing was very successful, more than half of the loci were typed during multiple amplifications (10 loci in one reaction) in 23 cases. Polymerase chain reaction (PCR) inhibition was observed in five cases including three samples that were found buried in soil. This inhibitory effect was identified as the result of unbalanced multiple PCR during the profiler test. These results revealed that DNA typing targeting nuclear DNA is a potentially powerful tool for bone identification.

Detecting hemifacial asymmetries in emotional expression with three-dimensional computerized image analysis.

Emotions are expressed more clearly on the left side of the face than the right: an asymmetry that probably stems from right hemisphere dominance for emotional expression (right hemisphere model). More controversially, it has been suggested that the left hemiface bias is stronger for negative emotions and weaker or reversed for positive emotions (valence model). We examined the veracity of the right hemisphere and valence models by measuring asymmetries in: (i) movement of the face; and (ii) observer's rating of emotionality. The study uses a precise three-dimensional (3D) imaging technique to measure facial movement and to provide images that simultaneously capture the left or right hemifaces. Models (n = 16) with happy, sad and neutral expressions were digitally captured and manipulated. Comparison of the neutral and happy or sad images revealed greater movement of the left hemiface, regardless of the valence of the emotion, supporting the right hemisphere model. There was a trend, however, for left-sided movement to be more pronounced for negative than positive emotions. Participants (n = 357) reported that portraits rotated so that the left hemiface was featured, were more expressive of negative emotions whereas right hemiface portraits were more expressive for positive emotions, supporting the valence model. The effect of valence was moderated when the images were mirror-reversed. The data demonstrate that relatively small rotations of the head have a dramatic effect on the expression of positive and negative emotions. The fact that the effect of valence was not captured by the movement analysis demonstrates that subtle movements can have a strong effect on the expression of emotion.

A Japanese computer-assisted facial identification system successfully identifies non-Japanese faces.

The method developed by Yoshino et al. in [Forensic Sci. Int. 109 (2000) 225 and Jpn. J. Sci. Tech. Iden. 5 (2000) 9] and already being applied in Japan utilizes a three-dimensional (3D) physiognomic rangefinder combined with a computer-assisted superimposition system. Facial outlines can be compared between two-dimensional (2D) surveillance images and data extracted from 3D images obtained from the rangefinder. Also, the loci of potentially concordant features can be compared and differences measured. The method is largely objective and gives statistics for false positive/false negative findings. This recently developed method by Yoshino et al. is currently being introduced to the Japanese courts. To enable courts outside Japan to assess the admissibility of this new method, studies of non-Japanese faces have been undertaken and shown to produce similar low error rates. The present authors, therefore, consider the Yoshino method to be applicable in a non-Japanese context. As part of this study a comparison of morphological features between two ethnic groups has been undertaken using 3D measurements for the first time and will serve as the foundation for an anthropological database in the future.

Pralidoxime iodide (2-pAM) penetrates across the blood-brain barrier.

The in vivo rat brain microdialysis technique with HPLC/UV was used to determine the blood-brain barrier (BBB) penetration of pralidoxime iodide (2-PAM), which is a component of the current nerve agent antidote therapy. After intravenous dosage of 2-PAM (10, 50, 100 mg/kg), 2-PAM appeared dose-dependently in the dialysate; the striatal extracellular/blood concentration ratio at 1 h after 50 mg/kg dosage was 0.093 +/- 0.053 (mean +/- SEM). This finding offered conclusive evidence of the BBB penetration of 2-PAM. We also examined whether the BBB penetration of 2-PAM was mediated by a certain specific transporter, such as a neutral or basic amino acid transport system. Although it was unclear, the neural uptake of 2-PAM was Na+ dependent. The mean BBB penetration by 2-PAM was approximately 10%, indicating the intravenous administration of 2-PAM might be to a degree effective to reactivation of the blocked cholinesterase in the brain.