A vertebrate-specific qPCR assay as an endogenous internal control for robust species identification.

The identification of vertebrate species is important in numerous fields including archaeology, ecology, as well as food and forensic sciences. Real-time quantitative PCR (qPCR) assays specific for one vertebrate species are promising approaches for species identification, although there are several drawbacks such as difficulty determining whether the detected DNA is authentic or a contaminant. Here, we describe a qPCR assay specific for vertebrate mitochondrial DNA (mtDNA) which can overcome these drawbacks. Since we found that mitochondrial 16S rRNA contains regions that are perfectly (not highly) conserved across virtually all vertebrates, but are variable in invertebrates, we were able to design a vertebrate-specific qPCR assay by placing primers/probe within these regions. The specificity and accuracy of this assay were validated with representative vertebrate and invertebrate samples. This assay detected DNA from all vertebrate samples, but not from any invertebrate samples. In addition, this assay was able to quantify vertebrate mtDNAs as accurately as previously reported species-specific qPCR assays. The results demonstrated it is feasible to quantify vertebrate mtDNA specifically and accurately in a sample. This means that it is possible to determine the ratio of specific vertebrate species mtDNA to total vertebrate mtDNA in a sample. In conjunction with this assay as an endogenous internal control, species-specific qPCR assays will allow for the robust identification of vertebrate species.

Automatic landmark annotation in 3D surface scans of skulls: Methodological proposal and reliability study.

BACKGROUND AND OBJECTIVES: Craniometric landmarks are essential in many biomedical applications, such as morphometric analysis or forensic identification. The process of locating landmarks is usually a manual and slow task, highly influenced by fatigue, skills and the experience of the practitioner. Localization errors are propagated and magnified in subsequent steps, which can result in incorrect measurements or assumptions. Thereby, standardization, reliability and reproducibility lay the foundations for the necessary accuracy in subsequent measurements or anatomical analysis. In this paper, we present an automatic method to annotate 3D surface skull models taking into account anatomical and geometrical features. METHODS: The proposed method follows a hybrid structure where a deformable template is used to initialize the landmark positions. Then, a refinement stage is applied using prior anatomical knowledge to ensure a correct placement. Our proposal is validated over thirty 3D skull scans of male Caucasians, acquired by hand-held surface scanning, and a set of 58 craniometric landmarks. A statistical analysis was carried out to analyze the inter- and intra-observer variability of manual annotations and the automatic results, along with a visual assessment of the final results. RESULTS: Inter-observer errors show significant differences, which are reflected in the expert consensus used as reference. The average localization error was 2.19±1.5 mm when comparing the automatic landmarks to the reference location. The subsequent visual analysis confirmed the reliability of the refinement method for most landmarks. CONCLUSIONS: Repeated manual annotations show a high variability depending on both skills and expertise of the observer, and landmarks' location and characteristics. In contrast, the automatic method provides an accurate, robust and reproducible alternative to the tedious and error-prone task of manual landmarking.

Evaluation of Parameters for Estimating the Postmortem Interval of Skeletal Remains Using Bovine Femurs: A Pilot Study.

The postmortem interval (PMI) of victims is a key parameter in criminal investigations. However, effective methods for estimating the PMI of skeletal remains have not been established because it is determined by various factors, including environmental conditions. To identify effective parameters for estimating the PMI of skeletal remains, we investigated the change in bone focusing on the amount of DNA, element concentrations, and bone density that occurred in the bone samples of bovine femurs, each maintained under one of five simulated environmental conditions (seawater, freshwater, underground, outdoors, and indoors) for 1 year. The amount of extracted mitochondrial DNA (mtDNA; 404 bp fragment) decreased over time, and significant DNA degradation (p < 0.01), as estimated by a comparison with amplification results for a shorter fragment (128 bp), was detected between 1 month and 3 months. Eleven of 30 elements were detected in samples by inductively coupled plasma optical emission spectrometry, and Na and Ba showed significant quantitative differences in terms of environmental conditions and time (p < 0.01). This preliminary study suggests that the level of DNA degradation determined by real-time polymerase chain reaction and element concentrations determined by inductively coupled plasma optical emission may be useful indices for estimating the PMI of victims under a wide range of environmental conditions. However, this study is a limited experimental research and not applicable to forensic cases as it is. Further studies of human bone with longer observation periods are required to verify these findings and to establish effective methods for PMI estimation.

Validation of mammographic x-ray spectra generated using Particle and Heavy Ion Transport code System.

A Monte Carlo (MC) code is a robust method to generate a mammographic x-ray spectrum because the geometry of a mammography system can be flexible and directly modeled in MC simulation. However, simulations from MC code need to be validated before it can be reliably used for specific applications. This study aimed to generate and validate the x-ray spectra of relevant anodes used in mammography and breast tomosynthesis using Particle and Heavy Ion Transport code System (PHITS). PHITS version 3.08 was used to generate the x-ray spectra of molybdenum (Mo), rhodium (Rh), and tungsten (W) anodes. The Mo anode spectrum derived using PHITS was compared with those obtained using other MC codes. The generated spectra of all anodes were compared with the literature. Parameters including spectral shape, K characteristic x-ray yield, heel effect, and half-value layer (HVL) were used for a comparative assessment. The differences in these assessment parameters conducted by PHITS and PHITSEGS5 simulations were studied. Regarding the comparative parameters, PHITSEGS5 simulation improved the accuracy of mammographic x-ray generation compared to PHITS simulation; K x-ray and bremsstrahlung yields of the Mo anode spectrum generated by PHITSEGS5 simulation were a better agreement with those generated by other MC code simulations. The PHITSEGS5 spectra overestimated K x-ray and low-energy bremsstrahlung photons in comparison with measured spectra. Subsequently, HVLs calculated from PHITSEGS5 spectra were 1.0% (Mo/Mo) and 7.0% (W/Al) lower than those derived from measured spectra. For Mo and Rh anodes, relative difference of HVLs calculated from PHITSEGS5 spectra and those obtained from literature and measurement were within the TRS 457 acceptance criteria (±0.02 mm Al). The observed difference exceeded the acceptance criteria for W anode. Regarding existed consistency in HVL between simulation and measurement, PHITSEGS5 simulation can be reliably used to generate x-ray spectra of Mo and Rh anodes. However, its accuracy should be improved for generating W anode spectrum.

Three-dimensional shape variation and sexual dimorphism of the face, nose, and mouth of Japanese individuals.

Three-dimensional (3D) shape variations of the face and facial parts in Japanese adults were examined to collect basic data to be used for facial comparison in forensics. In total, 1000 3D facial scans (500 males, 500 females) of Japanese individuals were re-meshed into anatomically homologous shape models and analyzed by principal component analysis (PCA) after Procrustes superimposition. Facial parts (the nose and the mouth) were segmented from homologous face models and analyzed by PCA, too. Among all kinds of objects (the face, the nose, and the mouth), the most predominant shape variation represented by the first principal component (PC1) was the height-width proportion. The second largest variation (PC2) in the face and the nose was depth; for the mouth, it was the relative protrusion of the upper and lower lips. We interpreted predominant shape variations represented by the first five principal components (PCs) in each object. Asymmetric shape variations were observed within these PCs for the nose and the mouth. Sexual dimorphism of the face and the facial parts was also examined by testing the significance of sex-linked differences in PC scores. A significant difference was found between males and females for many PCs. Sexual dimorphism was examined also by emphasizing the shape difference between average male and female faces. Our results revealed predominant 3D shape variations and sexual dimorphism of the face and facial parts. The results may be informative for performing facial comparison in police investigations, an increasingly used technique.

Assessment of scatter radiation dose and absorbed doses in eye lens and thyroid gland during digital breast tomosynthesis.

Digital breast tomosynthesis (DBT) is an alternative tool for breast cancer screening; however, the magnitude of peripheral organs dose is not well known. This study aimed to measure scattered dose and estimate organ dose during mammography under conventional (CM) and Tomo (TM) modes in a specific DBT system. Optically stimulated luminescence dosimeters (OSLDs), whose responses were corrected using a parallel-plate ionization chamber, were pasted on the surface of custom-made polymethyl methacrylate (PMMA) and RANDO phantoms to measure entrance surface air kerma (ESAK). ESAK measurements were also acquired with a 4.5-cm thick breast phantom for a standard mammogram. Organ dose conversion factors (CFD ) were determined as ratio of air kerma at a specific depth to that at the surface for the PMMA phantom and multiplied by the ratio of mass energy absorption coefficients of tissue to air. Normalized eye lens and thyroid gland doses were calculated using the RANDO phantom by multiplying CFD and ESAK values. Maximum variability in OSLD response to scatter radiation from the DBT system was 33% in the W/Rh spectrum and variations in scattered dose distribution were observed between CM and TM. The CFD values for eye lens and thyroid gland ranged between 0.58 to 0.66 and 0.29 to 0.33, respectively. Mean organ doses for two-view unilateral imaging were 0.24 (CM) and 0.18 (TM) µGy/mAs for the eye lens and 0.24 (CM) and 0.25 (TM) µGy/mAs for the thyroid gland. Higher organ doses were observed during TM compared to CM as the automatic exposure control (AEC) system resulted in greater total mAs values in TM.

Three-dimensional analyses of aging-induced alterations in facial shape: a longitudinal study of 171 Japanese males.

We sought to generate data to facilitate forensic facial comparisons. Specifically, we conducted a longitudinal study of alterations in face shape induced by aging. We obtained two three-dimensional facial shape measurements in 171 Japanese males at intervals of approximately 10 years. With this data, we created a homologous model consisting of 10,741 data points for each face based on 33 anatomical landmarks. We averaged the movements of corresponding data points between the two homologous models for each individual and used this data to predict up to 30 years of face aging in an average Japanese male. We clearly identified aging-induced shape changes, such as drooping and denting of the facial folds, drooping of the upper lip, and projection of the lower eyelid, in the virtually aged model. A quantitative comparison of aging-induced shape alterations among three age groups (individuals in their 20's, 30's, and 40-50's) showed that these alterations accelerated more quickly as age increased. Using our predictive model, we conducted a preliminary study focused on facial shape alterations induced by reductions in body weight. Our findings indicated that our proposed method would also be valid for this purpose.

An evaluation of the effect of microwave irradiation on bone decalcification aimed to DNA extraction.

An effect of intermittent microwave irradiation on decalcification of compact bone followed by DNA extraction was verified. In order to perform quantitative analysis regarding the degree of decalcification, Cubic bone specimens were prepared from bovine metacarpal bone and micro-focus X-ray CT imaging was applied to measure precise volume of decalcified area in the cubes. Microwave irradiation was performed under strict control of temperature using commercially available experimental device which is designed for advancing tissue fixation, decalcification, and antigen-antibody reaction by intermittent microwave. The integrity of the DNA obtained from irradiated specimen was also examined by PCR analysis. The results of morphological analysis with CT imaging showed that microwave irradiation has a positive effect on decalcification though that effect is not so drastic. The results obtained from PCR analysis showed that microwave irradiation decrease amplifiable DNA, suggesting that we should be careful to use microwave for the purpose of bone DNA extraction.

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.

[Evaluation of pain reduction upon injecting goserelin acetate using an ice pack].

LH-RH agonist is the key drug in hormonal therapy for premenopausal patients with breast cancer. It is important to reduce the pain related to injecting the LH-RH agonist, because patients must continue the medication for several years. We developed a way to reduce the pain by cooling the needle injecting site with a frozen ice pack. 18 premenopausal postoperated women filled out a questionnaire on the severity of pain upon injecting Goserelin acetate compared with the pain without this cooling method and pretreatment. We estimated the pain by a Numerical Rating Scale(NRS). The NRS scores of this cooling method revealed the pain to be significantly less than by control method(p<0. 005). This cooling method could be useful for the reduction of pain upon injecting the LH-RH agonist.

Evaluation of a new experimental kit for the extraction of DNA from bones and teeth using a non-powder method.

An experimental DNA extraction kit (new kit) was recently developed to extract DNA from degraded skeletal remains without the need for powdering the samples. We compared the utility of the new kit with the conventional phenol/chloroform method using real-time quantitative PCR and multiplex STR analysis. The new kit yielded large amounts of DNA from a compact bone fragment compared with the conventional phenol/chloroform method. We were able to extract sufficient DNA for STR analysis from 75% (3 of 4) and 60% (3 of 5) of the un-powdered tooth and bone samples, respectively, using the new kit. We were able to perform mini-STR analysis of the remaining samples using DNA extracted with the new kit. Furthermore, we successfully performed mitochondrial DNA sequencing of every sample. The new kit simplifies the DNA extraction procedure as it does not require powdering samples. Decreasing the number of procedural steps in DNA extraction will be beneficial in controlling DNA contamination in laboratories. Our results suggest that the new kit may be used for the simple, simultaneous extraction of DNA from multiple samples.

Mitochondrial DNA population data of HV1 and HV2 sequences from Japanese individuals.

Mitochondrial DNA sequences of the hypervariable regions HV1 and HV2 were determined for 1204 unrelated Japanese individuals. A total of 741 different mtDNA haplotypes were found, 157 of which were seen in multiple individuals. Twenty-seven of these individuals showed point heteroplasmy. The most frequent haplotype (16223T-16362C-73G-263G-315.1C) was found in 31 individuals and the second most frequent haplotypes (16129A-16223T- 16362C-73G-152C-263G-309.1C-315.1C) was found in 24 individuals. The haplotypes diversity and random match probability were calculated to be 0.9969 and 0.0040, respectively.

Gene injections of vascular endothelial growth factor and growth differentiation factor-9 stimulate ovarian follicular development in immature female rats.

OBJECTIVE: To examine the effect of vascular endothelial growth factor (VEGF) and growth differentiation factor-9 (GDF-9) on follicular development of the ovaries in immature female rats. DESIGN: Superovulation and gene injection. SETTING: Animal reproduction laboratory in Tohoku University, Sendai, Japan. ANIMAL(S): Wister-Imamichi female rats. INTERVENTION(S): The ovulated oocytes from rats with injected VEGF and GDF-9 gene fragments were counted, and the ovaries removed from those rats were used in the histologic observation. MAIN OUTCOME MEASURE(S): Follicular dynamics and angiogenesis after VEGF and GDF-9 gene fragments injection. RESULT(S): A single injection of the VEGF gene led to the production of a large number of oocytes (approximately 110 oocytes) from an individual animal that was injected with the gene at 21 days after birth, and after mating most of the oocytes were fertilized. Direct ovarian injection of GDF-9 stimulated the development of medium-sized antral follicles. The number of ovulated oocytes after injection of the VEGF plus GDF-9 gene fragments was the same as with a single injection of the VEGF gene. CONCLUSION(S): A single injection of the VEGF or GDF-9 gene stimulated follicular development, and injection of both genes did increase the number of ovulated oocytes from individual animals. An exogenous gene fragments injection promoted the maximum potential of ovarian function in immature female rats.

Effect of direct ovarian injection of vascular endothelial growth factor gene fragments on follicular development in immature female rats.

Vascular endothelial growth factor (VEGF) expression in granulosa cells is associated with the thecal vasculature growth during ovarian follicular development. We hypothesized that injection of VEGF gene fragments directly into the rat ovary would induce production of a large number of ovulatory follicles and that these follicles would ovulate. To test this hypothesis, we treated immature female rats with combinations of hormones and VEGF gene fragments. The animals were divided into two groups: one group received solution containing transfection reagents as a control (n = 5), while the other group received direct ovarian injection of VEGF gene fragments at 19 (n = 5), 21 (n = 5), 23 (n = 5), or 25 (n = 5) days after birth followed by i.p. administration of 20 IU equine chorionic gonadotropin (eCG) at the age of 26 days. Forty-eight hours after eCG injection, animals were given 20 IU human chorionic gonadotropin (hCG) i.p. and then the oocytes in both groups were counted. The maximum number of ovulated oocytes was obtained when the VEGF gene fragments were injected into the rat ovary at 21 days after birth. Histological examination revealed that the injection of VEGF gene fragments markedly increased the vascular density around the preovulatory follicles and also the number of these follicles. Our data provide the first reported evidence that most ovulatory follicles generated by injection of VEGF gene fragments are able to ovulate upon hCG treatment. These results demonstrate that injection of VEGF gene fragments directly into the ovary stimulates the development of antral follicles by inducing the formation of thecal vasculature in immature female rats.

Changes in blood viscosity with heavy and light exercise.

To clarify the relationship of the intensity of acute exercise to sudden cardiac death, we examined the effects of short-term heavy and light exercise on whole blood viscosity. Nine healthy sedentary male volunteers performed ten minutes of heavy (more than 95% of maximum oxygen consumption) or light (60% to 65% of maximum oxygen consumption) exercise. Blood samples were obtained before, immediately after, and one hour after exercise. The whole blood viscosity was immediately examined with an oscillation-type viscometer and was found to increase significantly after exercise and subsequently return to baseline levels within one hour after exercise. The whole blood viscosity increased by a similar degree after heavy or light exercise. Therefore, our results suggest that there is a similar risk of sudden cardiac death, due to increased whole blood viscosity, after short-term heavy or light exercise.

Induction of follicular development by direct single injection of vascular endothelial growth factor gene fragments into the ovary of miniature gilts.

Perifollicular angiogenesis is closely associated with ovarian follicular development. To investigate whether additional induction of perifollicular angiogenesis would support subsequent follicular development, we directly injected vascular endothelial growth factor (VEGF) gene fragments into the ovaries of miniature gilts, followed by gonadotroph treatment to stimulate follicle growth. In addition, to confirm extraexpression of the VEGF gene after injection, we assessed the expression of two isoforms of VEGF (VEGF 120 and VEGF 164) in granulosa cells and expression of fms-like tyrosine kinase (Flt-1), expression of fetal liver kinase (Flk-1), and density of capillary networks in theca cells. Direct injection of VEGF gene fragments into the ovaries was performed 7 days before eCG treatment. The ovaries in miniature gilts were removed 72 h after eCG treatment for histological examination. Granulosa cells and thecal tissues in the antral follicles (diameter, >4 mm) were collected to detect the mRNA expression of VEGF isoforms in the granulosa cells and of Flt-1 and Flk-1 in the thecal tissues by semiquantitative reverse transcription-polymerase chain reaction. The VEGF levels were measured in the follicular fluid by enzyme immunoassay. Injection of VEGF gene fragments increased the level of mRNA expression of VEGF 120 and 164 isoforms in the granulosa cells and VEGF protein contents in the follicular fluid. The number of preovulatory follicles and the capillary density in the theca interna increased significantly in the ovaries injected with VEGF gene fragments compared with those treated with eCG alone. The Flt-1, but not the Flk-1, mRNA expression show a tendency toward increasing in the thecal tissues of antral follicles in the ovaries injected with VEGF gene fragments. These results demonstrate that Flt-1 may be predominantly involved in the regulation of the capillary network in the theca interna during follicular development. Our data suggest that the regulation of perifollicular angiogenesis during follicular development is a very important factor in the development of ovulatory follicles. Our findings may offer an innovative technique for enhanced induction of follicular development in the ovary through gene and hormonal treatment, which may lead to prevention of infertility caused by ovarian dysfunction.