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We evaluated whether MinION, an inexpensive portable sequencer, can be used to identify the origin of crude drugs derived from animals. Standard and nonstandard crude drugs with different species of origin were examined. In addition, standards mixed with nonstandard samples were used. As a target gene, cytochrome c oxidase I was amplified and sequenced. The fast mode results had a slightly lower match ratio than high-accuracy mode, but the animals of origin were correctly determined by BLAST for all samples. For antler velvet derived from Rangifer tarandus, even when the sequences were aligned based on Cervus elaphus, the animal of origin was determined correctly. Minor contents could be detected from mixtures of two animals, if the mixtures contained at least 19:1 mtDNA when the coverage allele-fraction threshold was 0.05. By contrast, in fast mode, two sequences could not be separated due to the low accuracy of the base-calling for each read. For fieldwork, the species of origin of crude drugs could be identified with only simple DNA extraction and library preparation. Therefore, MinION appears to be a convenient tool for identifying the origins of crude drugs derived from animals. [ABSTRACT FROM AUTHOR]

We tried to estimate individual mtDNA haplotypes in mixed DNA samples by combining MinION and MiSeq. The BAM files produced by MiSeq were viewed using Integrative Genomics Viewer (IGV) to verify mixed bases. By sorting the reads according to base type for each mixed base, partial haplotypes were determined. Then, the BAM files produced by MinKNOW were viewed using IGV. To determine haplotypes with IGV, only mixed bases determined by MiSeq were used as target bases. By sorting the reads according to base type for each target base, each contributor's haplotype was estimated. In mixed samples from two contributors, even a haplotype with a minor contribution of 5% could be distinguished from the haplotype of the major contributor. In mixed samples of three contributors (mixture ratios of 1:1:1 and 4:2:1), each haplotype could also be distinguished. Sequences of C-stretches were determined very inaccurately in the MinION analysis. Although the analysis method was simple, each haplotype was correctly detected in all mixed samples with two or three contributors in various mixture ratios by combining MinION and MiSeq. This should be useful for identifying contributors to mixed samples. [ABSTRACT FROM AUTHOR]

We developed a method that can detect each animal species of origin for crude drugs derived from multiple animal species based on massively parallel sequencing analysis of mitochondrial genes. The crude drugs derived from animals investigated in this study were Cervi Parvum Cornu and Trogopterorum feces, which are derived from a mix of different animal species, two chopped cicada sloughs, and two commercial Kampo drugs. The mitochondrial 12S rRNA, 16S rRNA, and cytochrome oxidase subunit I gene regions were amplified and sequenced using MiSeq. The ratios of haplotype to total number of sequences reads were calculated after sequence extraction and trimming. Haplotypes that exceeded the threshold were defined as positive haplotypes, which were compared with all available sequences using BLAST. In the Cervi Parvum Cornu and Trogopterorum feces samples, the haplotype ratios corresponded roughly to the mixture ratios, although there was a slight difference from mixture ratios depending on the gene examined. This method could also roughly estimate the compositions of chopped cicada sloughs and Kampo drugs. This analysis, whereby the sequences of several genes are elucidated, is better for identifying the included animal species. This method should be useful for quality control of crude drugs and Kampo drugs. [ABSTRACT FROM AUTHOR]

We evaluated whether the number of contributors to mixed DNA samples can be estimated by analyzing the D-loop of mitochondrial DNA using massively parallel sequencing. The A- (positions 16,209–16,400) and B- (positions 30–284) amplicons in hypervariable regions 1 and 2, respectively, were sequenced using MiSeq with 2 × 251 cycles. Sequence extraction and trimming were performed using CLC Genomics Workbench 11 and the number of observed haplotypes was counted for each amplicon type using Microsoft Excel. The haplotype ratios were calculated by dividing the number of counted reads of the corresponding haplotype by the total number of sequence reads. Haplotypes that were over the threshold (5%) were defined as positive haplotypes. The number of larger positive haplotypes in either of the two amplicon types was defined as the number of contributors. Samples were collected from seven individuals. Seventeen mixed samples were prepared by mixing DNA from two to five contributors at various ratios. The number of contributors was correctly estimated from almost all of the mixed samples containing equal amounts of DNA from two to five people. In mixed samples of two or three people, the minor components were detected down to a ratio of 20:1 or 8:2:1. However, heteroplasmy, base deletions, and sharing of the same haplotypes caused incorrect estimations of the number of contributors. Although this method still has room for improvement, it may be useful for estimating the number of contributors in a mixed sample, as it does not rely on forensic mathematics. [ABSTRACT FROM AUTHOR]

We evaluated whether the origins of crude drugs derived from arthropods and annelids could be identified using molecular biological techniques. DNA was extracted from 20 crude drugs prepared from different animals using a commercial kit with added phenol treatment. The target regions used to identify origin were the mitochondrial 16S ribosomal RNA (rRNA), 12S rRNA, and cytochrome oxidase subunit I (COI) gene regions. Extracted DNA was amplified by polymerase chain reaction, and then sequenced by the Sanger method. The aligned sequences were compared with all available sequences using BLAST to estimate the origins of the crude drugs. The origin of crude drugs used in this study could be estimated using this method. The COI region was the best for identifying origin among three regions examined, based on the success rate of PCR amplification and analysis. Moreover, the 12S rRNA region was also useful for origin identification, with the exception of the earthworm. However, the origin of some crude drugs could not be strictly identified due to matches to various species in all three regions. One likely cause was that the species of origin of a crude drug has not been registered in DNA databases. We found that even the same crude drug from the same pharmaceutical company had different origins by production lot or import source country. Therefore, this method is useful not only for DNA-based origin identification but also quality control of production lots. [ABSTRACT FROM AUTHOR]

We established widely applicable DNA extraction methods to identify the origins of crude drugs derived from animals. Twenty-one samples including 17 kinds of crude drug derived from animals were examined. DNA was extracted from most of the crude drugs by adjustment of the QIAamp® DNA Mini Kit. DNA extraction was performed successfully using phenol to remove impurities after applying a proteinase treatment. DNA extraction was performed successfully by decalcification treatment using ethylenediaminetetraacetic acid (EDTA), before applying the proteinase treatment for crude drugs having high calcium content, such as those from oyster shell and cuttlefish bone. DNA could not be extracted from sea-ear shell using the EDTA decalcification treatment, but was extracted successfully using a TBONE EX KIT. The mitochondrial 16S ribosomal RNA (rRNA) gene region was amplified, and Basic Local Alignment Search Tool (BLAST) analysis was performed after sequencing. Polymerase chain reaction (PCR) products of approximately 600 bp in length were obtained from all samples except donkey glue, one of the two seahorses, and longgu. Drug origins were determined in all samples by sequence analysis based on the BLAST results, and match rates were >97 %. Moreover, 16 samples had a match rate >99 %. Our DNA extraction methods were widely applicable to evaluation of many crude drugs derived from animals, and proved very useful for identifying the origins of such drugs. [ABSTRACT FROM AUTHOR]

Mosquitoes occur almost worldwide, and females of some species feed on blood from humans and other animals to support ovum maturation. In warm and hot seasons, such as the summer in Japan, fed mosquitoes are often observed at crime scenes. The current study attempted to estimate the time that elapsed since feeding from the degree of human DNA digestion in mosquito blood meals and also to identify the individual human sources of the DNA using genotyping in two species of mosquito: Culex pipiens pallens and Aedes albopictus. After stereomicroscopic observation, the extracted DNA samples were quantified using a human DNA quantification and quality control kit and were genotyped for 15 short tandem repeats using a commercial multiplexing kit. It took about 3 days for the complete digestion of a blood meal, and genotyping was possible until 2 days post-feeding. The relative peak heights of the 15 STRs and DNA concentrations were useful for estimating the post-feeding time to approximately half a day between 0 and 2 days. Furthermore, the quantitative ratios derived from STR peak heights and the quality control kit (Q129/Q41, Q305/Q41, and Q305/Q129) were reasonably effective for estimating the approximate post-feeding time after 2–3 days. We suggest that this study may be very useful for estimating the time since a mosquito fed from blood meal DNA, although further refinements are necessary to estimate the times more accurately. [ABSTRACT FROM AUTHOR]

A dog saliva analysis in addition to a bite-mark analysis may be important for evidence when a crime involves a dog bite. In this study, the utility of detecting canine saliva-specific mRNAs to identify canine saliva was evaluated. Canine saliva swabs ( n = 20), urine swabs ( n = 20), body surface swabs ( n = 20), whole blood samples ( n = 10), human saliva ( n = 20), human skin surface swabs ( n = 20), and human whole blood ( n = 20) were tested. The saliva-specific genes encoding statherin (STATH), carbonic anhydrase VI (CA-VI), and dog allergens (Canf1 and Canf2) were analyzed as candidate genes. Moreover, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as confirmation of canine mRNA extraction. STATH, CA-VI, Canf1, Canf2, and GAPDH mRNAs were detected in 19/20, 1/20, 11/20, 4/20, and 20/20 saliva samples, respectively. The STATH, CA-VI, Canf1, Canf2, and GAPDH mRNAs did not exhibit cross-reactivity with samples of human origin. This mRNA-based assay was also able to detect canine saliva in mock forensic samples. The results of this study indicated that the detection of STATH mRNA is useful for the identification of canine saliva, and GAPDH is a suitable marker for canine mRNA extraction. [ABSTRACT FROM AUTHOR]

A novel screening method for shed skin cells by detecting Staphylococcus epidermidis ( S. epidermidis), which is a resident bacterium on skin, was developed. Staphylococcus epidermidis was detected using real-time PCR. Staphylococcus epidermidis was detected in all 20 human skin surface samples. Although not present in blood and urine samples, S. epidermidis was detected in 6 of 20 saliva samples, and 5 of 18 semen samples. The ratio of human DNA to S. epidermidis DNA was significantly smaller in human skin surface samples than in saliva and semen samples in which S. epidermidis was detected. Therefore, although skin cells could not be identified by detecting only S. epidermidis, they could be distinguished by measuring the S. epidermidis to human DNA ratio. This method could be applied to casework touch samples, which suggests that it is useful for screening whether skin cells and human DNA are present on potential evidentiary touch samples. [ABSTRACT FROM AUTHOR]

A novel method for sex determination, based on the detection of the number of X chromosomes, was established. Current methods, based on the detection of the Y chromosome, can directly identify an unknown sample as male, but female gender is determined indirectly, by not detecting the Y chromosome. Thus, a direct determination of female gender is important because the quality (e.g., fragmentation and amelogenin-Y null allele) of the Y chromosome DNA may lead to a false result. Thus, we developed a novel sex determination method by analyzing the number of X chromosomes using a copy number variation (CNV) detection technique (the comparative Ct method). In this study, we designed a primer set using the amelogenin-X gene without the CNV region as the target to determine the X chromosome copy number, to exclude the influence of the CNV region from the comparative Ct value. The number of X chromosomes was determined statistically using the CopyCaller software with real-time PCR. All DNA samples from participants (20 males, 20 females) were evaluated correctly using this method with 1-ng template DNA. A minimum of 0.2-ng template DNA was found to be necessary for accurate sex determination with this method. When using ultraviolet-irradiated template DNA, as mock forensic samples, the sex of the samples could not be determined by short tandem repeat (STR) analysis but was correctly determined using our method. Thus, we successfully developed a method of sex determination based on the number of X chromosomes. Our novel method will be useful in forensic practice for sex determination. [ABSTRACT FROM AUTHOR]

Tubal choriocarcinoma is an extremely rare condition and can be of gestational or non-gestational origin. The appropriate management of choriocarcinoma begins with the categorization of the tumor. However, it is difficult to discriminate the two types by routine histological examination. We report the first case of gestational choriocarcinoma of the Fallopian tube to be confirmed by a combination of p57KIP2 immunostaining and DNA polymorphism analysis at 15 short tandem repeat loci, along with X and Y chromosome markers. The patient had no detectable metastasis or evidence of recurrence 15 months after treatment, which involved surgery without adjuvant chemotherapy. This case demonstrates the usefulness of a combination of p57KIP2 immunostaining and DNA polymorphism analysis in determining the origin of extrauterine choriocarcinoma (i.e. gestational or non-gestational), which helps to determine the strategy for treatment of the disease. [ABSTRACT FROM AUTHOR]

We previously reported that detection of Streptococcus salivarius is feasible for proving the presence of saliva in a forensic sample. Here, a simple and rapid method for the detection of S. salivarius in forensic samples was developed that uses loop-mediated isothermal amplification (LAMP). The LAMP primer set was designed using S. salivarius-specific sequences of glucosyltransferase K. To simplify the procedure, the sample was prepared by boiling and mutanolysin treatment only, and the entire analytical process was completed within 2.5 h. The cut-off value was set at 0.1 absorbance units, measured at 660 nm, upon termination of the reaction. S. salivarius was identified in all saliva samples, but was not detected in other body fluids or on the skin surface. Using this method, S. salivarius was successfully detected in various mock forensic samples. We therefore suggest that this approach is useful for the identification of saliva in forensic practice. [ABSTRACT FROM AUTHOR]

BACKGROUND: Recently, a more accurate and reliable screening test has been investigated for noninvasive prenatal fetal RHD genotyping from D– women. The objective of this study was to perform the new method of noninvasive fetal RHD genotyping with maternal plasma from D– women by use of capillary electrophoresis. STUDY DESIGN AND METHODS: Blood samples were obtained from 8 D+ and 8 D– nonpregnant donors and mixed to make test plasma samples. DNA was extracted and the appropriate conditions relating to the initial sample volume as well as polymerase chain reaction cycle numbers were analyzed to detect the RHD gene with RHD exon 10 primer. Blood samples were also obtained from 13 D– pregnant women ranging from the 12th to 39th weeks of gestation. The presence of the RHD gene and Y-chromosome–specific STR (Y-STR) derived from the fetus was analyzed. The results were compared with the D status of newborns. RESULTS: In samples from 12 D– pregnant women, the RHD gene was detected. In one sample, the RHD gene was not detected but Y-STR loci were demonstrated in this sample, indicating a D– male baby. The results of fetal genotyping were all in concordance with the postpartum samples by serologic tests on D as well as with the sex of newborns. CONCLUSION: Capillary electrophoresis can be used for the determination of fetal RHD status in D– women. This diagnostic method is useful for the noninvasive prenatal diagnosis of the fetal RHD genotyping from D– women. [ABSTRACT FROM AUTHOR]

Describes an algorithm called Path Limited Partial Response and Maximum Likelihood (PRML) that has been used to implement a high speed Viterbi decoder large scale integration (LSI). Importance to digital magnetic recording systems; Pre-detection and limited path; Development of viterbi decoder LSI; Experimental high-definition television full bit digital video-cassette recorder.

Focuses on a study which presented a controller design framework for cooperating manipulators based on a reliable controller design concept. Formula of reliable stabilization problem; Details on the controller design; Conclusion.

POPULATION: Seventy-three unrelated Africans living in Cape Town, south region of South Africa. [ABSTRACT FROM AUTHOR]

Examines genomic DNA extracted from blood samples taken from unrelated Indonesian males using the phenol-chloroform method. Detection of the amplified products with ABI PRISM 310 Genetic Analyzer; Haplotype diversity; Allele frequencies; Gene diversity.

Focuses on the extraction of genomic DNA from blood and semen samples taken from unrelated Japanese males through the phenol-chloroform method for forensic analysis. Amplification of the Y-chromosomal short tandem repeat loci; Detection of the amplified products with the genetic analyzer.

The authors have measured the potential of electrostatic discharge using a commercial VCR drive with metal powder (MP) tapes that are widely used in business. They set up an experimental drive, which has a surface voltage meter and a magneto-resistive read (MR) head with a current-measuring circuit. Using this system, they can measure the surface voltage and current while the tape is running and can investigate how the electrostatic potential from the tape can damage the head. In their assumption that if the media surface voltage is low and current flowing into the head is lower than the sensing current of the head, the MR head can be used in a VCR. It can be not only AMR but may also be giant magnetoresistive and TMR in the future. The results of their experiments show that the peak surface voltage is low and the peak current is only about 3.6 µA with a middle-resistance tape. The peak current is lower than the breakdown current since the sensing current is typically 10 mA. Therefore, an MR head can be used with an MP tape. The authors found that the optimum surface resistance of middle-resistance tape is 10[sup6] to 10[sup7] · Ωsq. [ABSTRACT FROM AUTHOR]

Background: A hydatidiform mole with a coexisting fetus is a rare condition that commonly occurs as either a partial mole with fetus or a twin pregnancy comprising a complete mole and normal fetus. In the former case, the fetus is triploid, and in the latter case, the fetus is diploid with different alleles from those of the mole. Because there is a difference in the persistent trophoblastic disease incidence between the two, an accurate diagnosis is required.Case Presentation: We present a case of a 34-year-old Japanese woman who was pregnant with a hydatidiform mole and two coexisting fetuses. At 17 weeks of gestation, hemorrhage-induced progressive anemia in the mother prompted the decision to terminate the pregnancy, after which no complications occurred. Molecular cytogenetic analysis revealed that one of the fetuses was a normal diploid fetus with the same allele in the fetus and placenta. The hydatidiform mole was revealed to be a mosaic of two diploids, and the other coexisting fetus was a normal diploid that shared one of the mole alleles.Conclusions: This was presumed to be a rare case of twin pregnancy by triploid embryo formation, followed by loss of an allele due to postzygotic diploidization, development of a diploid fetus, and development of another fetus from a separate embryo. Because of the existence of cases such as this one with a diploid fetus, but without a normal pregnancy coexistent with a complete hydatidiform mole, diagnosis by genetic analysis is required for prognosis. [ABSTRACT FROM AUTHOR]

A linear channel model and a linear signal processing technique are available for a holographic data storage channel when our proposed coherent addition technique is applied to the reproduction process. As a result, the signal-to-noise ratio (SNR) after zero-interpixel-interference (IPI) equalization is markedly improved. Moreover, we can achieve a higher transfer rate at a lower oversampling rate owing to the almost ideal linearity of the reproduced signal. [ABSTRACT FROM AUTHOR]

An improvement of the recording density in a coaxial holographic recording system is described. We employ a new optical setup into our coaxial system, where the numerical aperture (NA) of an objective lens and the data capacity in one page increase up to 0.85 and 135 kbits, respectively. The recording performances are evaluated experimentally and numerically. The obtained results demonstrate that implemented techniques successfully improve the recording density by a factor of 1.5, achieving a raw data density of 270 Gbit/in.2. [ABSTRACT FROM AUTHOR]

A holographic data storage channel is normally a nonlinear channel; however, it can be made linear. Using coherent addition of DC components in the reproduction process and calculating the square root of intensity, we can retrieve a linearly reproduced signal. Our simulation results revealed that a conventional equalizer works well to suppress interpixel interference so that a higher recording density can be achieved. [ABSTRACT FROM AUTHOR]