Developing forensic reference database by 18 autosomal STR for DNA identification in Republic of Belarus

For the Republic of Belarus, development of a forensic reference database on the basis of 18 autosomal microsatellites (STR) using a population dataset (N = 1040), “familial” genotypic dataset (N = 2550) obtained from expertise performance of paternity testing, and a dataset of genotypes from a criminal registration database (N = 8756) is described. Population samples studied consist of 80% ethnic Belarusians and 20% individuals of other nationality or of mixed origin (by questionnaire data). Genotypes of 12346 inhabitants of the Republic of Belarus from 118 regional samples studied by 18 autosomal microsatellites are included in the sample: 16 tetranucleotide STR (D2S1338, TPOX, D3S1358, CSF1PO, D5S818, D8S1179, D7S820, THO1, vWA, D13S317, D16S539, D18S51, D19S433, D21S11, F13B, and FGA) and two pentanucleotide STR (Penta D and Penta E). The samples studied are in Hardy–Weinberg equilibrium according to distribution of genotypes by 18 STR. Significant differences were not detected between discrete populations or between samples from various historical ethnographic regions of the Republic of Belarus (Western and Eastern Polesie, Podneprovye, Ponemanye, Poozerye, and Center), which indicates the absence of prominent genetic differentiation. Statistically significant differences between the studied genotypic datasets also were not detected, which made it possible to combine the datasets and consider the total sample as a unified forensic reference database for 18 “criminalistic” STR loci. Differences between reference database of the Republic of Belarus and Russians and Ukrainians by the distribution of the range of autosomal STR also were not detected, corresponding to a close genetic relationship of the three Eastern Slavic nations mediated by common origin and intense mutual migrations. Significant differences by separate STR loci between the reference database of Republic of Belarus and populations of Southern and Western Slavs were observed. The necessity of using original reference database for support of forensic expertise practice in the Republic of Belarus was demonstrated.     

Performance evaluation method for read mapping tool in clinical panel sequencing

In addition to the rapid advancement in Next-Generation Sequencing (NGS) technology, clinical panel sequencing is being used increasingly in clinical studies and tests. However, tools that are used in NGS data analysis have not been comparatively evaluated in performance for panel sequencing. This study aimed to evaluate the tools used in the alignment process, the first procedure in bioinformatics analysis, by comparing tools that have been widely used with ones that have been introduced recently. With the accumulated panel sequencing data, detected variant lists were cataloged and inserted into simulated reads produced from the reference genome (h19). The amount of unmapped reads and misaligned reads, mapping quality distribution, and runtime were measured as standards for comparison. As the most widely used tools, Bowtie2 and BWA–MEM each showed explicit performance with AUC of 0.9984 and 0.9970 respectively. Kart, maintaining superior runtime and less number of misaligned read, also similarly possessed high level of AUC (0.9723). Such selection and optimization method of tools appropriate for panel sequencing can be utilized for fields requiring error minimization, such as clinical application and liquid biopsy studies.     

STR polymorphisms in Philippine ethnolinguistic groups: evaluation of forensic utility

Population data was collected for the STR loci F13AO1, FES/FPS, HUMvWA, and HUMTHO1, in three major Philippine ethnolinguistic groups and used to estimate statistical parameters for identity testing in forensic work on Filipinos. The Cebuano, Ilocano, and Pampango populations in the Philippines were studied because they are among the biggest linguistic groups in the country, thus their genotypic profiles should substantially represent those of many Filipinos. The number of alleles varied from 4 to 9 at all loci, falling within the range observed for other local and world populations. Pairwise comparisons of the allele frequency distributions showed no statistical differences among the populations. The test for linkage equilibrium showed no evidence of non-random association of alleles across the physically unlinked loci in any of the three populations. The four loci combined gave an exclusion power of > or =0.9995 and a power of paternity exclusion of 0.8859-0.9389.     

Synthesis and evaluation of in vitro bioactivity for vesicular acetylcholine transporter inhibitors containing two carbonyl groups

To identify selective high-affinity ligands for the vesicular acetylcholine transporter (VAChT), we have incorporated a carbonyl group into the structures of trozamicol and prezamicol scaffolds, and also converted the secondary amines of the piperidines of trozamicols and prezamicols into amides. Of 18 new racemic compounds, 4 compounds displayed high affinity for VAChT (K(i)=10-20 nM) and greater than 300-fold selectivity for VAChT over σ(1) and σ(2) receptors, namely (4-(4-fluorobenzoyl)-4'-hydroxy-[1,3'-bipiperidin]-1'-yl)(3-methylthiophen-2-yl)methanone oxalate (9g) (K(i-VAChT)=11.4 nM, VAChT/σ(1)=1063, VAChT/σ(2)=370), (1'-benzoyl-4'-hydroxy-[1,3'-bipiperidin]-4-yl)(4-methoxyphenyl)methanone oxalate (10c) (K(i-VAChT)=15.4 nM, VAChT/σ(1)=374, VAChT/σ(2)=315), (4'-hydroxy-1'-(thiophene-2-carbonyl)-[1,3'-bipiperidin]-4-yl)(4-methoxyphenyl)methanone oxalate (10e) (K(i-VAChT)=19.0 nM, VAChT/σ(1)=1787, VAChT/σ(2)=335), and (4'-hydroxy-1'-(3-methylthiophene-2-carbonyl)-[1,3'-bipiperidin]-4-yl)(4-methoxyphenyl)methanone oxalate (10g) (K(i-VAChT)=10.2 nM, VAChT/σ(1)=1500, VAChT/σ(2)=2030). These four compounds can be radiosynthesized with C-11 or F-18 to validate their possibilities of serving as PET probes for quantifying the levels of VAChT in vivo.     

Isolation and identification of an alkyldiacylglycerol containing isovaleric acid

We have isolated and identified a unique subclass of alkyldiacylglycerols from the pink portion of the harderian gland of the New Zealand white rabbit. Using chemical, enzymic, chromatographic, and physical procedures, the lipid class has been identified as 1-alkyldiacylglycerol containing 1 mole of isovaleric acid. More than 50% of the O-alkyl moieties consist of 16:0 and 18:0 carbon chains, whereas the other major O-alkyl moieties are 15:0 and 17:0 branched chains ( approximately 30%). The long-chain acyl groups of the alkyldiacylglycerol subclass consist primarily of saturated fatty acids (60% 16:0 and 30% 18:0) and a small amount of branched-chain fatty acids ( approximately 5%), whereas the 3-position appears to be occupied by isovaleric acid.     

Genetic variations and forensic characteristics of Han Chinese population residing in the Pearl River Delta revealed by 23 autosomal STRs

Molecular Biology Reports - Pearl River Delta, known as the Chinese “South Gate”, locates adjacent to Hong Kong and Macao, and across the sea from the Southeast Asian region. Herein,...     

Use of in-house PCR for identification of Mycobacterium tuberculosis in BACTEC broth cultures of respiratory specimens

We evaluated the ability of a PCR assay to identify Mycobacterium tuberculosis complex (MTBC) from positive BACTEC(R) 12B broth cultures. A total of 107 sputum samples were processed and inoculated into Ogawa slants and BACTEC(R) 12B vials. At a growth index (GI) > 30, 1.0 ml of the 12B broth was removed, stored, and assayed with PCR. Molecular results were compared to those obtained by phenotypic identification methods, including the BACTEC(R) NAP method. The average times required to perform PCR and NAP were compared. Of the 107 broth cultures evaluated, 90 were NAP positive, while 91 were PCR positive for MTBC. Of particular interest were three contaminated BACTEC(R) 12B broth cultures yielding microorganisms other than acid-fast bacilli growth with a MTBC that were successfully identified by PCR, resulting in a mean time of 14 days to identify MTBC before NAP identification. These results suggest that PCR could be used as an alternative to the NAP test for the rapid identification of MTBC in BACTEC(R) 12B cultures, particularly in those that contained both MTBC and nontuberculous mycobacteria.     

Development and application of an in-house reverse hybridization method for Chlamydia trachomatis genotyping

Aim

To develop and evaluate an in‐house reverse hybridization technique for Chlamydia trachomatis genotype identification.

Methods and Results

The evaluation of the developed and optimized reverse hybridization method on reference strains showed the specific detection of all genotypes. This technique showed its ability to type one inclusion‐forming unit of C. trachomatis genotype E and equivalent sensitivity to the Cobas TaqMan assay. It was also able to detect mixed infections in vitro. Application of the reverse hybridization method on 38 isolated C. trachomatis strains and their respective swabs allowed the detection of six urogenital genotypes D, E, F, G, H and K and one trachoma genotype B. Genotype E was the most prevalent, detected in 73% of the swab samples. Mixed infections were detected in 26% of swab cases.

Conclusion

The reverse hybridization technique is simple and does not require specialized instruments. It is powerful in the diagnosis of mixed infections and is suitable for use in epidemiological studies.

Significance and Impact of the Study

This technique allowed rapid C. trachomatis genotype identification.     

Construction of a BAC library for Chinese amphioxus Branchiostoma belcheri and identification of clones containing Amphi-Pax genes

Amphioxus is a crucial organism for the study of vertebrate evolution. Although a genomic BAC library of Branchiostoma floridae has been constructed, we report here another BAC library construction of its distant relative species Branchiostoma belcheri. The amphioxus BAC library established in present study consists of 45,312 clones arrayed in one hundred and eighteen 384-well plates. The average insert fragment size was 120 kb estimated by Pulsed Field Gel Electrophoresis (PFGE) analysis of 318 randomly selected clones. The representation of the library is about 12 equivalent to the genome, allowing a 99.9995% probability of recovering any specific sequence of interest. We further screened the library with 4 single copied Amphi-Pax genes and identified total of 26 positive clones with average of 6.5 clones for each gene. The result indicates this library is well suited for many applications and should also serve as a useful complemental resource for the scientific community.     

Laser Raman identification of an interaction site on DNA for arginine containing histones in chromatin.

Comparisons of the Raman spectra of DNA, chromatin, and complexes of DNA with poly-L-arginine and N-α-acetylarginine have been made. Both in native chromatin and in complexes of DNA with the arginine derivatives there is a marked decreased in the Raman intensity of the 1490±2 cm^?1 band due to guanine. Considerable evidence is presented to show that a decrease in the intensity of the 1490 cm^?1 Raman band of quanine in DNA is strong indication of a hydrogen bond being attached to the N-7 position of quanine. A specific model is presented for the interaction of the arginine residues with the guanine residues in the major groove of DNA. The Raman frequency of the histone Amide 1 band indicates that these protein molecules have a high α-helical content while the phosphate diester stretch frequency of the DNA shows the DNA to be in the B-family.     

Use of an alpha-D-glucosidase for the specific cytochemical identification of lateral alpha-D-xylosyl end groups in plant xyloglucans

alpha-Linked D-xylosyl side chains represent the typical feature common to all xyloglucans not shared by other cell wall polysaccharides. Since no easily available alpha-D-xyloxidase is known, advantage was taken of the conformational and configurational homologies between alpha-D-xylopyranose and alpha-D-glucopyranose to make an alpha-D-glucosidase-gold complex which was able to recognize alpha-D-xylosyl terminal residues of xyloglucans. This marker was used together with alpha-L-fucosidase gold complex for the double labeling on two different structural features of the same macromolecule in plant primary cell wall.     

Reconstruction of facial features from the skull: an evaluation of its usefulness in forensic anthropology

Although facial reconstructions from the skull have been widely used in forensic anthropology, their effectiveness has not been objectively assessed. Photographs of two reconstructions produced in this laboratory were shown to FAA employees and local policemen who were asked to select the subject's in vivo photograph when included with those of six other randomly-selected individuals of the same sex, race, and general age. While in both tests the reconstruction subject was chosen with significantly greater frequency than the controls, the results ranged from 26% correct (N = 104) on the first subject, a 67-year-old female, to 67% correct (N = 200) on the second, a 36-year-old male. In both tests policemen and civilian females performed better than civilian males.     

Synthesis and evaluation of phosphopeptides containing iminodiacetate groups as binding ligands of the Src SH2 domain

Phosphopeptide pTyr-Glu-Glu-Ile (pYEEI) has been introduced as an optimal Src SH2 domain ligand. Peptides, Ac-K(IDA)pYEEIEK(IDA) (1), Ac-KpYEEIEK (2), Ac-K(IDA)pYEEIEK (3), and Ac-KpYEEIEK(IDA) (4), containing 0–2 iminodiacetate (IDA) groups at the N- and C-terminal lysine residues were synthesized and evaluated as the Src SH2 domain binding ligands. Fluorescence polarization assays showed that peptide 1 had a higher binding affinity (K_d = 0.6 μM) to the Src SH2 domain when compared with Ac-pYEEI (K_d = 1.7 μM), an optimal Src SH2 domain ligand, and peptides 2–4 (K_d = 2.9–52.7 μM). The binding affinity of peptide 1 to the SH2 domain was reduced by more than 2-fold (K_d = 1.6 μM) upon addition of Ni²⁺ (300 μM), possibly due to modest structural effect of Ni²⁺ on the protein as shown by circular dichroism experimental results. The binding affinity of 1 was restored in the presence of EDTA (300 μM) (K_d = 0.79 μM). These studies suggest that peptides containing IDA groups may be used for designing novel SH2 domain binding ligands.     

Breaking out of the biomed box: an audit assessment and recommendations for an in-house biomedical engineering program

In order to assess the current performance and to identify future growth opportunities of an in-house biomedical engineering (BME) program, senior management of Lehigh Valley Hospital (Allentown, Penn) engaged (in July 2001) the services of a clinical engineering consultant. Although the current in-house program was both functionally and financially sound, an independent audit had not been performed in over 4 years, and there were growing concerns by the BME staff related to the department's future leadership and long-term support from senior management. After an initial 2-month audit of the existing program, the consultant presented 41 separate recommendations for management's consideration. In order to refine and implement these recommendations, 5 separate committees were established to further evaluate a consolidated version of them, with the consultant acting as the facilitator for each group. Outcomes from each of the committees were used in the development of a formal business plan, which, upon full implementation, would not only strengthen and refine the current in-house service model but could also result in a substantial 3-year cost savings for the organization ($1,100,000 from existing operations, $500,000 in cost avoidance by in-sourcing postwarranty support of future capital equipment acquisitions). Another key outcome of the project was related to the development of a new master policy, titled the "Medical Equipment Management Program," complete with a newly defined state-of-the-art equipment scheduled inspection frequency model.     

Molecular cloning and in vitro evaluation of an infectious simian-human immunodeficiency virus containing env of a primary Chinese HIV-1 subtype C isolate

Human immunodeficiency virus (HIV) clade C is the most prevalent subtype and accounts for approximately 50% of all HIV infections worldwide. In China, the prevalent HIV strains are B'/C subtypes, in which the envelope belongs to subtype C. To evaluate potential AIDS vaccines targeting Chinese viral strains in non-human primate models, we constructed an infectious simian-human immunodeficiency virus (SHIV) that expresses most of the envelope of a primary HIV strain, which was isolated from a HIV-positive intravenous drug user from XinJiang province in China. The resulting chimeric SHIV-XJ02170 was infectious in human, rhesus monkey and cynomolgus monkey peripheral blood mononuclear cells (PBMC) and used CCR5 exclusively as coreceptor.