Forensic evaluation of the Asia Pacific ancestry-informative MAPlex assay.

DNA intelligence, and particularly the inference of biogeographical ancestry (BGA) is increasing in interest, and relevance within the forensic genetics community. The majority of current MPS-based forensic ancestry-informative assays focus on the differentiation of major global populations. The recently published MAPlex (Multiplex for the Asia Pacific) panel contains 144 SNPs and 20 microhaplotypes and aims to improve the differentiation of populations in the Asia Pacific region. This study reports the first forensic evaluation of the MAPlex panel using AmpliSeq technology and Ion S5 sequencing. This study reports on the overall performance of MAPlex including the assay's sequence coverage distribution and stability, baseline noise and description of problematic SNPs. Dilution series, artificially degraded and mixed DNA samples were also analysed to evaluate the sensitivity of the panel with challenging or compromised forensic samples. As the first panel to combine biallelic SNPs, multiple-allele SNPs and microhaplotypes, the MAPlex assay demonstrated an enhanced capacity for mixture detection, not easily performed with common binary SNPs. This performance evaluation indicates that MAPlex is a robust, stable and highly sensitive assay that is applicable to forensic casework for the prediction of BGA.

MAPlex - A massively parallel sequencing ancestry analysis multiplex for Asia-Pacific populations.

Current forensic ancestry-informative panels are limited in their ability to differentiate populations in the Asia-Pacific region. MAPlex (Multiplex for the Asia-Pacific), a massively parallel sequencing (MPS) assay, was developed to improve differentiation of East Asian, South Asian and Near Oceanian populations found in the extensive cross-continental Asian region that shows complex patterns of admixture at its margins. This study reports the development of MAPlex; the selection of SNPs in combination with microhaplotype markers; assay design considerations for reducing the lengths of microhaplotypes while preserving their ancestry-informativeness; adoption of new population-informative multiple-allele SNPs; compilation of South Asian-informative SNPs suitable for forensic AIMs panels; and the compilation of extensive reference and test population genotypes from online whole-genome-sequence data for MAPlex markers. STRUCTURE genetic clustering software was used to gauge the ability of MAPlex to differentiate a broad set of populations from South and East Asia, the West Pacific regions of Near Oceania, as well as the other globally distributed population groups. Preliminary assessment of MAPlex indicates enhanced South Asian differentiation with increased divergence between West Eurasian, South Asian and East Asian populations, compared to previous forensic SNP panels of comparable scale. In addition, MAPlex shows efficient differentiation of Middle Eastern individuals from Europeans. MAPlex is the first forensic AIM assay to combine binary and multiple-allele SNPs with microhaplotypes, adding the potential to detect and analyze mixed source forensic DNA.

A study of torrefied cardboard characterization and applications: Composition, oxidation kinetics and methane adsorption.

Torrefaction is proposed as a valorization process for non recycled cardboard. Torrefied cardboard was physically and chemically characterized and it was proposed for energy production and methane adsorption. The surface area and pore volume obtained were among 3.0-6.0m2/g and 5.7·10-3-2.3·10-2cm3/g, respectively. The carbon content increased with temperature and residence time of torrefaction. Oxidation kinetics of torrefied cardboard at different temperatures (250-300°C) and at different plateaus (60-120min) were tested. Torrefied cardboard was chemically treated with KOH in order to study the effect of K on thermal oxidation kinetics. It was observed that high torrefaction temperatures and residence times lead to a more stable char. Furthermore, kinetic parameters were obtained by iso-conversional methods and Coats and Redfern method. Attending to iso-conversional method, a decrease of Ea was observed with both, temperature and residence time of torrefaction. Whereas chemically treated presented highest Ea values than torrefied cardboard. In addition, regarding Coats and Redfern method, the oxidation model was not highly modified by torrefaction temperature and residence time. However, for chemically treated samples the oxidation model was modified by K presence. Finally, CH4 adsorption capacity of torrefied cardboard was studied at 30°C and atmospheric pressure. CH4 partial pressures tested were lower than 0.45kPa. It was observed that CH4 adsorption capacity increased with torrefaction time and decreased with chemical treatment. Thus, for the tested samples, the highest adsorption capacity observed was 5.70mgCH4/g of sample.

Towards simultaneous individual and tissue identification: A proof-of-principle study on parallel sequencing of STRs, amelogenin, and mRNAs with the Ion Torrent PGM.

DNA-based individual identification and RNA-based tissue identification represent two commonly-used tools in forensic investigation, aiming to identify crime scene sample donors and helping to provide links between DNA-identified sample donors and criminal acts. Currently however, both analyses are typically performed separately. In this proof-of-principle study, we developed an approach for the simultaneous analysis of forensic STRs, amelogenin, and forensic mRNAs based on parallel targeted DNA/RNA sequencing using the Ion Torrent Personal Genome Machine(®) (PGM™) System coupled with the AmpliSeq™ targeted amplification. We demonstrated that 9 autosomal STRs commonly used for individual identification (CSF1PO, D16S539, D3S1358, D5S818, D7S820, D8S1179, TH01, TPOX, and vWA), the AMELX/AMELY system widely applied for sex identification, and 12 mRNA markers previously established for forensic tissue identification (ALAS2 and SPTB for peripheral blood, MMP10 and MMP11 for menstrual blood, HTN3 and STATH for saliva, PRM1 and TGM4 for semen, CYP2B7P1 and MUC4 for vaginal secretion, CCL27 and LCE1C for skin) together with two candidate reference mRNA markers (HPRT1 and SDHA) can all be successfully combined. Unambiguous mRNA-based tissue identification was achieved in all samples from all forensically relevant tissues tested, and STR sequencing analysis of the tissue sample donors was 100% concordant with conventional STR profiling using a commercial kit. Successful STR analysis was obtained from 1ng of genomic DNA and mRNA analysis from 10ng total RNA; however, sensitivity limits were not investigated in this proof-of-principle study and are expected to be much lower. Since dried materials with noticeable RNA degradation and small DNA/RNA amplicons with high-coverage sequencing were used, the achieved correct individual and tissue identification demonstrates the suitability of this approach for analyzing degraded materials in future forensic applications. Overall, our study demonstrates the feasibility of simultaneously obtaining multilocus STR, amelogenin, and multilocus mRNA information for combined individual and tissue identification from a small sample of degraded biological material. Moreover, our study marks the first step towards combining many DNA/RNA markers for various forensic purposes to increase the effectiveness of molecular forensic analysis and to allow more forensically relevant information to be obtained from limited forensic material.

Predicting nitrate leaching under potato crops using transfer functions.

Nitrate leaching is a major issue in many cultivated soils. Models that predict the major processes involved at the field scale could be used to test and improve management practices. This study aims to evaluate a simple transfer function approach to predict nitrate leaching in sandy soils. A convective lognormal transfer (CLT) function is convoluted with functional equations simulating N mineralization, plant N uptake, N fertilizer dissolution, and nitrification at the soil surface to predict solute concentrations under potato (Solanum tuberosum L.) and barley (Hordeum vulgare L.) fields as a function of drainage water. Using this approach, nitrate flux concentrations measured in drainable lysimeters (1-m soil depth) were reasonably predicted from 29 Apr. 1996 to 3 Dec. 1996. With average application rates of 16.9 g m(-2) of N fertilizer in potato crops, mean nitrate-leaching losses measured under potato were 8.5 g N m(-2). Tuber N uptake averaged 9.7 g N m(-2) and soil mineral N at start (spring) and end (fall) of N mass balance averaged 1.7 and 4.5 g N m(-2), respectively. Soil N mineralization was estimated by difference (4.3 g N m(-2) on average) and was small compared with N fertilization. Small nitrate flux concentrations at the beginning of the cropping season (May) resulted mainly from initial soil nitrate concentrations. Measured and predicted nitrate flux concentrations significantly increased at mid-season (July-August) following important drainage events coupled with complete dissolution and nitrification of N fertilizers, and declining N uptake by potato plants. Decreases in nitrate concentrations before the end of year (November-December) underlined the predominant effect of N fertilizers applied for the most part at planting acting as a pulse input of solute.

Solute transport modeling under cultivated sandy soils and transient water regime.

Drainable lysimeters offer the possibility to integrate heterogeneous solute leaching conditions caused by row crops and transient water regime, and to conveniently measure water and solute fluxes at the drainage outlet. To compare solute leaching behavior in and around drainable lysimeters operating under a transient water regime in potato (Solanum tuberosum L.) fields, parameters of the convective lognormal transfer (CLT) function model were fitted using bromide (Br-) flux concentrations (Cf) measured in lysimeters and from Br- resident concentrations (Cr) measured in adjacent soil cores. Expected mean values Ez(I) obtained from Cr and Cf CLT parameters were equivalent and well correlated (R2 = 0.78). However, estimated median values mu of the CLT function were smaller when derived from Cr (1.05 to 1.28) compared with Cf (1.23 to 2.14). Most mu values were also smaller than previously reported values for a 30-cm reference depth, indicating that 50% of solute mass would leach more readily in these coarse sandy soils. Higher variance and dispersion of Cr compared with those of Cf could be related to a smaller sampling support (sample size/sampling area) in the case of Cr measured by soil coring, or to disruption of solute transport mechanisms in the repacked lysimeter. Retained Br- in the top soil layer after 12 to 17 cm of cumulative drainage was indicated by measured Cr. Neither CLT function simulated well residual topsoil Cr values, indicating that Br- plant cycling or preferential flow probably interfered even though tuber Br- uptake was relatively small.

Genome of the bacterium Streptococcus pneumoniae strain R6.

Streptococcus pneumoniae is among the most significant causes of bacterial disease in humans. Here we report the 2,038,615-bp genomic sequence of the gram-positive bacterium S. pneumoniae R6. Because the R6 strain is avirulent and, more importantly, because it is readily transformed with DNA from homologous species and many heterologous species, it is the principal platform for investigation of the biology of this important pathogen. It is also used as a primary vehicle for genomics-based development of antibiotics for gram-positive bacteria. In our analysis of the genome, we identified a large number of new uncharacterized genes predicted to encode proteins that either reside on the surface of the cell or are secreted. Among those proteins there may be new targets for vaccine and antibiotic development.

Consistent DNA losses on the short arm of chromosome 1 in a series of malignant gastrointestinal stromal tumors.

Fourteen malignant gastrointestinal stromal tumors (GISTs), characterized by immunohistochemistry, were analyzed by comparative genomic hybridization (CGH). The most striking feature was the detection of consistent DNA losses on the short arm of chromosome 1 in these 14 malignant tumors. Additional recurrent imbalances were also found: significant gains, which could be indicative of tumor progression, were frequent on the long arm of chromosome 1, as were losses of DNA copy number detected in chromosomes 13, 14, 15 and 22.

Fine-needle sampling of primary neuroendocrine carcinomas of salivary glands: cytohistological correlations and clinical analysis.

Fine-needle samplings of nine examples of primary neuroendocrine carcinoma of salivary glands were evaluated for their cytologic characteristics and were correlated with the corresponding histological sections. Consistent cytological findings were dispersed or loose clusters of poorly differentiated small- to intermediate-sized cells and occasional smudged nuclei. Mild to moderate nuclear pleomorphism, scant or absent cytoplasm, and nuclear molding were also observed. Rosette-like patterns and multinucleated cells were occasionally seen. Immunostaining of one recent case showed positivity for chromogranin and keratin. The differential diagnosis of primary and metastatic tumors with neuroendocrine features of the salivary glands is discussed.

Leiomyosarcomas and most malignant fibrous histiocytomas share very similar comparative genomic hybridization imbalances: an analysis of a series of 27 leiomyosarcomas.

Twenty-seven tumor samples with a diagnosis of leiomyosarcomas (LMS) were characterized by comparative genomic hybridization. The results were compared with immunohistochemical analysis of the smooth muscle profile of the tumors and expression of the RB1 gene protein. The comparative genomic hybridization profiles suggested that 7 of the 27 tumors might have been misclassified. High levels of DNA amplification were detected in 20 different small regions and recurrently involved bands 1p34, q21, 12q13-15, 17p, and 22q. Most recurrent simple gains were noted at sites such as 1p3, 1q21, 15q12-15, 16p, 17p and 17q, 19, 20q, 22q, and Xp. Significant losses of chromosome 13 were detected in 19 of the 27 tumors with a putative common region of loss in bands 13q14-21. Losses of chromosomes 1q, 2p and 2q, 4q, 9p, 10p and 10q, 11p and 11q23, and 16q were also highly recurrent. A comparative analysis between the most frequent genomic imbalances observed in this study of LMS and the genomic imbalances observed in a large proportion of malignant fibrous histiocytomas (MFH) from a previous study demonstrated that both types of tumors had similar recurrent imbalances. Although MFH were once thought to be a separate member of the soft tissue sarcoma family, our observations support the hypothesis that MFH are a morphologic modulation in the tumoral progression of other sarcomas, particularly LMS.

A germline mutation at the extreme 3' end of the APC gene results in a severe desmoid phenotype and is associated with overexpression of beta-catenin in the desmoid tumor.

Desmoid tumors arise sporadically or as part of the extraintestinal manifestations of familial adenomatous polyposis (FAP). In FAP, two distinct clinical presentations of the desmoid phenotype are seen: 1) one or a few desmoid tumors present predominantly in the abdominal wall or the abdomen; 2) a florid proliferation of tumors early in life, mostly near the axial skeleton or extremities. These different phenotypes have been associated with different sites of germline mutations in the adenomatous polyposis coli gene (APC gene). We present a large, French-Canadian kindred with a florid desmoid tumor phenotype caused by a germline mutation at codon 2643-2644 of the APC gene. The phenotype was characterized by the early onset of multiple tumors, arising near the axial skeleton and in proximal extremities. The penetrance of desmoid tumors was near 100% in this kindred. However, the expression of the disease was variable amongst the different affected relatives. Many gene carriers had cutaneous cysts. Polyposis of the colon was rarely observed in the affected individuals and we did not document upper gastro-intestinal polyps. The mutant APC allele did not express a stable truncated protein in vivo. Molecular analysis of the proband's tumor DNA revealed a somatic inactivating mutation of the wild-type allele. Immunohistochemistry on the tumor also demonstrated elevated levels of beta-catenin. The present study demonstrates that this extreme 3' APC mutation is associated with a severely penetrant desmoid phenotype and attenuated polyposis coli. It also suggests the involvement of the beta-catenin pathway in the development of desmoid tumors in FAP. The natural history of the disease is variable between individuals, and surgical interventions have to be timed appropriately due to the frequent recurrences.

Pleomorphic soft tissue myogenic sarcomas of adulthood. A reappraisal in the mid-1990s.

325 diverse sarcomas, 39 rhabdomyosarcomas (RMS), including all histologic variants, and 135 leiomyosarcomas (LMS) were identified. Within these two groups, 18 (46%) of the RMS and 14 (10%) of the LMS represented pleomorphic variants. These neoplasms were studied by morphology (histology and ultrastructure) and by immunohistochemical methods employing antibodies to intermediate filaments (vimentin and desmin) and actin isoforms [alpha-smooth (sm) and alpha-sarcomeric (sr) actins]. Twenty-four pleomorphic malignant fibrous histiocytomas (MFH) and eight pleomorphic liposarcomas (LS) were examined in a similar fashion. By light microscopy, the pleomorphic RMS, LMS, and MFH were indistinguishable, as each was dominated by pleomorphic cells disposed in a haphazard growth pattern; moreover, many featured fascicular, storiform, and sclerotic zones. The distinction between these neoplasms became apparent only following immunohistochemistry and/or ultrastructural study. All pleomorphic RMS disclosed rudimentary sarcomeres and exhibited the following cytoskeletal profile: vimentin (+) (18 of 18), desmin (+) (14 of 18), alpha-sr actin (+) (18 of 18) and alpha-sm actin (+) (five of 18). All the pleomorphic LMS featured smooth-muscle differentiation of variable degrees in the form of cytoplasmic bundles of microfilaments and associated dense bodies; their cytoskeletal profile was vimentin (+) (14 of 14), desmin (+) (seven of 14), alpha-sr actin (+) (none of 14), and alpha-sm actin (+) (eight of 14). The latter was demonstrated in all moderately differentiated, but absent or only focally expressed in poorly differentiated variants. All pleomorphic MFH and LS were devoid of myogenic (skeletal or smooth) ultrastructural features and expressed vimentin solely. This combined morphological and immunohistochemical study illustrates the following: First, these pleomorphic sarcomas are often indistinguishable by histologic growth pattern alone; thus, an accurate diagnosis requires study with all of these techniques. Second, pleomorphic myogenic sarcomas are restricted to adults and are not uncommon neoplasms among pleomorphic sarcomas: RMS (28%), LMS (21%), MFH (38%), and LS (13%). Third, the study defines desmin-negative and alpha-sm actin-positive pleomorphic RMS, and desmin-negative and alpha-sm-actin-negative pleomorphic LMS.

Isolation of yeast artificial chromosome clones from 54 polymorphic loci mapped with high odds on human chromosome 4.

We constructed a yeast artificial chromosome (YAC) framework map of human chromosome 4 by screening a YAC library with 63 polymorphic DNA markers located on the chromosome. These genetic markers are from two framework meiotic maps that had previously been constructed by two research groups, and are placed on the two maps with odds for their order of 1000:1 or greater. In addition to isolating and determining the sizes of 141 YAC clones for 54 of these markers, we combined the two framework meiotic maps to produce a single integrated map. These combined maps and the YAC clones provide a set of extended DNA loci ordered at high odds that can be used to isolate additional polymorphic loci and genes, and to serve as a framework for obtaining a higher resolution physical map of the chromosome.

The human MUC2 intestinal mucin has cysteine-rich subdomains located both upstream and downstream of its central repetitive region.

The human MUC2 mucin is a large secretory glycoconjugate that coats the epithelia of the intestines, airways, and other mucus membrane-containing organs. Previous work has shown that this mucin contains an extended tandem repeat-containing domain rich in Thr and Pro. In the present work we describe two additional regions of this mucin located both upstream and downstream of the tandem repeat array. The carboxyl-terminal domain contains 984 residues and can be divided into mucin-like (139 residues) and cysteine-rich (845 residues) subdomains. This latter subdomain exhibits varying degrees of sequence similarity to a wide range of mucins and mucin-like proteins including those isolated from rats, pigs, cows, and frogs. We also report here the sequence of 1270 residues lying immediately upstream of the tandem repeats. This region contains a repetitive, mucin-like subdomain and a second cysteine-rich stretch of more than 700 residues. Both cysteine-rich subdomains of this mucin have sequence similarity with von Willebrand factor, a serum protein that exists as a disulfide-linked polymer. This suggests that these cysteine-rich subdomains are important in the catenation of mucin monomers into oligomers, the structures that confer viscoelasticity upon mucus.

Prognostic influence of HSP-27 expression in malignant fibrous histiocytoma: a clinicopathological and immunohistochemical study.

Malignant fibrous histiocytoma is a very aggressive sarcoma. After the tumor has disseminated, chemotherapy is of little influence on the course of the disease because of the resistance to most chemotherapy regimens. We evaluated by immunohistochemistry the prognostic influence of the expression of a member of the stress polypeptides family, the heat-shock protein of 27 KDa (HSP-27). HSP-27 was found to be associated with an aggressive behavior in breast carcinoma and was related to chemoresistance in cell cultures. Forty-three malignant fibrous histiocytomas with no evidence of metastases at the time of diagnosis and resected between 1974 and 1985 were retrieved from the files of the Pathology Department of L'Hôtel-Dieu de Québec hospital. The immunostaining was performed on Bouin-fixed, paraffin-embedded tissue. Regardless of the percentage of positive cells, HSP-27 was expressed in the cytoplasm of 25 (58.1%) cases. HSP-27 expression was associated with a more favorable prognosis, and a significant correlation was observed with overall survival (P less than 0.025) and metastasis-free survival (P less than 0.05). HSP-27 expression was found to be the strongest prognostic factor, and multivariate analysis revealed that it was independent of tumor size, necrosis, and histological subtype. However, in the 13 patients with recurrent disease who underwent chemotherapy, the antigenic expression did not help to predict the treatment response. HSP-27 expression is one of the rare prognostic markers in this tumor type.

Molecular cloning of rat intestinal mucin. Lack of conservation between mammalian species.

We have prepared antisera to deglycosylated rat intestinal mucin and used it to obtain immunoreactive clones from a rat jejunum cDNA library. Four of these clones were sequenced, and all were found to be partial cDNAs that contained 18-base pair tandem repeats characteristic of a mucin. These cDNAs encoded a repetitive peptide with a consensus sequence of TTTPDV. Thus, they bear little resemblance to either of the two human intestinal mucin cDNAs isolated previously (Gum, J. R., Byrd, J. C., Hicks, J. W., Toribara, N. W., Lamport, D. T. A., and Kim, Y. S. (1989) J. Biol. Chem. 264, 6480-6487 and Gum, J. R., Hicks, J. W., Swallow, D. M., Lagace, R. E., Byrd, J. C., Lamport, D. T. A., Siddiki, B., and Kim, Y. S. (1990) Biochem. Biophys. Res. Commun. 171, 407-415). One of these rat mucin clones, designated RMUC 176, was chosen for further analysis. This clone recognized a band of approximately 9 kilobases when used to probe RNA blots. A strong hybridization band was present using rat small intestine and colon RNA but was not detectable when RNA isolated from heart, liver, or kidney was tested. The RMUC 176 clone and the two previously isolated human intestinal mucin cDNA clones were used to probe blots prepared from BamHI-digested DNA of various species. Here, the human probes detected fragments present only in human and chimpanzee DNA, whereas the RMUC 176 clone recognized fragments only in rat and mouse DNA. Thus, the repetitive portions of intestinal mucin genes are apparently not well conserved between phylogenetically distant species.

MUC-2 human small intestinal mucin gene structure. Repeated arrays and polymorphism.

MUC-2, the first described intestinal mucin gene, has become important as a prototype for secreted mucins in several organ systems. However, little is known about its protein backbone structure and hence its role in diseases such as colon cancer, ulcerative colitis, and cystic fibrosis, which are known to have mucin abnormalities. Studies in this manuscript show that MUC-2 contains two distinct regions with a high degree of internal homology, but the two regions bear no significant homology to each other. Region 1 consists mostly of 48-bp repeats which are interrupted in places by 21-24-bp segments. Several of these interrupting sequences show similarity to each other, creating larger composite repeat units. Region 1 has no length polymorphisms. Region 2 is composed of 69-bp tandem repeats arranged in an uninterrupted array of up to 115 individual units. Southern analysis of genomic DNA samples using TaqI and HinfI reveals both length and sequence polymorphisms which occur within region 2. The sequence polymorphisms have different ethnic distributions, while the length polymorphisms are due to variable numbers of tandem repeats.

The impact of source credibility characteristics on physicians' satisfaction with pharmaceutical salespeople.

Pharmaceutical salespeople are important sources of information for physicians about new and existing drugs. This study examines the relative impact of various source characteristics (i.e., expertise, trustworthiness and attractiveness) on physicians' satisfaction with the salesperson as an information source. The results suggest that although perceived expertise and trustworthiness are significant predictors of satisfaction, attractiveness is not. Implications are offered.