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.

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.

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.

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.

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.

Malignant fibrous histiocytoma. Clinicopathologic and ultrastructural study of 12 cases.

The clinical and pathologic findings of 12 cases of malignant fibrous histiocytoma of the soft tissues are presented. The mean age of the patients (eight males and four females) was 64 years, and there was a strong predilection for localization in the extremities (10 cases). The lesion is of rather low-grade malignancy, with metastases in only a minority of cases (three cases) but with nonetheless a marked tendency for local recurrence (nine cases). Histologically, the essential feature of the tumor is its biphasic composition with both histiocytic and fibroblastic components, the latter showing a characteristic storiform pattern. In addition, a variable number of xanthomatous cells and multinucleated giant cells were observed. Ultrastructural studies confirmed the presence of the aforementioned cells and in addition revealed a primitive mesenchymal cell. It is suggested that this cell may be a primitive histiocyte from which the two main cellular components of the tumor arise.

Myofibroblasts in the stroma of invasive and metastatic carcinoma: a possible host response to neoplasia.

After observing the presence of numerous stromal myofibroblasts in scirrhous mammary carcinomas, a series of invasive and metastatic carcinomas from diverse sites was examined by electron microscopy to determine whether myofibroblasts might also be present in their stroma. Myofibroblasts were identified in each instance and were most abundant in neoplasms which were hard, sclerotic, and retracted. This finding suggests that myofibroblasts represent a component of the stromal reaction to many carcinomas and contribute to the desmoplasia and retraction which characterize many of these neoplasms. The host commands several responses to neoplasia. As a result of the expression of tumor-associated antigens, the immune system contributes lymphocytes, macrophages, and antibodies, a reflection of immunologic surveillance against neoplasia. In contrast to experimental systems tumor neoantigens are poorly expressed or even lacking in many human neoplasms; thus, the immune system may be weakly stimulated or not activated at all. Tumor neovascularization induced by a tumor-angiogenesis factor represents a second host response, possibly deleterious, for it may facilitate tumor dissemination. The stromal myofibroblast reaction to many invasive and metastatic carcinomas may constitute a third, albeit more primitive response. The density of collagen produced and contractile state of such tissue may signify an attempt by the host stroma to contain the neoplasm and impede vascular invasion. If so, myofibroblast induction may complement immune surveillance or constitute a separate mechanism of response to invasive neoplasia in man.

Desmoplastic fibroma of bone. An ultrastructural study.

The ultrastructure of three cases of desmoplastic fibroma of bone is presented. The lesion is principally characterized by myofibroblasts admixed with lesser numbers of fibroblasts and primitive mesenchymal cells. Thus, the cellular composition is similar to that described in a variety of nonneoplastic proliferative processes of soft tissue. It is postulated that the myofibroblastic proliferation develops in response to unknown factors acting on marrow fibroblasts or primitive mesenchymal cells.

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.

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.

Paraganglioma of the filum terminale.

An unusual tumor arising in the filum terminale is described. The clinical data revealed an extensive and slowly growing lesion. The histologic picture was characterized by a proliferation of lobules and sheets of regular cells within a rich vascular network. Electron microscopic studies showed light and dark cells with sustentacular extensions. Typical neurosecretory granules were observed in both cell types, establishing the diagnosis of para-ganglioma. The glomus coccygeum could be the site of this tumor.

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.

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.

[Polypoid pseudosarcomatous carcinoma. Histogenesis with evidence of myofibroblastic response (author's tranls)]. / Carcinoma polypoïde pseudosarcomateux. Histogenèse. Evidence d'une résponse myofibroblastique.

Three polypoid pseudosarcomatous carcinomas (PPSC) of the oesophagus, larynx, and urinary bladder were studied by electron microscopy to determine their histogenesis and the cellular associations which might contribute to their relative clinical benignity. In addition to a polypoid, exophytic growth, the three shared similar light microscopy features : surface ulceration, a minimal invasive epithelial component and prominent spindle-cell proliferation in association with reticulin and collagen. Three cell types were identified ultrastructurally : 1. - spindle and polygonal cells demonstrating tonofilaments and plasma membrane specialization, denoting an epithelial origin; 2. - cells identical to the first type, but with cytoplasm containing intracellular collagen ; and 3. - myofibroblasts characterized by a rough endoplasmic reticulum, prominent Golgi complexes and well formed bundles of myofilaments. The latter constituted the principal cellular component of the tumors. These findings demonstrate that the stroma of the PPSC is dominated by myofibroblasts, which probably synthesized much of the collagen associated with these neoplasms. It is suggested that the myofibroblasts signify an expression of host reactivity to the invasive carcinoma and constitute a beneficial response which contains and restricts local and vascular invasion by neoplastic cells, this contributing to the relative benignity of the neoplasms.

[Evaluation of the differences of opinion in the histopathologic diagnosis of soft tissue tumors]. / Evaluation des divergences d'opinion sur le diagnostic histopathologique des tumeurs des tissus mous.

Evaluation of observers variation in the histopathologic diagnosis of soft tissue tumors. 260 cases of soft tissue tumors referred to the CTRC (Canadian Tumour Reference Center) for diagnostic opinion were reviewed. These tumors were collected from 98 different hospitals within the 10 canadian provinces and had been submitted to a specialized panel of pathologists having a particular interest in this field. 22 tumors of nonmesenchymal origin have been discarded. The main problem posed by the contributors related to the histologic typing of overtly malignant and sarcomatous tumors (122/238). Comparing the diagnoses proposed by contributors and the panel, a consensus in term of benign and malignant tumor was reached in 89% of the cases. Within this panel, there was consensus in 84% of the cases. However, regarding the histologic typing, a consensus was reached between contributors and the panel in only 65% of the studied cases. Within the panel, there was a majority diagnosis in merely 62% of them. A mean of three different diagnoses were proposed for each case. A review of the literature shows that histologic typing of soft tissue tumors is of only limited prognostic significance. On the other hand, the clinical staging comprising histologic grade, size, depth, local growth and metastases is essential to establish prognosis and treatment. However, since certain tumors respond differently to treatment, a precise histopathologic diagnosis using immunohistochemistry and electron microscopy is mandatory.