Multiple Tissue Samples Research Articles

A novel method to scale up fungal endophyte isolations

Studies involving fungal endophytes very often rely on surface-sterilisation of plant samples to enable endophyte isolations. However, surface-sterilisation can be very time-consuming, potentially limiting the number of samples processed. To overcome this limitation, a novel method was developed to bulk surface-sterilise multiple plant tissue samples simultaneously and separately. The method relies on 24 perforated Falcon™ tubes, each containing a sample, sequentially transferred through a series of containers holding the sterilants. The samples that can be surface-sterilised using this method include roots, stems and leaves or entire seedlings. This method increased our throughput by a factor of 24 relative to conventional surface-sterilisation methods.

KeyGenes, a Tool to Probe Tissue Differentiation Using a Human Fetal Transcriptional Atlas

SummaryDifferentiated derivatives of human pluripotent stem cells in culture are generally phenotypically immature compared to their adult counterparts. Their identity is often difficult to determine with certainty because little is known about their human fetal equivalents in vivo. Cellular identity and signaling pathways directing differentiation are usually determined by extrapolating information from either human adult tissue or model organisms, assuming conservation with humans. To resolve this, we generated a collection of human fetal transcriptional profiles at different developmental stages. Moreover, we developed an algorithm, KeyGenes, which uses this dataset to quantify the extent to which next-generation sequencing or microarray data resemble specific cell or tissue types in the human fetus. Using KeyGenes combined with the human fetal atlas, we identified multiple cell and tissue samples unambiguously on a limited set of features. We thus provide a flexible and expandable platform to monitor and evaluate the efficiency of differentiation in vitro.

A Multiscale Model Evaluates Screening for Neoplasia in Barrett's Esophagus.

Barrett’s esophagus (BE) patients are routinely screened for high grade dysplasia (HGD) and esophageal adenocarcinoma (EAC) through endoscopic screening, during which multiple esophageal tissue samples are removed for histological analysis. We propose a computational method called the multistage clonal expansion for EAC (MSCE-EAC) screening model that is used for screening BE patients in silico to evaluate the effects of biopsy sampling, diagnostic sensitivity, and treatment on disease burden. Our framework seamlessly integrates relevant cell-level processes during EAC development with a spatial screening process to provide a clinically relevant model for detecting dysplastic and malignant clones within the crypt-structured BE tissue. With this computational approach, we retain spatio-temporal information about small, unobserved tissue lesions in BE that may remain undetected during biopsy-based screening but could be detected with high-resolution imaging. This allows evaluation of the efficacy and sensitivity of current screening protocols to detect neoplasia (dysplasia and early preclinical EAC) in the esophageal lining. We demonstrate the clinical utility of this model by predicting three important clinical outcomes: (1) the probability that small cancers are missed during biopsy-based screening, (2) the potential gains in neoplasia detection probabilities if screening occurred via high-resolution tomographic imaging, and (3) the efficacy of ablative treatments that result in the curative depletion of metaplastic and neoplastic cell populations in BE in terms of the long-term impact on reducing EAC incidence.

Brain somatic mutations in MTOR cause focal cortical dysplasia type II leading to intractable epilepsy.

Focal cortical dysplasia type II (FCDII) is a sporadic developmental malformation of the cerebral cortex characterized by dysmorphic neurons, dyslamination and medically refractory epilepsy. It has been hypothesized that FCD is caused by somatic mutations in affected regions. Here, we used deep whole-exome sequencing (read depth, 412-668×) validated by site-specific amplicon sequencing (100-347,499×) in paired brain-blood DNA from four subjects with FCDII and uncovered a de novo brain somatic mutation, mechanistic target of rapamycin (MTOR) c.7280T>C (p.Leu2427Pro) in two subjects. Deep sequencing of the MTOR gene in an additional 73 subjects with FCDII using hybrid capture and PCR amplicon sequencing identified eight different somatic missense mutations found in multiple brain tissue samples of ten subjects. The identified mutations accounted for 15.6% of all subjects with FCDII studied (12 of 77). The identified mutations induced the hyperactivation of mTOR kinase. Focal cortical expression of mutant MTOR by in utero electroporation in mice was sufficient to disrupt neuronal migration and cause spontaneous seizures and cytomegalic neurons. Inhibition of mTOR with rapamycin suppressed cytomegalic neurons and epileptic seizures. This study provides, to our knowledge, the first evidence that brain somatic activating mutations in MTOR cause FCD and identifies mTOR as a treatment target for intractable epilepsy in FCD.

Role of double-balloon enteroscopy in malignant small bowel tumors.

To assess the double-balloon enteroscopy (DBE) role in malignant small bowel tumors (MSBT). This is a retrospective descriptive study performed in a single center. All consecutive patients who underwent a DBE with final diagnosis of a malignant neoplasm from 2004 to 2014 in our referral center were included. Patient demographic and clinical pathological characteristics were recorded and reviewed. MSBT diagnosis was achieved either by DBE directed biopsy with multiple tissue sampling, endoscopic findings or histological analysis of surgical specimen. We have analyzed double-balloon enteroscopy impact in outcome and clinical course of these patients. Of 627 patients, 28 (4.5%) (mean age = 60 ± 17.3 years) underwent 30 procedures (25 anterograde, 5 retrograde) and were diagnosed of a malignant tumor. Patients presented with obscure gastrointestinal bleeding (n = 19, 67.9%), occlusion syndrome (n = 7, 25%) and diarrhea (n = 1, 3.6%). They were diagnosed by DBE biopsy (n = 18, 64.3%), histological analysis of surgical specimen (n = 7, 25%) and unequivocal endoscopic findings (n = 2, 7.1%). Gastrointestinal stromal tumor (n = 8, 28.6%), adenocarcinoma (n = 7, 25%), lymphoma (n = 4, 14.3%), neuroendocrine tumor (n = 4, 14.3%), metastatic (n = 3, 10.7%) and Kaposi sarcoma (n = 1, 3.6%) were identified. DBE modified outcome in 7 cases (25%), delaying or avoiding emergency surgery (n = 3), modifying surgery approach (n = 2) and indicating emergency SB partial resection instead of elective approach (n = 2). DBE may be critical in the management of MSBT providing additional information that may be decisive in the clinical course of these patients.

Multi-residues determination of antimicrobials in fish tissues by HPLC-ESI-MS/MS method.

A rapid, simple, sensitive and specific LC-MS/MS method was developed and validated for the simultaneous quantification of four antimicrobials commonly used in aquaculture, namely ciprofloxacin (CPX), trimethoprim (TMP), sulphadimethoxine (SDM) and florphenicol (FLOR) in fish tissues. The LC-MS/MS was operated under the multiple-reaction monitoring mode using electrospray ionization. Sample preparation involves simple liquid extraction step followed by post-extraction clean-up step with n-hexane. The purified extracts were chromatographed on Agilent Poroshell 120 EC, C18 (50 mm × 3 mm, 2.7 μm) column by pumping an isocratic mobile phase consisting of 0.1% formic acid in water:0.1% formic acid in methanol (20:80, by volume) at a flow rate of 0.4 mL/min. A detailed validation of the method was performed as per FDA guidelines and the standard curves were found to be linear in the range of 1-100 ng/g for both CPX and TMP, 0.5-100 ng/g for SDM and 1-50 ng/g for FLOR. The intra-day and inter-day precision and accuracy of the results were within the acceptable limits. A run time of 1.5 min for each sample made it possible to analyze multiple fish tissue samples per day. The developed assay method was successfully applied for the detection of antimicrobials in real fish tissue samples obtained from different fish farms.

Abstract 3346: Development of a multiplex screening panel for Akt signaling pathway biomarkers in cell and tissue lysate models

Abstract Major challenges face the large-scale implementation of intracellular tumor biomarkers for clinical diagnosis and therapeutic development. These challenges include non-quantitative results, insufficient assay sensitivity, and lack of multiplexing capability. For many studies, sample volumes are limited (e.g., tumor lysates); however, it is essential to extract as much biomarker information as possible from a given sample while maintaining the quality and consistency necessary to sustain ongoing studies. To address these challenges, MSD® has developed multiplex panels for assaying cell signaling biomarkers using fit-for-purpose methods that emphasize optimal multiplex combinations and rigorous development of critical reagents. Here we report the development and verification of a multiplex screening panel of immunoassays for simultaneous measurement of total and phosphorylated analytes of the Akt signalling pathway using MSD technology. Markers in the panel include phosphorylated and total forms of GSK3B, p70S6K, FOXO3a, PTEN, Akt, S6RP, PRAS40, and ERK1. We demonstrate the ability of these assays to measure analyte levels in multiple tumor-derived cell lines and human tissue samples (normal and tumor) with excellent sensitivity and performance. Most analytes can be quantified using no more than 10 µg of sample. By using recombinant proteins to calibrate some of the assays, we were able to quantify analytes in cultured cell lysates, including MCF-7 and Jurkat T-cells. Lack of analyte specificity is a well-known issue when multiplexing intracellular signaling analytes, especially those in a common signaling pathway such as the Akt pathway. Through rigorous optimization, we achieved non-specific binding less than or equal to 1% for all analytes in this panel. Spike recovery and dilution linearity were characterized to demonstrate matrix tolerance in tumor cell line and tissue lysates as well as dynamic ranges between 3 and 4 logs, allowing quantification of analytes at different abundance levels without using multiple dilutions. In conclusion, MSD MULTI-SPOT® Akt signaling assay panels have been developed and verified for measurement of intracellular tumor biomarkers of relevance to clinical diagnosis, therapeutic development, and treatment of various cancers. The accuracy, reliability, ease of use, and high-throughput features of these multiplex assays make them ideally suited for use in large-scale clinical studies. Citation Format: Thomas W. Miller, Karen Tressler, Jill Dunty, Paula Eason, Leonid Dzantiev, Sripriya Ranganathan, Laura Schaefer, David Stewart, Pankaj Oberoi, Jacob Wohlstadter. Development of a multiplex screening panel for Akt signaling pathway biomarkers in cell and tissue lysate models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3346. doi:10.1158/1538-7445.AM2014-3346

Current antibiotic management of prosthetic joint infections in Italy: the 'Udine strategy'.

The rate of prosthetic joint infections followed and cured at our institution is constantly increasing, in line with epidemiological data from the recent literature. This is probably related to the greater number of knee and hip prostheses implanted every year. For intermediate and late infections, only the two-stage approach is applied, as this demonstrates the best outcome in our experience. Particular attention is paid to microbiological isolation of the pathogen: multiple samples of tissue are collected during the interventions, and kept in culture for a longer period of time than usual. Sonication of prosthetic devices is used to enhance the sensitivity and specificity of the microbiological cultures. Histological examination influences surgical choices either towards implantation of a new prosthesis or replacement of the spacer. An empirical antibiotic backbone of a glycopeptide/lipopeptide and rifampicin is chosen, due to the leading role of Gram-positive bacteria in this setting and the high incidence of methicillin resistance in our centre (>30%), followed by an antibiotic regimen containing linezolid. If specific risk factors are present, an anti-Gram-negative drug is added to the regimen. Duration of therapy depends upon the approach that is chosen, usually being 6 weeks when the prosthesis is removed. Despite at the moment being limited by its small sample size, data from our experience confirms that our empirical approach may represent a valid choice during the early phase of treatment, by keeping linezolid for a step-down therapy of shorter duration (4 weeks).

'All in a box' a concept for optimizing microbiological diagnostic sampling in prosthetic joint infections.

BackgroundAccurate microbial diagnosis is crucial for effective management of prosthetic joint infections. Culturing of multiple intraoperative tissue samples has increased diagnostic accuracy, but new preparatory techniques and molecular methods hold promise of further improvement. The increased complexity of sampling is, however, a tough challenge for surgeons and assistants in the operation theatre, and therefore we devised and tested a new concept of pre-packed boxes with a complete assortment of swabs, vials and additional tools needed in the operating theatre for non-standard samples during a clinical study of prosthetic joint infections.FindingsThe protocol for the clinical study required triplicate samples of joint fluid, periprosthetic tissue, bone tissue, and swabs from the surface of the prosthesis. Separate boxes were prepared for percutaneous joint puncture and surgical revision; the latter included containers for prosthetic components or the entire prosthesis. During a 2-year project period 164 boxes were used by the surgeons, 98 of which contained a complete set of samples. In all, 1508 (89%) of 1685 scheduled samples were received.ConclusionWith this concept a high level of completeness of sample sets was achieved and thus secured a valid basis for evaluation of new diagnostics. Although enthusiasm for the project may have been a contributing factor, the extended project period suggests that the ‘All in a box’ concept is equally applicable in routine clinical settings with standardized but complex diagnostic sampling.

Prognostic values of ETS-1, MMP-2 and MMP-9 expression and co-expression in breast cancer patients

The aim of this study was to analyse expression of ETS-1 protein and two gelatinases (MMP-2 and MMP-9) and their possible prognostic value in breast carcinoma patients, as well as correlation of their expression with other known prognostic factors such as tumor size, grade, vascular invasion, steroid receptor values, HER2 values and proliferative index. The expression of MMP-2, MMP-9 and ETS-1 was immunohistochemicaly analysed in 121 consecutive primary breast carcinoma patients who underwent surgery at the Clinical Hospital Centre Zagreb during 2002. Three representative areas from each tumor paraffin blocks were taken and arranged on a recipient paraffin block with predefined coordinates for simultaneous analyses of multiple tissue samples (TMA). ETS-1, MMP-2 and MMP-9 expression and co-expression were correlated with other clinico-pathological parameters and based on the available clinical follow up data survival analysis was performed. The ETS-1 protein is found to be expressed in tumor cell nuclei and cytoplasm as well as in stromal lymphocytes, fibroblasts and endothelial cells. MMP-2 and MMP-9 were found to be expressed in cytoplasm of both, tumor and stromal cells. For our analysis only tumor cell expression was used for statistical analysis. We found 56,2% ETS-1 positive tumors, 77,7% were MMP-2 positive, and MMP-9 was expressed in 90% of primary breast carcinomas. There were no significant correlations between MMP-s expression and other patohistological prognostic factors, but expression of ETS-1 was significantly correlated with higher tumor size and grade, as well as with negative steroid receptors. Co-expression of MMP-2, MMP-9 and ETS-1 was found in 40,5 % of tumors, and more commonly was found in tumors larger than 2 cm, high grade tumors, and steroid receptor negative tumors. In univariate analysis, statistically significant negative impact on overall survival (OS) had tumor size, nuclear and tumor grade, ETS-1 expression in tumor cells, co-expression of ETS-1 either with MMP-2 or MMP-9, as well as co-expression of ETS-1, MMP-2 and MMP-3. Disease free survival (DFS) was significantly shorter in patients with tumors greater than 2 cm, ETS-1 positive tumors, ETS-1 and MMP-2 or MMP-9 co-expressed tumors, and additionally in tumors with ETS-1, MMP-2 and MMP-9 co-expression. These results suggest that expression of ETS-1 as well as MMP-2, MMP-9 and ETS-1 co-expression might be used as a poor prognostic factor in breast cancer patients.

Acquisition of Mouse Tumor Biopsies through Abdominal Laparotomy

The static analysis of tumor tissues at a single, terminal end point has been the mainstay of studies in mouse models of cancer. However, with the development of model systems that reproduce the intra- and intertumoral heterogeneity of human tumors, such studies are limited by the need for large numbers of animals to overcome increased intragroup variance. The shortcomings of a single-timepoint approach to molecular analysis are especially apparent in the context of therapeutic studies, in which the dynamic response to treatment is of particular consequence. To mitigate the effects of intertumoral heterogeneity, multiple tissue samples may be harvested from the same tumor at different time points through the use of surgical biopsies. For abdominal tumors, preprocedure imaging may be used to assess the suitability of tumors for biopsy. Sterile surgical techniques are used to access the abdominal cavity, and customized instruments facilitate the immobilization and retrieval of tissue samples from hard or fibrous tumors. Thermoregulation, hemostasis, and wound closure techniques are critical to successful surgical outcomes, whereas appropriate anesthetic, analgesic, and postoperative recovery regimens are important for maintenance of animal welfare. Using a mouse model of pancreatic ductal adenocarcinoma, we present a comprehensive protocol suitable for the routine acquisition of abdominal tumor biopsies.

Evaluating real-time immunohistochemistry on multiple tissue samples, multiple targets and multiple antibody labeling methods

BackgroundImmunohistochemistry (IHC) is a well-established method for the analysis of protein expression in tissue specimens and constitutes one of the most common methods performed in pathology laboratories worldwide. However, IHC is a multi-layered method based on subjective estimations and differences in staining and interpretation has been observed between facilities, suggesting that the analysis of proteins on tissue would benefit from protocol optimization and standardization. Here we describe how the emerging and operator independent tool of real-time immunohistochemistry (RT-IHC) reveals a time resolved description of antibody interacting with target protein in formalin fixed paraffin embedded tissue. The aim was to understand the technical aspects of RT-IHC, regarding generalization of the concept and to what extent it can be considered a quantitative method.ResultsThree different antibodies labeled with fluorescent or radioactive labels were applied on nine different tissue samples from either human or mouse, and the results for all RT-IHC analyses distinctly show that the method is generally applicable. The collected binding curves showed that the majority of the antibody-antigen interactions did not reach equilibrium within 3 hours, suggesting that standardized protocols for immunohistochemistry are sometimes inadequately optimized. The impact of tissue size and thickness as well as the position of the section on the glass petri dish was assessed in order for practical details to be further elucidated for this emerging technique. Size and location was found to affect signal magnitude to a larger extent than thickness, but the signal from all measurements were still sufficient to trace the curvature. The curvature, representing the kinetics of the interaction, was independent of thickness, size and position and may be a promising parameter for the evaluation of e.g. biopsy sections of different sizes.ConclusionsIt was found that RT-IHC can be used for the evaluation of a number of different antibodies and tissue types, rendering it a general method. We believe that by following interactions over time during the development of conventional IHC assays, it becomes possible to better understand the different processes applied in conventional IHC, leading to optimized assay protocols with improved sensitivity.

Histology-derived volumetric annotation of the human hippocampal subfields in postmortem MRI

Recently, there has been a growing effort to analyze the morphometry of hippocampal subfields using both in vivo and postmortem magnetic resonance imaging (MRI). However, given that boundaries between subregions of the hippocampal formation (HF) are conventionally defined on the basis of microscopic features that often lack discernible signature in MRI, subfield delineation in MRI literature has largely relied on heuristic geometric rules, the validity of which with respect to the underlying anatomy is largely unknown. The development and evaluation of such rules are challenged by the limited availability of data linking MRI appearance to microscopic hippocampal anatomy, particularly in three dimensions (3D). The present paper, for the first time, demonstrates the feasibility of labeling hippocampal subfields in a high resolution volumetric MRI dataset based directly on microscopic features extracted from histology. It uses a combination of computational techniques and manual post-processing to map subfield boundaries from a stack of histology images (obtained with 200μm spacing and 5μm slice thickness; stained using the Kluver–Barrera method) onto a postmortem 9.4Tesla MRI scan of the intact, whole hippocampal formation acquired with 160μm isotropic resolution. The histology reconstruction procedure consists of sequential application of a graph-theoretic slice stacking algorithm that mitigates the effects of distorted slices, followed by iterative affine and diffeomorphic co-registration to postmortem MRI scans of approximately 1cm-thick tissue sub-blocks acquired with 200μm isotropic resolution. These 1cm blocks are subsequently co-registered to the MRI of the whole HF. Reconstruction accuracy is evaluated as the average displacement error between boundaries manually delineated in both the histology and MRI following the sequential stages of reconstruction. The methods presented and evaluated in this single-subject study can potentially be applied to multiple hippocampal tissue samples in order to construct a histologically informed MRI atlas of the hippocampal formation.

A novel device to stretch multiple tissue samples with variable patterns: Application for mRNA regulation in tissue-engineered constructs

A broad range of cells are subjected to irregular time varying mechanical stimuli within the body, particularly in the respiratory and circulatory systems. Mechanical stretch is an important factor in determining cell function; however, the effects of variable stretch remain unexplored. In order to investigate the effects of variable stretch, we designed, built and tested a uniaxial stretching device that can stretch three-dimensional tissue constructs while varying the strain amplitude from cycle to cycle. The device is the first to apply variable stretching signals to cells in tissues or three dimensional tissue constructs. Following device validation, we applied 20% uniaxial strain to Gelfoam samples seeded with neonatal rat lung fibroblasts with different levels of variability (0%, 25%, 50% and 75%). RT-PCR was then performed to measure the effects of variable stretch on key molecules involved in cell-matrix interactions including: collagen 1α, lysyl oxidase, α-actin, β1 integrin, β3 integrin, syndecan-4, and vascular endothelial growth factor-A. Adding variability to the stretching signal upregulated, downregulated or had no effect on mRNA production depending on the molecule and the amount of variability. In particular, syndecan-4 showed a statistically significant peak at 25% variability, suggesting that an optimal variability of strain may exist for production of this molecule. We conclude that cycle-by-cycle variability in strain influences the expression of molecules related to cell-matrix interactions and hence may be used to selectively tune the composition of tissue constructs.

ERCP with Probe-Based Confocal Laser Endomicroscopy for the Evaluation of Dominant Biliary Stenoses in Primary Sclerosing Cholangitis Patients

The incidence of cholangiocarcinoma (CCA) in primary sclerosing cholangitis (PSC) ranges between 7 and 14 %. Despite using multiple tissue sampling modalities, detection of CCA remains a challenge. Probe-based confocal laser endomicroscopy (pCLE) has been utilized to visualize subepithelial biliary mucosa in patients with indeterminate strictures. We assessed the technical feasibility and operating characteristics of pCLE in a cohort of PSC patients with dominant biliary strictures (DS). This was a chart review of a prospectively maintained database at a single tertiary referral center of 15 PSC patients with 21 dominant stenoses undergoing pCLE. A data collection sheet included demographics, ERCP, cholangioscopy, pCLE (Miami criteria), tissue sampling results, and follow-up to 12 months or liver transplantation. Operating characteristics for pCLE and ERCP tissue sampling were calculated. Sufficient visualization of DS by pCLE was achieved in 20/21 (95 %). pCLE sensitivity, specificity, PPV, and NPV were 100 % (95 % CI 19.3-100 %), 61.1 % (95 % CI 35.8-82.6 %), 22.2 % (95 % CI 3.5-59.9 %), and 100 % (95 % CI 71.3-100 %), respectively, in detecting neoplasia. In comparison, concomitant tissue sampling yielded sensitivity, specificity, PPV, and NPV of 0 % (95 % CI 0-80.7 %), 94.4 % (95 % CI 72.6-99.1 %), 0 % (95 % CI 0-83.5 %), and 89.5 % (95 % CI 66.8-98.4 %), respectively. pCLE achieves a high technical success rate in patients with PSC and DS. This single center, small series, suggests that pCLE may have a high sensitivity and negative predictive value to exclude neoplasia. If verified in larger prospective studies, the technology may be utilized to risk stratify dominant strictures in patients with PSC.

PBOV1 Is a Human De Novo Gene with Tumor-Specific Expression That Is Associated with a Positive Clinical Outcome of Cancer

PBOV1 is a known human protein-coding gene with an uncharacterized function. We have previously found that PBOV1 lacks orthologs in non-primate genomes and is expressed in a wide range of tumor types. Here we report that PBOV1 protein-coding sequence is human-specific and has originated de novo in the primate evolution through a series of frame-shift and stop codon mutations. We profiled PBOV1 expression in multiple cancer and normal tissue samples and found that it was expressed in 19 out of 34 tumors of various origins but completely lacked expression in any of the normal adult or fetal human tissues. We found that, unlike the cancer/testis antigens that are typically controlled by CpG island-containing promoters, PBOV1 was expressed from a GC-poor TATA-containing promoter which was not influenced by CpG demethylation and was inactive in testis. Our analysis of public microarray data suggests that PBOV1 activation in tumors could be dependent on the Hedgehog signaling pathway. Despite the recent de novo origin and the lack of identifiable functional signatures, a missense SNP in the PBOV1 coding sequence has been previously associated with an increased risk of breast cancer. Using publicly available microarray datasets, we found that high levels of PBOV1 expression in breast cancer and glioma samples were significantly associated with a positive outcome of the cancer disease. We also found that PBOV1 was highly expressed in primary but not in recurrent high-grade gliomas, suggesting the presence of a negative selection against PBOV1-expressing cancer cells. Our findings could contribute to the understanding of the mechanisms behind de novo gene origin and the possible role of tumors in this process.

EZH2 and CD79B mutational status over time in B‐cell non‐Hodgkin lymphomas detected by high‐throughput sequencing using minimal samples

BACKGROUND: Numerous genomic abnormalities in B-cell non-Hodgkin lymphomas (NHLs) have been revealed by novel high-throughput technologies, including recurrent mutations in EZH2 (enhancer of zeste homolog 2) and CD79B (B cell antigen receptor complex-associated protein beta chain) genes. This study sought to determine the evolution of the mutational status of EZH2 and CD79B over time in different samples from the same patient in a cohort of B-cell NHLs, through use of a customized multiplex mutation assay.METHODS: DNA that was extracted from cytological material stored on FTA cards as well as from additional specimens, including archived frozen and formalin-fixed histological specimens, archived stained smears, and cytospin preparations, were submitted to a multiplex mutation assay specifically designed for the detection of point mutations involving EZH2 and CD79B, using MassARRAY spectrometry followed by Sanger sequencing.RESULTS: All 121 samples from 80 B-cell NHL cases were successfully analyzed. Mutations in EZH2 (Y646) and CD79B (Y196) were detected in 13.2% and 8% of the samples, respectively, almost exclusively in follicular lymphomas and diffuse large B-cell lymphomas. In one-third of the positive cases, a wild type was detected in a different sample from the same patient during follow-up.CONCLUSIONS: Testing multiple minimal tissue samples using a high-throughput multiplex platform exponentially increases tissue availability for molecular analysis and might facilitate future studies of tumor progression and the related molecular events. Mutational status of EZH2 and CD79B may vary in B-cell NHL samples over time and support the concept that individualized therapy should be based on molecular findings at the time of treatment, rather than on results obtained from previous specimens. Cancer (Cancer Cytopathol) 2013;121:377–386. © 2013 American Cancer Society.

Cellular interference in craniofrontonasal syndrome: males mosaic for mutations in the X-linked EFNB1 gene are more severely affected than true hemizygotes

Craniofrontonasal syndrome (CFNS), an X-linked disorder caused by loss-of-function mutations of EFNB1, exhibits a paradoxical sex reversal in phenotypic severity: females characteristically have frontonasal dysplasia, craniosynostosis and additional minor malformations, but males are usually more mildly affected with hypertelorism as the only feature. X-inactivation is proposed to explain the more severe outcome in heterozygous females, as this leads to functional mosaicism for cells with differing expression of EPHRIN-B1, generating abnormal tissue boundaries—a process that cannot occur in hemizygous males. Apparently challenging this model, males occasionally present with a more severe female-like CFNS phenotype. We hypothesized that such individuals might be mosaic for EFNB1 mutations and investigated this possibility in multiple tissue samples from six sporadically presenting males. Using denaturing high performance liquid chromatography, massively parallel sequencing and multiplex-ligation-dependent probe amplification (MLPA) to increase sensitivity above standard dideoxy sequencing, we identified mosaic mutations of EFNB1 in all cases, comprising three missense changes, two gene deletions and a novel point mutation within the 5′ untranslated region (UTR). Quantification by Pyrosequencing and MLPA demonstrated levels of mutant cells between 15 and 69%. The 5′ UTR variant mutates the stop codon of a small upstream open reading frame that, using a dual-luciferase reporter construct, was demonstrated to exacerbate interference with translation of the wild-type protein. These results demonstrate a more severe outcome in mosaic than in constitutionally deficient males in an X-linked dominant disorder and provide further support for the cellular interference mechanism, normally related to X-inactivation in females.

Assessment of Mercury and Selenium Concentrations in Tissues of Stranded Leatherback Sea Turtles (Dermochelys coriacea)

The impact of mercury (Hg) and selenium (Se) compounds on wildlife health is an issue of concern, especially because anthropogenic practices (e.g., fossil fuel combustion, metal processing) release these trace elements and their compounds into the environment where they become incorporated into food chains. Little is known about the concentrations of these elements in tissues (other than in blood) of most wild reptiles, especially for pelagic species. The purpose of this study was to document Hg and Se concentrations in leatherback sea turtles, Dermochelys coriacea. To assess Hg and Se concentrations in leatherbacks, multiple tissue samples (whole blood, enteric contents from the small intestine, feces, yolked follicles, liver, and salt gland) were collected from dead, stranded male and female leatherbacks that were assigned to three life stage classes (juvenile, subadult, adult) identified at gross necropsy. The tissues were tested for Hg and Se concentrations. The liver exhibited the highest concentrations of both Hg and Se compared with other tissues. Adults had the highest concentrations of Hg in their livers compared with juveniles and subadults; however, Se concentrations did not differ between subadults and adults. Leatherbacks with a larger curved carapace length had greater liver Hg and Se concentrations. The Se:Hg ratio in the liver was highest in juveniles, followed by subadults, and then adults. Because these animals are long-lived, Hg or Se accumulation may become a physiological challenge, especially because of ongoing anthropogenic input of these elements and their compounds into the environment. Further population monitoring of toxicants in marine turtles is warranted to determine whether tissue concentrations of these elements continue to increase long term or vary with age, life stage, sex, or reproductive status.

Reference Gene Selection in the Desert Plant Eremosparton songoricum

Eremosparton songoricum (Litv.) Vass. (E. songoricum) is a rare and extremely drought-tolerant desert plant that holds promise as a model organism for the identification of genes associated with water deficit stress. Here, we cloned and evaluated the expression of eight candidate reference genes using quantitative real-time reverse transcriptase polymerase chain reactions. The expression of these candidate reference genes was analyzed in a diverse set of 20 samples including various E. songoricum plant tissues exposed to multiple environmental stresses. GeNorm analysis indicated that expression stability varied between the reference genes in the different experimental conditions, but the two most stable reference genes were sufficient for normalization in most conditions. EsEF and Esα-TUB were sufficient for various stress conditions, EsEF and EsACT were suitable for samples of differing germination stages, and EsGAPDHand EsUBQ were most stable across multiple adult tissue samples. The Es18S gene was unsuitable as a reference gene in our analysis. In addition, the expression level of the drought-stress related transcription factor EsDREB2 verified the utility of E. songoricum reference genes and indicated that no single gene was adequate for normalization on its own. This is the first systematic report on the selection of reference genes in E. songoricum, and these data will facilitate future work on gene expression in this species.