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Journal of clinical microbiology, 56
2018

Automated Interpretation of Blood Culture Gram Stains by Use of a Deep Convolutional Neural Network.

Smith, Kenneth P, Kang, Anthony D, Kirby, James E

Microscopic interpretation of stained smears is one of the most operator-dependent and time-intensive activities in the clinical microbiology laboratory. Here, we investigated application of an automated image acquisition and convolutional neural network (CNN)-based approach for automated Gram stain classification. Using an automated microscopy platform, uncoverslipped slides were scanned with a 40× dry objective, generating images of sufficient resolution for interpretation. We collected 25,488 images from positive blood culture Gram stains prepared during routine clinical workup. These images were used to generate 100,213 crops containing Gram-positive cocci in clusters, Gram-positive cocci in chains/pairs, Gram-negative rods, or background (no cells). These categories were targeted for proof-of-concept development as they are associated with the majority of bloodstream infections. Our CNN model achieved a classification accuracy of 94.9% on a test set of image crops. Receiver operating characteristic (ROC) curve analysis indicated a robust ability to differentiate between categories with an area under the curve of >0.98 for each. After training and validation, we applied the classification algorithm to new images collected from 189 whole slides without human intervention. Sensitivity and specificity were 98.4% and 75.0% for Gram-positive cocci in chains and pairs, 93.2% and 97.2% for Gram-positive cocci in clusters, and 96.3% and 98.1% for Gram-negative rods. Taken together, our data support a proof of concept for a fully automated classification methodology for blood-culture Gram stains. Importantly, the algorithm was highly adept at identifying image crops with organisms and could be used to present prescreened, classified crops to technologists to accelerate smear review. This concept could potentially be extended to all Gram stain interpretive activities in the clinical laboratory.

Digital object identifier (DOI): 10.1128/JCM.01521-17

Journal of Pathology Informatics, 9(1), 13
2018

Constant quest for quality: Digital cytopathology

Van Es, Simone L., Greaves, Janelle, Gay, Stephanie, Ross, Jennifer, Holzhauser, Derek, Badrick, Tony

Background: Special consideration should be given when creating and selecting cytopathology specimens for digitization to maximize quality. Advances in scanning and viewing technology can also improve whole-slide imaging (WSI) output quality. Methods: Accumulated laboratory experience with digitization of glass cytopathology slides was collected. Results: This paper describes characteristics of a cytopathology glass slide that can reduce quality on resulting WSI. Important points in the glass cytopathology slide selection process, preparation, scanning, and WSI-editing process that will maximize the quality of the resulting acquired digital image are covered. The paper outlines scanning solutions which have potential to predict issues with a glass cytopathology slide before image acquisition, allowing for adjustment of the scanning approach. WSI viewing solutions that better simulate the traditional microscope experience are also discussed. Conclusion: In addition to taking advantage of technical advances, practical steps can taken to maximize quality of cytopathology WSI.

Digital object identifier (DOI): 10.4103/jpi.jpi_6_18

TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik, 131, 389--406
2018

Characterisation of Thinopyrum bessarabicum chromosomes through genome-wide introgressions into wheat.

Grewal, Surbhi, Yang, Caiyun, Edwards, Stella Hubbart, Scholefield, Duncan, Ashling, Stephen, Burridge, Amanda J, King, Ian P, King, Julie

Genome-wide introgressions of Thinopyrum bessarabicum into wheat resulted in 12 recombinant lines. Cytological and molecular techniques allowed mapping of 1150 SNP markers across all seven chromosomes of the J genome. Thinopyrum bessarabicum (2n = 2x = 14, JJ) is an important source for new genetic variation for wheat improvement due to its salinity tolerance and disease resistance. Its practical utilisation in wheat improvement can be facilitated through development of genome-wide introgressions leading to a variety of different wheat-Th . bessarabicum translocation lines. In this study, we report the generation of 12 such wheat-Th . bessarabicum recombinant lines, through two different crossing strategies, which were characterized using sequential single colour and multi-colour genomic in situ hybridization (sc-GISH and mc-GISH), multi-colour fluorescent in situ hybridization (mc-FISH) and single nucleotide polymorphic (SNP) DNA markers. We also detected 13 lines containing different Th. bessarabicum chromosome aberrations through sc-GISH. Through a combination of molecular and cytological analysis of all the 25 lines containing Th. bessarabicum recombinants and chromosome aberrations we were able to physically map 1150 SNP markers onto seven Th. bessarabicum J chromosomes which were divided into 36 segmental blocks. Comparative analysis of the physical map of Th. bessarabicum and the wheat genome showed that synteny between the two species is highly conserved at the macro-level and confirmed that Th. bessarabicum contains the 4/5 translocation also present in the A genome of wheat. These wheat-Th . bessarabicum recombinant lines and SNP markers provide a useful genetic resource for wheat improvement with the latter having a wider impact as a tool for detection of introgressions from other Thinopyrum species containing the J or a closely-related genome such as Thinopyrum intermedium (J J J J StSt) and Thinopyrum elongatum (E E ), respectively.

Digital object identifier (DOI): 10.1007/s00122-017-3009-y

Scientific reports, 8, 3122
2018

Phaeophleospora vochysiae Savi & Glienke sp. nov. Isolated from Vochysia divergens Found in the Pantanal, Brazil, Produces Bioactive Secondary Metabolites.

Savi, Daiani C, Shaaban, Khaled A, Gos, Francielly Maria Wilke Ramos, Ponomareva, Larissa V, Thorson, Jon S, Glienke, Chirlei, Rohr, Jürgen

Microorganisms associated with plants are highly diverse and can produce a large number of secondary metabolites, with antimicrobial, anti-parasitic and cytotoxic activities. We are particularly interested in exploring endophytes from medicinal plants found in the Pantanal, a unique and widely unexplored wetland in Brazil. In a bio-prospecting study, strains LGMF1213 and LGMF1215 were isolated as endophytes from Vochysia divergens, and by morphological and molecular phylogenetic analyses were characterized as Phaeophleospora vochysiae sp. nov. The chemical assessment of this species reveals three major compounds with high biological activity, cercoscosporin (1), isocercosporin (2) and the new compound 3-(sec-butyl)-6-ethyl-4,5-dihydroxy-2-methoxy-6-methylcyclohex-2-enone (3). Besides the isolation of P. vochysiae as endophyte, the production of cercosporin compounds suggest that under specific conditions this species causes leaf spots, and may turn into a pathogen, since leaf spots are commonly caused by species of Cercospora that produce related compounds. In addition, the new compound 3-(sec-butyl)-6-ethyl-4,5-dihydroxy-2-methoxy-6-methylcyclohex-2-enone showed considerable antimicrobial activity and low cytotoxicity, which needs further exploration.

Digital object identifier (DOI): 10.1038/s41598-018-21400-2

British journal of pharmacology
2018

CO-EXPRESSION OF μ AND ∂ OPIOID RECEPTORS BY MOUSE COLONIC NOCICEPTORS.

Guerrero-Alba, Raquel, Valdez-Morales, Eduardo Emmanuel, Jiménez-Vargas, Nestor Nivardo, Bron, Romke, Poole, Daniel, Reed, David, Castro, Joel, Campaniello, Melissa, Hughes, Patrick A, Brierley, Stuart M, Bunnett, Nigel, Lomax, Alan E, Vanner, Stephen

To better understand opioid signaling in visceral nociceptors, we examined the expression and selective activation of mu (MOR) and delta opioid receptors (DOR) by dorsal root ganglia (DRG) neurons innervating the mouse colon. DRG neurons projecting to the colon were identified by retrograde tracing. DOR-GFP reporter mice, in situ hybridization, single-cell RT-PCR, and MOR-specific antibodies were used to characterize expression of MOR and DOR. Voltage-gated Ca currents and neuronal excitability were recorded in small diameter nociceptive neurons (capacitance < 30pF) by patch clamp and ex vivo single-unit afferent recordings were obtained from the colon. In situ hybridization of oprm1 expression in Fast Blue-labeled DRG neurons was observed in 61% of neurons. MOR and DOR were expressed by 36-46% of colon DRG neurons, and co-expressed by ~25 % of neurons. MOR and DOR agonists inhibited Ca currents in DRG and these effects were blocked by opioid antagonists. One or both agonists inhibited action potential firing by colonic afferent endings. Incubation of neurons with supernatants from inflamed colon segments inhibited Ca currents and neuronal excitability. The MOR but not the DOR antagonist, inhibited the supernatant effects on Ca currents, whereas both antagonists inhibited their actions on neuronal excitability. A significant number of small diameter colonic nociceptors co-express MOR and DOR and are inhibited by agonists and endogenous opioids in inflamed tissues. Thus, opioids that act at MOR or DOR or their heterodimers may be effective in the treatment of visceral pain.

Digital object identifier (DOI): 10.1111/bph.14222

Scientific Reports, 8(1), 1141
2018

First experimental proof of Proton Boron Capture Therapy (PBCT) to enhance protontherapy effectiveness

Cirrone, GAP, Manti, L, Margarone, D, Petringa, G, Giuffrida, L, Minopoli, A, Picciotto, A, Russo, G, Cammarata, F, Pisciotta, P, others

Protontherapy is hadrontherapy’s fastest-growing modality and a pillar in the battle against cancer. Hadrontherapy’s superiority lies in its inverted depth-dose profile, hence tumour-confined irradiation. Protons, however, lack distinct radiobiological advantages over photons or electrons. Higher LET (Linear Energy Transfer) 12C-ions can overcome cancer radioresistance: DNA lesion complexity increases with LET, resulting in efficient cell killing, i.e. higher Relative Biological Effectiveness (RBE). However, economic and radiobiological issues hamper 12C-ion clinical amenability. Thus, enhancing proton RBE is desirable. To this end, we exploited the p + 11B → 3α reaction to generate high-LET alpha particles with a clinical proton beam. To maximize the reaction rate, we used sodium borocaptate (BSH) with natural boron content. Boron-Neutron Capture Therapy (BNCT) uses 10B-enriched BSH for neutron irradiation-triggered alpha particles. We recorded significantly increased cellular lethality and chromosome aberration complexity. A strategy combining protontherapy’s ballistic precision with the higher RBE promised by BNCT and 12C-ion therapy is thus demonstrated.

Diagnostics (Basel, Switzerland), 8
2018

Aneuploid CTC and CEC.

Lin, Peter Ping

Conventional circulating tumor cell (CTC) detection technologies are restricted to large tumor cells (> white blood cells (WBCs)), or those unique carcinoma cells with double positive expression of surface epithelial cell adhesion molecule (EpCAM) for isolation, and intracellular structural protein cytokeratins (CKs) for identification. With respect to detecting the full spectrum of highly heterogeneous circulating rare cells (CRCs), including CTCs and circulating endothelial cells (CECs), it is imperative to develop a strategy systematically coordinating all tri-elements of nucleic acids, biomarker proteins, and cellular morphology, to effectively enrich and comprehensively identify CRCs. Accordingly, a novel strategy integrating subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH), independent of cell size variation and free of hypotonic damage as well as anti-EpCAM perturbing, has been demonstrated to enable in situ phenotyping multi-protein expression, karyotyping chromosome aneuploidy, and detecting cytogenetic rearrangements of the gene in non-hematologic CRCs. Symbolic non-synonymous single nucleotide variants (SNVs) of both the gene (P33R) in each single aneuploid CTCs, and the cyclin-dependent kinase inhibitor 2A ( ) tumor suppressor gene in each examined aneuploid CECs, were identified for the first time across patients with diverse carcinomas. Comprehensive co-detecting observable aneuploid CTCs and CECs by SE-iFISH, along with applicable genomic and/or proteomic single cell molecular profiling, are anticipated to facilitate elucidating how those disparate categories of aneuploid CTCs and CECs cross-talk and functionally interplay with tumor angiogenesis, therapeutic drug resistance, tumor progression, and cancer metastasis.

Digital object identifier (DOI): 10.3390/diagnostics8020026

Molecular cytogenetics, 11, 4
2018

Is cancer progression caused by gradual or simultaneous acquisitions of new chromosomes?

Bloomfield, Mathew, Duesberg, Peter

Foulds defined, "Tumor progression (as a) permanent, irreversible qualitative change in one or more of its characters" (Cancer Res. 1954). Accordingly progressions, such as metastases and acquired drug-resistance, were since found to be subspecies of cancers with conserved and numerous new chromosomes. Here we ask whether cancers acquire numerous new chromosomes gradually or simultaneously in progressions. The currently prevailing theory of Nowell (Science, 1976) holds that unexplained "genetic instability" generates "variant sublines (with) changes in chromosome number" and that "clonal" progressions arise by "stepwise selection of more aggressive sublines". The literature, however, contains many examples of "immediate" selections of progressions with numerous new chromosomes - notably experimentally initiated fusions between cancers and heterologous cells. Furthermore, the stepwise progression theory predicts intermediate sublines of cancers with multiple non-clonal additions of new chromosomes. However, the literature does not describe such intermediates. In view of these inconsistencies with stepwise progression we test here a saltational theory, in which the inherent variability of cancer-specific aneuploidy generates "immediate" progressions with individual clonal karyotypes, transcriptomes and phenotypes in single steps. Using cell fusion as an established controllable model of "immediate" progression, we generated seven immortal murine hybridomas by fusing immortal murine myeloma cells and normal antibody-producing B-cells with polyethylene glycol within a few minutes. These immortal hybridomas contained individual sets of 71 to 105 clonal chromosomes, compared to the 52 chromosomes of the parental myeloma. Thus the myeloma had gained 19 to 53 new clonal chromosomes in seven individual hybridomas in a single step. Furthermore, no stable intermediates were found, as would be predicted by a saltational process. We conclude that random fusions between myelomas and normal B-cells generate clonal hybridomas with multiple, individual chromosomes in single steps. Similar single-step mechanisms may also generate the "late" clonal progressions of cancers with gains of numerous new chromosomes and thus explain the absence of intermediates. Latency would reflect the low probability of rare stochastic progressions. In conclusion, the karyotypic clonality of hybridomas and spontaneous progressions suggests karyotypic alterations as proximate causes of neoplastic progressions. Since cancer-specific aneuploidy catalyzes karyotypic variation, the degree of aneuploidy predicts the clinical risk of neoplastic progression onfirming classical predictions based on DNA content

Digital object identifier (DOI): 10.1186/s13039-017-0350-4

Cancer letters, 412, 99--107
2018

Quantified postsurgical small cell size CTCs and EpCAM, [email protected], circulating tumor stem cells with cytogenetic abnormalities in hepatocellular carcinoma patients determine cancer relapse.

Wang, Liang, Li, Yilin, Xu, Jing, Zhang, Aiqun, Wang, Xuedong, Tang, Rui, Zhang, Xinjing, Yin, Hongfang, Liu, Manting, Wang, Daisy Dandan, Lin, Peter Ping, Shen, Lin, Dong, Jiahong

Detection of hepatocellular carcinoma circulating tumor cells performed with conventional strategies, is significantly limited due to inherently heterogeneous and dynamic expression of EpCAM, as well as degradation of cytokeratins during epithelial-to-mesenchymal transition, which inevitably lead to non-negligible false negative detection of such "uncapturable and invisible" CTCs. A novel SE-iFISH strategy, improved for detection of HCC CTCs in this study, was applied to comprehensively detect, in situ phenotypically and karyotypically characterize hepatocellular and cholangiocarcinoma CTCs (CD45 /CD31 ) in patients subjected to surgical resection. Clinical significance of diverse subtypes of CTC was systematically investigated. Existence of small cell size CTCs (≤5 μm of WBCs) with cytogenetic abnormality of aneuploid chromosome 8, which constituted majority of the detected CTCs in HCC patients, was demonstrated for the first time. The stemness marker EpCAM aneuploid circulating tumor stem cells (CTSCs), and EpCAM small CTCs with trisomy 8, promote tumor growth. Postsurgical quantity of small triploid CTCs (≥5 cells/6 ml blood), multiploid (≥pentasomy 8) CTSCs or CTM (either one ≥ 1) significantly correlated to HCC patients' poor prognosis, indicating that detection of those specific subtypes of CTCs and CTSCs in post-operative patients help predict neoplasm recurrence.

Digital object identifier (DOI): 10.1016/j.canlet.2017.10.004

Journal of Alzheimer's disease : JAD, 61, 899--905
2018

A Novel Antibody Targeting Tau Phosphorylated at Serine 235 Detects Neurofibrillary Tangles.

Brici, David, Götz, Jürgen, Nisbet, Rebecca M

Alzheimer's disease is characterized by two main pathological hallmarks in the human brain: the extracellular deposition of amyloid-β as plaques and the intracellular accumulation of the hyperphosphorylated protein tau as neurofibrillary tangles (NFTs). Phosphorylated tau (p-tau) specific-antibodies and silver staining have been used to reveal three morphological stages of NFT formation: pre-NFTs, intraneuronal NFTs (iNFTs), and extraneuronal NFTs (eNFTs). Here we characterize a novel monoclonal antibody, RN235, which is specific for tau phosphorylated at serine 235, and detects iNFTs and eNFTs in brain tissue, suggesting that phosphorylation at this site is indicative of late stage changes in tau.

Digital object identifier (DOI): 10.3233/JAD-170610

International journal of radiation biology, 93, 48--57
2017

Dose assessment intercomparisons within the RENEB network using G0-lymphocyte prematurely condensed chromosomes (PCC assay).

Terzoudi, Georgia I, Pantelias, Gabriel, Darroudi, Firouz, Barszczewska, Katarzyna, Buraczewska, Iwona, Depuydt, Julie, Georgieva, Dimka, Hadjidekova, Valeria, Hatzi, Vasiliki I, Karachristou, Ioanna, Karakosta, Maria, Meschini, Roberta, M'Kacher, Radhia, Montoro, Alegria, Palitti, Fabrizio, Pantelias, Antonio, Pepe, Gaetano, Ricoul, Michelle, Sabatier, Laure, Sebastià, Natividad, Sommer, Sylwester, Vral, Anne, Zafiropoulos, Demetre, Wojcik, Andrzej

Dose assessment intercomparisons within the RENEB network were performed for triage biodosimetry analyzing G0-lymphocyte PCC for harmonization, standardization and optimization of the PCC assay. Comparative analysis among different partners for dose assessment included shipment of PCC-slides and captured images to construct dose-response curves for up to 6 Gy γ-rays. Accident simulation exercises were performed to assess the suitability of the PCC assay by detecting speed of analysis and minimum number of cells required for categorization of potentially exposed individuals. Calibration data based on Giemsa-stained fragments in excess of 46 PCC were obtained by different partners using galleries of PCC images for each dose-point. Mean values derived from all scores yielded a linear dose-response with approximately 4 excess-fragments/cell/Gy. To unify scoring criteria, exercises were carried out using coded PCC-slides and/or coded irradiated blood samples. Analysis of samples received 24 h post-exposure was successfully performed using Giemsa staining (1 excess-fragment/cell/Gy) or centromere/telomere FISH-staining for dicentrics. Dose assessments by RENEB partners using appropriate calibration curves were mostly in good agreement. The PCC assay is quick and reliable for whole- or partial-body triage biodosimetry by scoring excess-fragments or dicentrics in G0-lymphocytes. Particularly, analysis of Giemsa-stained excess PCC-fragments is simple, inexpensive and its automation could increase throughput and scoring objectivity of the PCC assay.

Digital object identifier (DOI): 10.1080/09553002.2016.1234725

Oncotarget, 8, 26269--26280
2017

Opposite effects of GCN5 and PCAF knockdowns on the alternative mechanism of telomere maintenance.

Jeitany, Maya, Bakhos-Douaihy, Dalal, Silvestre, David C, Pineda, Jose R, Ugolin, Nicolas, Moussa, Angela, Gauthier, Laurent R, Busso, Didier, Junier, Marie-Pierre, Chneiweiss, Hervé, Chevillard, Sylvie, Desmaze, Chantal, Boussin, François D

Cancer cells can use a telomerase-independent mechanism, known as alternative lengthening of telomeres (ALT), to elongate their telomeres. General control non-derepressible 5 (GCN5) and P300/CBP-associated factor (PCAF) are two homologous acetyltransferases that are mutually exclusive subunits in SAGA-like complexes. Here, we reveal that down regulation of GCN5 and PCAF had differential effects on some phenotypic characteristics of ALT cells. Our results suggest that GCN5 is present at telomeres and opposes telomere recombination, in contrast to PCAF that may indirectly favour them in ALT cells.

Digital object identifier (DOI): 10.18632/oncotarget.15447

International journal of radiation biology, 93, 58--64
2017

The second gamma-H2AX assay inter-comparison exercise carried out in the framework of the European biodosimetry network (RENEB).

Moquet, Jayne, Barnard, Stephen, Staynova, Albena, Lindholm, Carita, Monteiro Gil, Octávia, Martins, Vanda, Rößler, Ute, Vral, Anne, Vandevoorde, Charlot, Wojewódzka, Maria, Rothkamm, Kai

Within the EU RENEB project, seven laboratories have taken part in training and harmonisation activities to strengthen triage gamma-H2AX-based radiation exposure assessment. This has culminated in a second triage biodosimetry exercise. Whole blood and separated lymphocyte samples were homogenously irradiated with (60)Co gamma rays at 0.5, 2.5 (blind samples), 0 and 2 Gy (reference samples). Following post-exposure incubations of 4 and 24 h, 16 samples were shipped on ice packs to each partner. The samples were stained and scored for gamma-H2AX foci, using manual and/or automated fluorescence microscope scoring strategies. Dose estimates were obtained and used to assign triage categories to the samples. Average dose estimates across all the laboratories correlated well with true doses. The most accurate assignment of triage category was achieved by manual scoring of the 4-h blood and lymphocyte samples. Only three samples out of a total of 46 were miscategorized in a way that could have adversely effected the clinical management of a radiation casualty. This inter-comparison exercise has demonstrated that following a recent acute radiation exposure, the gamma-H2AX assay could be a useful triage tool that can be successfully applied across a network of laboratories.

Digital object identifier (DOI): 10.1080/09553002.2016.1207822

PloS one, 12, e0178877
2017

Depletion of ATP and glucose in advanced human atherosclerotic plaques.

Ekstrand, Matias, Widell, Emma, Hammar, Anna, Akyürek, Levent M, Johansson, Martin, Fagerberg, Björn, Bergström, Göran, Levin, Malin C, Fogelstrand, Per, Borén, Jan, Levin, Max

Severe hypoxia develops close to the necrotic core of advanced human atherosclerotic plaques, but the energy metabolic consequences of this hypoxia are not known. In animal models, plaque hypoxia is also associated with depletion of glucose and ATP. ATP depletion may impair healing of plaques and promote necrotic core expansion. To investigate if ATP depletion is present in human plaques, we analyzed the distribution of energy metabolites (ATP, glucose, glycogen and lactate) in intermediate and advanced human plaques. Snap frozen carotid endarterectomies from 6 symptomatic patients were analyzed. Each endarterectomy included a large plaque ranging from the common carotid artery (CCA) to the internal carotid artery (ICA). ATP, glucose, and glycogen concentrations were lower in advanced (ICA) compared to intermediate plaques (CCA), whereas lactate concentrations were higher. The lowest concentrations of ATP, glucose and glycogen were detected in the perinecrotic zone of advanced plaques. Our study demonstrates severe ATP depletion and glucose deficiency in the perinecrotic zone of human advanced atherosclerotic plaques. ATP depletion may impair healing of plaques and promote disease progression.

Digital object identifier (DOI): 10.1371/journal.pone.0178877

Scientific reports, 7, 3291
2017

Transmission of Induced Chromosomal Aberrations through Successive Mitotic Divisions in Human Lymphocytes after In Vitro and In Vivo Radiation.

Kaddour, Akram, Colicchio, Bruno, Buron, Diane, El Maalouf, Elie, Laplagne, Eric, Borie, Claire, Ricoul, Michelle, Lenain, Aude, Hempel, William M, Morat, Luc, Al Jawhari, Mustafa, Cuceu, Corina, Heidingsfelder, Leonhard, Jeandidier, Eric, Deschênes, Georges, Dieterlen, Alain, El May, Michèle, Girinsky, Theodore, Bennaceur-Griscelli, Annelise, Carde, Patrice, Sabatier, Laure, M'kacher, Radhia

The mechanisms behind the transmission of chromosomal aberrations (CA) remain unclear, despite a large body of work and major technological advances in chromosome identification. We reevaluated the transmission of CA to second- and third-division cells by telomere and centromere (TC) staining followed by M-FISH. We scored CA in lymphocytes of healthy donors after in vitro irradiation and those of cancer patients treated by radiation therapy more than 12 years before. Our data demonstrate, for the first time, that dicentric chromosomes (DCs) decreased by approximately 50% per division. DCs with two centromeres in close proximity were more efficiently transmitted, representing 70% of persistent DCs in ≥M3 cells. Only 1/3 of acentric chromosomes (ACs), ACs with four telomeres, and interstitial ACs, were paired in M2 cells and associated with specific DCs configurations. In lymphocytes of cancer patients, 82% of detected DCs were characterized by these specific configurations. Our findings demonstrate the high stability of DCs with two centromeres in close proximity during cell division. The frequency of telomere deletion increased during cell cycle progression playing an important role in chromosomal instability. These findings could be exploited in the follow-up of exposed populations.

Digital object identifier (DOI): 10.1038/s41598-017-03198-7

eLife, 6
2017

Epigenetic regulation of lateralized fetal spinal gene expression underlies hemispheric asymmetries.

Ocklenburg, Sebastian, Schmitz, Judith, Moinfar, Zahra, Moser, Dirk, Klose, Rena, Lor, Stephanie, Kunz, Georg, Tegenthoff, Martin, Faustmann, Pedro, Francks, Clyde, Epplen, Jörg T, Kumsta, Robert, Güntürkün, Onur

Lateralization is a fundamental principle of nervous system organization but its molecular determinants are mostly unknown. In humans, asymmetric gene expression in the fetal cortex has been suggested as the molecular basis of handedness. However, human fetuses already show considerable asymmetries in arm movements before the motor cortex is functionally linked to the spinal cord, making it more likely that spinal gene expression asymmetries form the molecular basis of handedness. We analyzed genome-wide mRNA expression and DNA methylation in cervical and anterior thoracal spinal cord segments of five human fetuses and show development-dependent gene expression asymmetries. These gene expression asymmetries were epigenetically regulated by miRNA expression asymmetries in the TGF-β signaling pathway and lateralized methylation of CpG islands. Our findings suggest that molecular mechanisms for epigenetic regulation within the spinal cord constitute the starting point for handedness, implying a fundamental shift in our understanding of the ontogenesis of hemispheric asymmetries in humans.

Digital object identifier (DOI): 10.7554/eLife.22784

Environmental and molecular mutagenesis
2017

Sesamol ameliorates radiation induced DNA damage in hematopoietic system of whole body γ-irradiated mice.

Kumar, Arun, Choudhary, Sandeep, Adhikari, Jawahar S, Chaudhury, Nabo K

Ionizing radiation exposure is harmful and at high doses can lead to acute hematopoietic radiation syndrome. Therefore, agents that can protect hematopoietic system are important for development of radioprotector. Sesamol is a potential molecule for development of radioprotector due to its strong free radical scavenging and antioxidant properties. In the present study, sesamol was evaluated for its role in DNA damage and repair in hematopoietic system of γ-irradiated CB57BL/6 mice and compared with amifostine. C57BL/6 male mice were administered with sesamol 20 mg/kg (i.p.) followed by 2 Gy whole body irradiation (WBI) at 30 min. Mice were sacrificed at 0.5, 3, 24 h postirradiation; bone marrow, splenocytes, and peripheral blood lymphocytes were isolated to measure DNA damages and repair using alkaline comet,γ-H2AXand micronucleus assays. An increase in % of tail DNA was observed in all organs of WBI mice. Whereas in pre-administered sesamol reduced %DNA in tail (P ≤ 0.05). Sesamol has also reduced formation of radiation induced γ-H2AX foci after 0.5 h in these organs and further lowered to respective control values at 24 h of WBI. Similar reduction of % DNA in tail and γ-H2AX foci were observed with amifostine (P ≤ 0.05). Analysis of mnPCE frequency at 24 h has revealed similar extent of protection by sesamol and amifostine. Interestingly, both sesamol and amifostine, alone and with radiation, also increased the granulocytes count significantly compared to the control (P ≤ 0.05). These findings suggest that sesamol has strong potential to protect hematopoietic system by lowering radiation induced DNA damages and can prevent acute hematopoietic syndrome in mice. Environ. Mol. Mutagen., 2017. © 2017 Wiley Periodicals, Inc.

Digital object identifier (DOI): 10.1002/em.22118

Molecular cell
2017

DNA Double-Strand Break Resection Occurs during Non-homologous End Joining in G1 but Is Distinct from Resection during Homologous Recombination.

Biehs, Ronja, Steinlage, Monika, Barton, Olivia, Juhász, Szilvia, Künzel, Julia, Spies, Julian, Shibata, Atsushi, Jeggo, Penny A, Löbrich, Markus

Canonical non-homologous end joining (c-NHEJ) repairs DNA double-strand breaks (DSBs) in G1 cells with biphasic kinetics. We show that DSBs repaired with slow kinetics, including those localizing to heterochromatic regions or harboring additional lesions at the DSB site, undergo resection prior to repair by c-NHEJ and not alt-NHEJ. Resection-dependent c-NHEJ represents an inducible process during which Plk3 phosphorylates CtIP, mediating its interaction with Brca1 and promoting the initiation of resection. Mre11 exonuclease, EXD2, and Exo1 execute resection, and Artemis endonuclease functions to complete the process. If resection does not commence, then repair can ensue by c-NHEJ, but when executed, Artemis is essential to complete resection-dependent c-NHEJ. Additionally, Mre11 endonuclease activity is dispensable for resection in G1. Thus, resection in G1 differs from the process in G2 that leads to homologous recombination. Resection-dependent c-NHEJ significantly contributes to the formation of deletions and translocations in G1, which represent important initiating events in carcinogenesis.

Digital object identifier (DOI): 10.1016/j.molcel.2016.12.016

Animal science journal = Nihon chikusan Gakkaiho, 88, 27--32
2017

Comparative genomic hybridization in detection of DNA changes in canine lymphomas.

Drážovská, Monika, Šiviková, Katarína, Dianovský, Ján, Horňák, Miroslav

In this study, chromosomal imbalances in tumor tissues (lymphomas) and nucleotide changes in tumor suppressor TP53 were studied in a Bernese Mountain dog bitch and a cross breed bitch. Using comparative genomic hybridization, numerous chromosomal rearrangements were detected, which indicated the heterogeneity in tumor growth: in the cross breed bitch, a deletion on the chromosome 9, and duplications on chromosomes 5, 8 and 17 have been found. In the Bernese Mountain Dog bitch, losses on chromosomes 1, 5, 8, 12, 18, 22, 27, 29 and gains on chromosomes 1, 2, 9, 11, 15, 16, 18, 20, 23, 24, 25, 28, 29, 30, 34, 36, 37 and 38 were identified. With the sequencing of the TP53 gene, one silent mutation, transition A/G at position 138 in exon 5 was detected, without changing the amino acid.

Digital object identifier (DOI): 10.1111/asj.12582

Nature Scientific Reports, 7(9789), 2-10
2017

Comprehensive in situ co-detection of aneuploid circulating endothelial and tumor cells

Peter Ping Lin, Olivier Gires, Daisy Dandan Wang, Linda, Li, Hongxia Wang

Conventional circulating tumor cell (CTC) detection strategies rely on cell surface marker EpCAM and intracellular cytokeratins (CKs) for isolation and identification, respectively. Application of such methods is considerably limited by inherent heterogeneous and dynamic expression or absence of EpCAM and/or CKs in CTCs. Here, we report a novel strategy, integrating antigen-independent subtraction enrichment and immunostaining-FISH (SE-iFISH), to detect a variety of aneuploid circulating rare cells (CRCs), including CTCs and circulating tumor endothelial cells (CECs). Enriched CRCs, maintained at high viability and suitable for primary tumor cell culture, are comprehensively characterized by in situ co-examination of chromosome aneuploidy by FISH and immunostaining of multiple biomarkers displayed in diverse fluorescence channels. We described and quantified for the first time the existence of individual aneuploid CD31+ CECs and co-existence of “fusion clusters” of endothelial-epithelial aneuploid tumor cells among enriched non-hematopoietic CRCs. Hence, SE-iFISH is feasible for efficient co-detection and in situ phenotypic and karyotypic characterization as well as quantification of various CRCs, allowing for their classification into diverse subtypes upon biomarker expression and chromosome ploidy. Enhanced SE-iFISH technology, assisted by the Metafer-iFISH automated CRC imaging system, provides a platform for the analysis of potential contributions of each subtype of CRCs to distinct clinical outcome.