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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

Nanoscale, 10, 4320--4331
2018

Super-resolution localization microscopy of radiation-induced histone H2AX-phosphorylation in relation to H3K9-trimethylation in HeLa cells.

Hausmann, Michael, Wagner, Emma, Lee, Jin-Ho, Schrock, Gerrit, Schaufler, Wladimir, Krufczik, Matthias, Papenfuß, Franziska, Port, Matthias, Bestvater, Felix, Scherthan, Harry

Ionizing radiation (IR)-induced damage confers functional and conformational changes to nuclear chromatin associated with DNA single and double strand breaks. This leads to the activation of complex DNA repair machineries that aim to preserve the integrity of the DNA molecule. Since hetero- and euchromatin are differentially accessible to DNA repair pathways, local chromatin re-arrangements and structural changes are among the consequences of an activated DNA damage response. Using super-resolution localization microscopy (SRLM), we investigated the X-ray-induced repositioning of γ-H2AX and histone H3K9me3 heterochromatin marks in the nuclei of HeLa cells. Aliquots of cells exposed to different IR doses (0.5, 1 and 2 Gy) were fixed at certain repair times for SRLM imaging. The number and size of nano-scale γ-H2AX molecule signal clusters detected increased with rising irradiation doses, with the number and size being the highest 0.5 h after irradiation. With growing repair time both the number and size of γ-H2AX nano-clusters decreased. Eight hours after irradiation, the number of clusters reached control levels, in agreement with the disappearance of most IR-induced foci seen by conventional microscopy. SRLM investigation of heterochromatin marks in spatial relation to γ-H2AX clusters showed that on average the heterochromatin density was high in the vicinity of γ-H2AX, which is in agreement with the observation that DSBs seem to relocate to the surface of heterochromatin clusters for DNA repair. The data demonstrate the potential of pointillist images obtained by SRLM for quantitative investigations of chromatin conformation changes and repair-protein recruitment on the nanoscale as measures for a radiation response.

Digital object identifier (DOI): 10.1039/c7nr08145f

Cancer genomics & proteomics, 15, 91--114
2018

Characterization of Camptothecin-induced Genomic Changes in the Camptothecin-resistant T-ALL-derived Cell Line CPT-K5.

Kjeldsen, Eigil, Nielsen, Christine J F, Roy, Amit, Tesauro, Cinzia, Jakobsen, Ann-Katrine, Stougaard, Magnus, Knudsen, Birgitta R

Acquisition of resistance to topoisomerase I (TOP1)-targeting camptothecin (CPT) derivatives is a major clinical problem. Little is known about the underlying chromosomal and genomic mechanisms. We characterized the CPT-K5 cell line expressing mutant CPT-resistant TOP1 and its parental T-cell derived acute lymphoblastic leukemia CPT-sensitive RPMI-8402 cell line by karyotyping and molecular genetic methods, including subtractive oligo-based array comparative genomic hybridization (soaCGH) analysis. Karyotyping revealed that CPT-K5 cells had acquired additional structural aberrations and a reduced modal chromosomal number compared to RPMI-8402. soaCGH analysis identified vast copy number alterations and >200 unbalanced DNA breakpoints distributed unevenly across the chromosomal complement in CPT-K5. In addition, the short tandem repeat alleles were found to be highly different between CPT-K5 and its parental cell line. We identified copy number alterations affecting genes important for maintaining genome integrity and reducing CPT-induced DNA damage. We show for the first time that short tandem repeats are targets for TOP1 cleavage, that can be differentially stimulated by CPT.

Digital object identifier (DOI): 10.21873/cgp.20068

International journal of radiation biology, 94, 664--670
2018

Evaluation of chromosomal aberrations induced by , [email protected], Re-dendrimer nanosystem on B16f1 melanoma cells.

Tassano, Marcos, Oddone, Natalia, Fernández, Marcelo, Porcal, Williams, García, María Fernanda, Martínez-López, Wilner, Benech, Juan Claudio, Cabral, Pablo

To study the rhenium-188 labeling of polyamidoamine (PAMAM) generation 4 (G4) dendrimer and its evaluation on biodistribution and chromosomal aberrations in melanoma cells induced by ionizing radiation as potential treatment agent. Dendrimers were first conjugated with Suc-HYNIC (succinimidyl 6-hydrazinopyridine-3-carboxylic acid hydrochloride). Dendrimer-HYNIC was then incubated with ReO . Biodistribution was performed administrating Re-dendrimer to normal (NM) or melanoma-bearing mice (MBM). Chromosome aberration test was conducted in order to measure treatment capacity of Re-dendrimer in melanoma cells. Radiolabeling yield of dendrimer was approx. 70%. Biodistribution studies in NM showed blood clearance with hepatic and renal depuration. MBM showed a similar pattern of biodistribution with tumor uptake of 6% of injected dose. Aberrant metaphases quantified in control cells were 7%, increasing to 29.5% in cells treated with 15μCi (0.555 MBq) of Re-dendrimer for 24 h. Re-dendrimer can produce double-stranded breaks in DNA induced by ionizing radiation in melanoma cells in vitro.

Digital object identifier (DOI): 10.1080/09553002.2018.1478161

Sexual development : genetics, molecular biology, evolution, endocrinology, embryology, and pathology of sex determination and differentiation
2018

Triploid Colubrid Snake Provides Insight into the Mechanism of Sex Determination in Advanced Snakes.

Rovatsos, Michail, Augstenová, Barbora, Altmanová, Marie, Sloboda, Michal, Kodym, Petr, Kratochvíl, Lukáš

The advanced snakes (Caenophidia), the important amniote lineage encompassing more than 3,000 living species, possess highly conserved female heterogamety across all families. However, we still lack any knowledge on the gene(s) and the molecular mechanism controlling sex determination. Triploid individuals spontaneously appear in populations of diploid species and can provide an important insight into the evolution of sex determination. Here, we report a case of spontaneous triploidy in a male of the twin-spotted ratsnake (Elaphe bimaculata) with ZZW sex chromosomes. We speculate that as both ZZ and ZZW individuals develop male gonads, the ratio between the number of Z chromosomes and autosomes, and not the presence of the W chromosome in the genome, drives sex determination in the advanced snakes.

Digital object identifier (DOI): 10.1159/000490124

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

Scientific reports, 8, 2286
2018

DNA damage in leukocytes after internal ex-vivo irradiation of blood with the α-emitter Ra-223.

Schumann, Sarah, Eberlein, Uta, Muhtadi, Razan, Lassmann, Michael, Scherthan, Harry

Irradiation with high linear energy transfer α-emitters, like the clinically used Ra-223 dichloride, severely damages cells and induces complex DNA damage including closely spaced double-strand breaks (DSBs). As the hematopoietic system is an organ-at-risk for the treatment, knowledge about Ra-223-induced DNA damage in blood leukocytes is highly desirable. Therefore, 36 blood samples from six healthy volunteers were exposed ex-vivo (in solution) to different concentrations of Ra-223. Absorbed doses to the blood were calculated assuming local energy deposition of all α- and β-particles of the decay, ranging from 0 to 142 mGy. γ-H2AX + 53BP1 co-staining and analysis was performed in leukocytes isolated from the irradiated blood samples. For DNA damage quantification, leukocyte samples were screened for occurrence of α-induced DNA damage tracks and small γ-H2AX + 53BP1 DSB foci. This revealed a linear relationship between the frequency of α-induced γ-H2AX damage tracks and the absorbed dose to the blood, while the frequency of small γ-H2AX + 53BP1 DSB foci indicative of β-irradiation was similar to baseline values, being in agreement with a negligible β-contribution (3.7%) to the total absorbed dose to the blood. Our calibration curve will contribute to the biodosimetry of Ra-223-treated patients and early after incorporation of α-emitters.

Digital object identifier (DOI): 10.1038/s41598-018-20364-7

Frontiers in neuroscience, 12, 55
2018

Safety and Efficacy of Scanning Ultrasound Treatment of Aged APP23 Mice.

Leinenga, Gerhard, Götz, Jürgen

Deposition of amyloid-β (Aβ) peptide leads to amyloid plaques that together with tau deposits characterize the brains of patients with Alzheimer's disease (AD). In modeling this pathology, transgenic animals such as the APP23 strain, that expresses a mutant form of the amyloid precursor protein found in familial cases of AD, have been instrumental. In previous studies, we have shown that repeated treatments with ultrasound in a scanning mode (termed scanning ultrasound or SUS) were effective in removing Aβ and restoring memory functions, without the need for a therapeutic agent such as an Aβ antibody. Considering that age is the most important risk factor for AD, we extended this study in which the mice were only 12 months old at the time of treatment by assessing a cohort of 2 year-old mice. Interestingly, at this age, APP23 mice are characterized by cerebral amyloid angiopathy (CAA) and the presence of occasional microbleeds. We found that SUS in aged mice that have been exposed to four SUS sessions that were spread out over 8 weeks and analyzed 4 weeks later did not show evidence of increased CAA or microbleeds. Furthermore, amyloid was reduced as assessed by methoxy-XO4 fluorescence. In addition, plaque-associated microglia were more numerous in SUS treated mice. Together this adds to the notion that SUS may be a treatment modality for human neurodegenerative diseases.

Digital object identifier (DOI): 10.3389/fnins.2018.00055

Postgraduate medical journal, 94, 398--403
2018

Adenotonsillar microbiome: an update.

Johnston, James Jordan, Douglas, Richard

Pathogenic bacteria associated with the adenoids and tonsils cause much morbidity in the paediatric population. Hyperplasia of the adenoids is associated with otitis media with effusion and hyperplasia of the palatine tonsils is associated with both recurrent tonsillitis and obstructive sleep apnoea. Most current knowledge of the microbiology of the upper airways has been derived from culture-based studies, which usually reflect only a small fraction of the bacteria present on the mucosal surface. Culture-independent molecular surveys based on 16S ribosomal RNA sequencing are now being employed to determine the microbiota on the surface and within the tissue of adenoids and palatine tonsils. This review describes the new techniques applied in determining the microbiome and summarises the results of studies employing these techniques.

Digital object identifier (DOI): 10.1136/postgradmedj-2018-135602

Evolution; international journal of organic evolution
2018

ZW, XY, and yet ZW: Sex chromosome evolution in snakes even more complicated.

Augstenová, Barbora, Johnson Pokorná, Martina, Altmanová, Marie, Frynta, Daniel, Rovatsos, Michail, Kratochvíl, Lukáš

Snakes are historically important in the formulation of several central concepts on the evolution of sex chromosomes. For over 50 years, it was believed that all snakes shared the same ZZ/ZW sex chromosomes, which are homomorphic and poorly differentiated in "basal" snakes such as pythons and boas, while heteromorphic and well differentiated in "advanced" (caenophidian) snakes. Recent molecular studies revealed that differentiated sex chromosomes are indeed shared among all families of caenophidian snakes, but that boas and pythons evolved likely independently male heterogamety (XX/XY sex chromosomes). The historical report of heteromorphic ZZ/ZW sex chromosomes in a boid snake was previously regarded as ambiguous. In the current study, we document heteromorphic ZZ/ZW sex chromosomes in a boid snake. A comparative approach suggests that these heteromorphic sex chromosomes evolved very recently and that they are poorly differentiated at the sequence level. Interestingly, two snake lineages with confirmed male heterogamety possess homomorphic sex chromosomes, but heteromorphic sex chromosomes are present in both snake lineages with female heterogamety. We point out that this phenomenon is more common across squamates. The presence of female heterogamety in non-caenophidian snakes indicates that the evolution of sex chromosomes in this lineage is much more complex than previously thought, making snakes an even better model system for the evolution of sex chromosomes.

Digital object identifier (DOI): 10.1111/evo.13543

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

Annals of diagnostic pathology, 34, 1--12
2018

Review with novel markers facilitates precise categorization of 41 cases of diagnostically challenging, "undifferentiated small round cell tumors". A clinicopathologic, immunophenotypic and molecular analysis.

Machado, Isidro, Yoshida, Akihiko, Morales, María Gema Nieto, Abrahão-Machado, Lucas Faria, Navarro, Samuel, Cruz, Julia, Lavernia, Javier, Parafioriti, Antonina, Picci, Piero, Llombart-Bosch, Antonio

Despite extensive immunohistochemical (IHC) and molecular studies combined with morphologic findings, a group of round/ovoid cell tumors histologically similar to Ewing sarcomas (ES) but lacking EWSR1-rearrangements may remain unclassifiable. We retrospectively analyzed 41 Ewing-like tumors (formalin-fixed, paraffin-embedded) previously determined as negative or non-informative for EWSR1-rearrangements by FISH and/or RT-PCR. A new histopathology revision and additional IHC and molecular analyses were carried out in order to investigate whether additional IHC and/or molecular testing in combination with the morphological findings may help in reaching a definitive diagnosis. Almost all the tumors (n=40) involved soft tissue and/or bone and half the patients died of disease. In the archival cases all diagnoses were Ewing sarcoma (ES), Ewing-like sarcoma (ELS), myoepithelial tumor and undifferentiated sarcoma (US). In the new review all the tumors were re-classified as, ES (n=16), Ewing-like tumor with EWSR1 rearrangement and amplification and possible EWSR1-NFATC2 gene fusion (n=1), CIC-rearranged sarcomas or undifferentiated sarcoma, most consistent with CIC-rearranged sarcoma (n=7), sarcoma with BCOR-alteration or undifferentiated sarcoma, consistent with BCOR-associated sarcoma (n=3), neuroblastoma (n=2), unclassifiable neoplasm with neuroblastic differentiation (n=1), malignant rhabdoid tumor (n=2), lymphoblastic lymphoma (n=1), clear cell sarcoma of the gastrointestinal tract (n=1), small cell carcinoma (n=1), sclerosing rhabdomyosarcoma (n=1), desmoplastic small round cell tumor (n=1), malignant peripheral sheath nerve tumor (n=1), poorly-differentiated synovial sarcoma (n=1), Possible gastrointestinal stromal tumor/GIST with predominant round cells (n=1) and possible SMARCA4-deficient-sarcoma (n=1). NKX2.2, ETV4 and BCOR immunoreactivity was observed in all ES, CIC-rearranged sarcomas and sarcomas with BCOR alteration, respectively. CIC-rearrangement by FISH was observed in many of the CIC-rearranged sarcomas. Our analysis of 41 Ewing-like tumors confirms that there may be a significant pathological and IHC overlap among Ewing-like tumors, with prognostic and therapeutic impacts. Additional IHC (NKX2.2, ETV4 and BCOR) and molecular studies including FUS, CIC or BCOR analysis may support the final diagnosis when FISH or RT-PCR fail to detect EWSR1-rearrangements. Any molecular findings should always be interpreted in relation to the specific clinical and pathological context.

Digital object identifier (DOI): 10.1016/j.anndiagpath.2017.11.011

British journal of cancer
2018

Heterogeneous MYCN amplification in neuroblastoma: a SIOP Europe Neuroblastoma Study.

Berbegall, Ana P, Bogen, Dominik, Pötschger, Ulrike, Beiske, Klaus, Bown, Nick, Combaret, Valérie, Defferrari, Raffaella, Jeison, Marta, Mazzocco, Katia, Varesio, Luigi, Vicha, Ales, Ash, Shifra, Castel, Victoria, Coze, Carole, Ladenstein, Ruth, Owens, Cormac, Papadakis, Vassilios, Ruud, Ellen, Amann, Gabriele, Sementa, Angela R, Navarro, Samuel, Ambros, Peter F, Noguera, Rosa, Ambros, Inge M

In neuroblastoma (NB), the most powerful prognostic marker, the MYCN amplification (MNA), occasionally shows intratumoural heterogeneity (ITH), i.e. coexistence of MYCN-amplified and non-MYCN-amplified tumour cell clones, called heterogeneous MNA (hetMNA). Prognostication and therapy allocation are still unsolved issues. The SIOPEN Biology group analysed 99 hetMNA NBs focussing on the prognostic significance of MYCN ITH. Patients <18 months (18?m) showed a better outcome in all stages as compared to older patients (5-year OS in localised stages: <18?m: 0.95?±?0.04, >18?m: 0.67?±?0.14, p?=?0.011; metastatic: <18?m: 0.76?±?0.15, >18?m: 0.28?±?0.09, p?=?0.084). The genomic 'background', but not MNA clone sizes, correlated significantly with relapse frequency and OS. No relapses occurred in cases of only numerical chromosomal aberrations. Infiltrated bone marrows and relapse tumour cells mostly displayed no MNA. However, one stage 4s tumour with segmental chromosomal aberrations showed a homogeneous MNA in the relapse. This study provides a rationale for the necessary distinction between heterogeneous and homogeneous MNA. HetMNA tumours have to be evaluated individually, taking age, stage and, most importantly, genomic background into account to avoid unnecessary upgrading of risk/overtreatment, especially in infants, as well as in order to identify tumours prone to developing homogeneous MNA.

Digital object identifier (DOI): 10.1038/s41416-018-0098-6

The American journal of surgical pathology, 42, 656--664
2018

Clarifying the Distinction Between Malignant Peripheral Nerve Sheath Tumor and Dedifferentiated Liposarcoma: A Critical Reappraisal of the Diagnostic Utility of MDM2 and H3K27me3 Status.

Makise, Naohiro, Sekimizu, Masaya, Kubo, Takashi, Wakai, Susumu, Hiraoka, Nobuyoshi, Komiyama, Motokiyo, Fukayama, Masashi, Kawai, Akira, Ichikawa, Hitoshi, Yoshida, Akihiko

Malignant peripheral nerve sheath tumor (MPNST) and dedifferentiated liposarcoma (DDLPS) are 2 major types of pleomorphic spindle cell sarcoma. The differentiation of MPNST and DDLPS by histomorphology alone can be problematic. Although MDM2 amplification and PRC2 alteration leading to H3K27me3 deficiency are genetic hallmarks of DDLPS and MPNST, respectively, a small number of MDM2-amplified MPNSTs and H3K27me3-deficient DDLPSs have been reported in the literature. We systematically compared MDM2 and H3K27me3 status in 68 MPNSTs and 47 DDLPSs. Of the 62 MPNSTs, 22 were immunopositive for MDM2, mostly in a weak and/or focal manner. Of the 21 MDM2-positive MPNSTs successfully tested by fluorescence in situ hybridization, high-level MDM2 amplification was observed in 1 case. In contrast, MDM2 staining and high-level MDM2 amplification were positive in all the DDLPS tested (28/28 and 20/20). Of the 68 MPNSTs, 42 cases (62%) exhibited complete loss of H3K27me3. All the 13 MPNSTs that showed heterologous differentiation were deficient in H3K27me3. Of the 47 DDLPSs, 3 cases (6%) had complete loss of H3K27me3, all of which exhibited heterologous differentiation. One case of H3K27me3-deficient DDLPS exhibited homozygous loss of EED according to targeted next-generation sequencing, whereas there were no alterations in NF1 and CDKN2A. In conclusion, high-level MDM2 amplification strongly suggests DDLPS over MPNST. Although a good marker for MPNST, H3K27me3 deficiency also uncommonly occurs in DDLPS in association with PRC2 mutational inactivation. Because both markers are imperfectly specific, rare sarcomas with dual features could be encountered, and their classification should integrate other parameters.

Digital object identifier (DOI): 10.1097/PAS.0000000000001014

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

Biological procedures online, 20, 13
2018

Optimization of Immunofluorescent Detection of Bone Marrow Disseminated Tumor Cells.

Axelrod, Haley D, Pienta, Kenneth J, Valkenburg, Kenneth C

Cancer metastasis is the primary cause of cancer-related deaths and remains incurable. Current clinical methods for predicting metastatic recurrence are not sensitive enough to detect individual cancer cells in the body; therefore, current efforts are directed toward liquid biopsy-based assays to capture circulating and disseminated tumor cells (CTCs and DTCs) in the blood and bone marrow, respectively. The most promising strategy is fluorescence-based immunostaining using cancer cell-specific markers. However, despite recent efforts to develop robust processing and staining platforms, results from these platforms have been discordant among groups, particularly for DTC detection. While the choice of cancer cell-specific markers is a large factor in this discordance, we have found that marker-independent factors causing false signal are just as critical to consider. Bone marrow is particularly challenging to analyze by immunostaining because endogenous immune cell properties and bone marrow matrix components typically generate false staining. For immunostaining of whole tumor tissue containing ample cancer cells, this background staining can be overcome. Application of fluorescent-based staining for rare cells, however, is easily jeopardized by immune cells and autofluorescence that lead to false signal. We have specifically found two types of background staining in bone marrow samples: autofluorescence of the tissue and non-specific binding of secondary antibodies. We systematically optimized a basic immunofluorescence protocol to eliminate this background using cancer cells spiked into human bone marrow. This enhanced the specificity of automated scanning detection software. Our optimized protocol also outperformed a commercial rare cell detection protocol in detecting candidate DTCs from metastatic patient bone marrow. Robust optimization to increase the signal-to-noise ratio of immunofluorescent staining of bone marrow is required in order to achieve the necessary sensitivity and specificity for rare cell detection. Background immunofluorescent staining in bone marrow causes uncertainty and inconsistency among investigators, which can be overcome by systematically addressing each contributing source. Our optimized assay eliminates sources of background signal, and is adaptable to automated staining platforms for high throughput analysis.

Digital object identifier (DOI): 10.1186/s12575-018-0078-5

PloS one, 13, e0193213
2018

Impact of 9p deletion and p16, Cyclin D1, and Myc hyperexpression on the outcome of anaplastic oligodendrogliomas.

Michaud, Karine, de Tayrac, Marie, D'Astous, Myreille, Paquet, Claudie, Gould, Peter Vincent, Saikali, Stéphan

To study the presence of 9p deletion and p16, cyclin D1 and Myc expression and their respective diagnostic and prognostic interest in oligodendrogliomas. We analyzed a retrospective series of 40 consecutive anaplastic oligodendrogliomas (OIII) from a single institution and compared them to a control series of 10 low grade oligodendrogliomas (OII). Automated FISH analysis of chromosome 9p status and immunohistochemistry for p16, cyclin D1 and Myc was performed for all cases and correlated with clinical and histological data, event free survival (EFS) and overall survival (OS). Chromosome 9p deletion was observed in 55% of OIII (22/40) but not in OII. Deletion was highly correlated to EFS (median = 29 versus 53 months, p<0.0001) and OS (median = 48 versus 83 months, p<0.0001) in both the total cohort and the OIII population. In 9p non-deleted oligodendrogliomas, p16 hyperexpression correlated with a shorter OS (p = 0.02 in OII and p = 0.0001 in OIII) whereas lack of p16 expression was correlated to a shorter EFS and OS in 9p deleted OIII (p = 0.001 and p = 0.0002 respectively). Expression of Cyclin D1 was significantly higher in OIII (median expression 45% versus 14% for OII, p = 0.0006) and was correlated with MIB-1 expression (p<0.0001), vascular proliferation (p = 0.002), tumor necrosis (p = 0.04) and a shorter EFS in the total cohort (p = 0.05). Hyperexpression of Myc was correlated to grade (median expression 27% in OII versus 35% in OIII, p = 0.03), and to a shorter EFS in 9p non-deleted OIII (p = 0.01). Chromosome 9p deletion identifies a subset of OIII with significantly worse prognosis. The combination of 9p status and p16 expression level identifies two distinct OIII populations with divergent prognosis. Hyperexpression of Bcl1 and Myc appears highly linked to anaplasia but the prognostic value is unclear and should be investigated further.

Digital object identifier (DOI): 10.1371/journal.pone.0193213

Environmental science and pollution research international
2018

Toxicological evaluation of nail polish waste discarded in the environment.

Felzenszwalb, Israel, Fernandes, Andreia da Silva, Brito, Lara Barroso, Oliveira, Gisele Augusto Rodrigues, Silva, Paula Aquino Soeiro, Arcanjo, Maria Elena, Marques, Monica Regina da Costa, Vicari, Taynah, Leme, Daniela Morais, Cestari, Marta Margarete, Ferraz, Elisa Raquel Anastacio

Nail polish has been widely used around the world. However, the hazards of nail polishes discarded in the environment are still poorly investigated. Thus, the toxicogenetic effects of solubilized (SE) and leached (LE) extracts from nail polishes were investigated, simulating their disposal on water and landfill, respectively, and identifying their physicochemical properties and chemical constituents. Organic compounds and metals were detected in both extracts. SE and LE only induced mutagenic effects in TA98 Salmonella strain in the presence and absence of exogenous metabolic activation. Although both extracts did not significantly increase the frequency of micronucleated HepG2 cells, the cell viability was affected by 24-h exposure. No DNA damage was observed in gonad fish cells (RTG-2) exposed to both extracts; however, the highest SE and LE concentrations induced significant lethal and sublethal effects on zebrafish early-life stages during 96-h exposure. Based on our findings, it can be concluded that if nail polishes enter aquatic systems, it may cause negative impacts to the environment.

Digital object identifier (DOI): 10.1007/s11356-018-1880-y

Mutation research, 826, 47--52
2018

Folate modulates guanine-quadruplex frequency and DNA damage in Werner syndrome.

Tavakoli Shirazi, Paniz, Leifert, Wayne Richard, Fenech, Michael Felix, François, Maxime

Guanine-quadruplexes (G4) are stable tetra-stranded DNA structures that may cause DNA replication stress and inhibit gene expression. Defects in unwinding these structures by DNA helicases may result in telomere shortening and DNA damage. Furthermore, due to mutations in WRN helicase genes in Werner syndrome, G4 motifs are likely to be key elements in the expression of premature aging phenotypes. The methylation of DNA plays a significant role in the stability and occurrence of G4. Thus, G4 frequency and DNA methylation mechanisms may be affected by excesses or deficiencies in methyl donors such as folate. B-Lymphocytes from Werner patients (n?=?5) and healthy individuals (n?=?5) were cultured in RPMI medium under condition of folate deficiency (20?nM) or sufficiency (200?nM) for 14 days. Cells were fixed on microscope slides for immunofluorescent staining to measure G4 frequency and ?H2AX (a marker of DNA strand breaks) intensity, using automated quantitative imaging fluorescent microscopy. There was a significant increase (p?<?0.05) in G4 levels in Werner syndrome patients compared to healthy controls. Werner and control cells grown in 20?nM folate media also showed significant increases in G4 (p?<?0.001) and ?H2AX (p?<?0.01) signals compared with the same cells grown in 200?nM folate. Control cells grown in 20?nM folate also showed a significant reduction in DNA methylation levels (P?<?0.05). The results of this study suggest that the occurrence of DNA G4 structures can be modulated in vitro via nutrients with important roles in methylation.

Digital object identifier (DOI): 10.1016/j.mrgentox.2017.12.002

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