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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

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

Journal of neurotrauma, 35, 671--680
2018

Erythropoietin Attenuates the Brain Edema Response after Experimental Traumatic Brain Injury.

Blixt, Jonas, Gunnarson, Eli, Wanecek, Michael

Erythropoietin (EPO) has neuroprotective effects in multiple central nervous system (CNS) injury models; however EPO's effects on traumatic brain edema are elusive. To explore EPO as an intervention in traumatic brain edema, male Sprague-Dawley (SD) rats were subjected to blunt, controlled traumatic brain injury (TBI). Animals were randomized to EPO 5000 IU/kg or saline (control group) intraperitoneally within 30 min after trauma and once daily for 4 consecutive days. Brain MRI, immunohistofluorescence, immunohistochemistry, and quantitative protein analysis were performed at days 1 and 4 post- trauma. EPO significantly prevented the loss of the tight junction protein zona occludens 1 (ZO-1) observed in control animals after trauma. The decrease of ZO-1 in the control group was associated with an immunoglobulin (Ig)G increase in the perilesional parenchyma, indicating blood-brain barrier (BBB) dysfunction and increased permeability. EPO treatment attenuated decrease in apparent diffusion coefficient (ADC) after trauma, suggesting a reduction of cytotoxic edema, and reduced the IgG leakage, indicating that EPO contributed to preserve BBB integrity and attenuated vasogenic edema. Animals treated with EPO demonstrated conserved levels of aquaporin 4 (AQP4) protein expression in the perilesional area, whereas control animals showed a reduction of AQP4. We show that post TBI administration of EPO decreases early cytotoxic brain edema and preserves structural and functional properties of the BBB, leading to attenuation of the vasogenic edema response. The data support that the mechanisms involve preservation of the tight junction protein ZO-1 and the water channel AQP4, and indicate that treatment with EPO may have beneficial effects on the brain edema response following TBI.

Digital object identifier (DOI): 10.1089/neu.2017.5015

Cancers, 10
2018

The Transition between Telomerase and ALT Mechanisms in Hodgkin Lymphoma and Its Predictive Value in Clinical Outcomes.

M'kacher, Radhia, Cuceu, Corina, Al Jawhari, Mustafa, Morat, Luc, Frenzel, Monika, Shim, Grace, Lenain, Aude, Hempel, William M, Junker, Steffen, Girinsky, Theodore, Colicchio, Bruno, Dieterlen, Alain, Heidingsfelder, Leonhard, Borie, Claire, Oudrhiri, Noufissa, Bennaceur-Griscelli, Annelise, Moralès, Olivier, Renaud, Sarah, Van de Wyngaert, Zoé, Jeandidier, Eric, Delhem, Nadira, Carde, Patrice

: We analyzed telomere maintenance mechanisms (TMMs) in lymph node samples from HL patients treated with standard therapy. The TMMs correlated with clinical outcomes of patients. : Lymph node biopsies obtained from 38 HL patients and 24 patients with lymphadenitis were included in this study. Seven HL cell lines were used as in vitro models. Telomerase activity (TA) was assessed by TRAP assay and verified through hTERT immunofluorescence expression; alternative telomere lengthening (ALT) was also assessed, along with EBV status. : Both TA and ALT mechanisms were present in HL lymph nodes. Our findings were reproduced in HL cell lines. The highest levels of TA were expressed in CD30-/CD15- cells. Small cells were identified with ALT and TA. Hodgkin and Reed Sternberg cells contained high levels of PML bodies, but had very low hTERT expression. There was a significant correlation between overall survival ( < 10 ), event-free survival ( < 10 ), and freedom from progression ( < 10 ) and the presence of an ALT profile in lymph nodes of EBV+ patients. : The presence of both types of TMMs in HL lymph nodes and in HL cell lines has not previously been reported. TMMs correlate with the treatment outcome of EBV+ HL patients.

Digital object identifier (DOI): 10.3390/cancers10060169

Health physics, 115, 77--89
2018

Investigation of Spatial Organization of Chromosome Territories in Chromosome Exchange Aberrations After Ionizing Radiation Exposure.

Balajee, Adayabalam S, Sanders, Jacob T, Golloshi, Rosela, Shuryak, Igor, McCord, Rachel Patton, Dainiak, Nicholas

Higher-order organization of the human genome is well established with chromosomes occupying distinct domains or territories in the interphase nucleus. Spatial organization of chromosome territories in the interphase nucleus occurs in a cell-type-specific manner. Since both stable and unstable aberrations induced by ionizing radiation involve the exchange of material between two or more chromosomes, this study investigated the role of spatial organization of chromosome domains in ionizing-radiation-induced chromosome translocation events. Using multicolor fluorescence in situ hybridization, the study characterized the positioning of each human chromosome relative to its neighborhood territories in the interphase nucleus of lymphocytes and B-lymphoblastoid cells before ionizing radiation and compared this interphase positioning with the spectrum of exchanges observed after ionizing radiation in the metaphase chromosomes. In addition to multicolor fluorescence in situ hybridization, the genome-wide chromosome conformation capture technique (Hi-C) was also performed in mock and x-ray-irradiated human B-lymphoblastoid and fibroblast cells to characterize the interactions among chromosomes and to assess the genome reorganization changes, if any, after ionizing radiation exposure. On average, 35-50% of the total translocations induced by x rays and neutrons correlated with proximity of chromosome territories detected by multicolor fluorescence in situ hybridization in both lymphocytes and lymphoblastoid cells. The translocation rate observed in proximally positioned chromosome territories was consistently higher than distally located territories and was found to be statistically significant (p = 0.01) in human lymphoblastoid cells after x rays. The interchromosome interaction frequencies detected by Hi-C correlate fairly well with ionizing-radiation-induced translocations detected by multicolor fluorescence in situ hybridization, suggesting the importance of chromosome proximity effects in ionizing-radiation-induced chromosomal translocation events.

Digital object identifier (DOI): 10.1097/HP.0000000000000840

Surgical oncology, 27, 106--113
2018

Detection of RET (rearranged during transfection) variants and their downstream signal molecules in RET rearranged lung adenocarcinoma patients.

Kim, Jeong-Oh, Shin, Jung-Young, Kim, Min Young, Son, Kyoung Hwa, Jung, Chan Kwon, Kim, Tae-Jung, Kim, Su Young, Park, Jae Kil, Sung, Sook Whan, Bae, Sang Ju, Min, Hyun Jung, Kang, Jin-Hyoung

We screened resected tumor tissues from patients with lung cancer for EGFR mutations, ALK rearrangements, and rearranged during transfection (RET) gene variants (including RET rearrangements and the Kinesin Family Member 5B (KIF5B)-RET fusion gene) using various methods including reverse transcription polymerase chain reaction (RT-PCR), transcript assays, fluorescence in situ hybridization (FISH), and immunohistochemistry (IHC). We also examined the protein expression of associated downstream signaling molecules to assess the effect of these variants on patient outcome. We constructed a tissue microarray (TMA) comprising 581 resected tumor tissues from patients with lung adenocarcinoma and analyzed the microarray by both FISH (using RET break-apart and KIF5B-RET SY translocation probes) and a commercial RET transcript assay. We evaluated the expression of RET and RET-related signaling molecules, including p-AKT and p-ERK, by TMA -based IHC staining. Among the 581 specimens, 51 (8.8%) specimens harbored RET rearrangements, including 12 cases (2.1%) carrying a KIF5B-RET fusion gene. Surprisingly, RET expression was lower in KIF5B-RET fusion gene-positive than in RET wild-type specimens. We detected activating EGFR mutations in 11 (21.6%) of the 51 RET variant-positive specimens. Among the KIF5B-RET fusion gene-positive specimens, p-ERK expression was significantly lower in the EGFR mutation subgroup showing RET expression than in the EGFR mutation subgroup that did not express RET. Similarly, the RET rearrangement group showed significant variation in the expression level of p-AKT (P?=?0.028) and p-ERK, whose expression remarkably increased in specimens not expressing RET. The expression of p-ERK markedly increased in the RET rearrangement group regardless of RET expression. This result suggests that a combination of RET and ERK inhibitors may be an effective treatment strategy for lung adenocarcinoma patients harboring RET variants.

Digital object identifier (DOI): 10.1016/j.suronc.2018.01.006

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