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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

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

Radiation protection dosimetry, 182, 139--145
2018

DEVELOPMENT OF A MINIATURIZED VERSION OF DICENTRIC CHROMOSOME ASSAY TOOL FOR RADIOLOGICAL TRIAGE.

Balajee, Adayabalam S, Smith, Tammy, Ryan, Terri, Escalona, Maria, Dainiak, Nicholas

Use of ionizing radiation (IR) in various industrial, medical and other applications can potentially increase the risk of medical, occupational or accidental human exposure. Additionally, in the event of a radiological or nuclear (R/N) incident, several tens of hundreds and thousands of people are likely to be exposed to IR. IR causes serious health effects including mortality from acute radiation syndrome and therefore it is imperative to determine the absorbed radiation dose, which will enable physicians in making an appropriate clinical 'life-saving' decision. The 'Dicentric Chromosome Assay (DCA)' is the gold standard for estimating the absorbed radiation dose but its performance is time consuming and laborious. Further, timely evaluation of dicentric chromosomes (DCs) for dose estimation in a large number of samples provides a bottleneck because of a limited number of trained personnel and a prolonged time for manual analysis. To circumvent some of these technical issues, we developed and optimized a miniaturized high throughput version of DCA (mini-DCA) in a 96-microtube matrix with bar-coded 1.4 ml tubes to enable the processing of a large number of samples. To increase the speed of DC analysis for radiation dose estimation, a semi-automated scoring was optimized using the Metafer DCScore algorithm. The accuracy of mini-DCA in dose estimation was verified and validated though comparison with conventional DCA performed in 15 ml conical tubes. The mini-DCA considerably reduced the sample processing time by a factor of 4 when compared to the conventional DCA. Further, the radiation doses estimated by mini-DCA using the triage mode of scoring (50 cells or 30 DCs) were similar to that of conventional DCA using 300-500 cells. The mini-DCA coupled with semi-automated DC scoring not only reduced the sample processing and analysis times by a factor of 4 but also enabled the processing of a large number of samples at once. Our mini-DCA method, once automated for high throughput robotic platforms, will be an effective radiological triage tool for mass casualty incidents.

Digital object identifier (DOI): 10.1093/rpd/ncy127

Radiology, 288, 529--535
2018

Abdominopelvic 1.5-T and 3.0-T MR Imaging in Healthy Volunteers: Relationship to Formation of DNA Double-Strand Breaks.

Suntharalingam, Saravanabavaan, Mladenov, Emil, Sarabhai, Theresia, Wetter, Axel, Kraff, Oliver, Quick, Harald H, Forsting, Michael, Iliakis, Georg, Nassenstein, Kai

Purpose To investigate the relationship between abdominopelvic magnetic resonance (MR) imaging and formation of DNA double-strand breaks (DSBs) in peripheral blood lymphocytes among a cohort of healthy volunteers. Materials and Methods Blood samples were obtained from 40 healthy volunteers (23 women and 17 men; mean age, 27.2 years [range, 21-37 years]) directly before and 5 and 30 minutes after abdominopelvic MR imaging performed at 1.5 T (n = 20) or 3.0 T (n = 20). The number of DNA DSBs in isolated blood lymphocytes was quantified after indirect immunofluorescent staining of a generally accepted DSB marker, ?-H2AX, by means of high-throughput automated microscopy. As a positive control of DSB induction, blood lymphocytes from six volunteers were irradiated in vitro with x-rays at a dose of 1 Gy (70-90 keV). Statistical analysis was performed by using a Friedman test. Results No significant alteration in the frequency of DNA DSB induction was observed after MR imaging (before imaging: 0.22 foci per cell, interquartile range [IQR] = 0.54 foci per cell; 5 minutes after MR imaging: 0.08 foci per cell, IQR = 0.39 foci per cell; 30 minutes after MR imaging: 0.09 foci per cell, IQR = 0.63 foci per cell; P = .057). In vitro radiation of lymphocytes with 1 Gy led to a significant increase in DSBs (0.22 vs 3.43 foci per cell; P = .0312). The frequency of DSBs did not differ between imaging at 1.5 T and at 3.0 T (5 minutes after MR imaging: 0.23 vs 0.06 foci per cell, respectively [P = .57]; 30 minutes after MR imaging: 0.12 vs 0.08 foci per cell [P = .76]). Conclusion Abdominopelvic MR imaging performed at 1.5 T or 3.0 T does not affect the formation of DNA DSBs in peripheral blood lymphocytes.

Digital object identifier (DOI): 10.1148/radiol.2018172453

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

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

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

Journal of clinical pathology
2018

KRAS fluorescence in situ hybridisation testing for the detection and diagnosis of pancreatic adenocarcinoma.

Shiroma, Noriyuki, Arihiro, Koji, Oda, Miyo, Orita, Makoto

The aim of our study was to analyse correlations between mutation status, chromosomal changes that affect status in cells from pancreatic tumours. We collected 69 cases of surgically resected pancreatic ductal adenocarcinoma (PDA) and seven cases of chronic pancreatitis (CP). Chromosomal abnormalities of and CEP12 were detected using fluorescence in situ hybridisation (FISH). The number of CEP12 signals per cell ranged from 1.78 to 2.04 and 1.46 to 4.88 in CP and PDA samples, respectively, while the number of signals per cell ranged from 1.94 to 2.06 and 1.88 to 8.18 in CP and PDA samples, respectively. The 'chromosomal instability index', which was defined as the percentage of cells with any chromosomal abnormality, was over 5.7 times greater in PDA than in CP. We performed mutation analysis by direct sequencing and found that tumours with mutations have a significantly higher mean signal per cell from PDA samples compared with tumours with wild-type amplification was noted in 10% of cases. Although we found that lymph node metastasis and distal metastasis of PDA were more frequent in cases with amplification, this was not correlated with overall survival. Using a threshold of 40%, we found that the chromosomal instability index robustly discriminated PDA cells from CP cells. Based on these findings, we concluded that FISH testing of using cytology samples may represent an accurate approach for the diagnosis of PDA.

Digital object identifier (DOI): 10.1136/jclinpath-2018-205002

Mutation research, 834, 35--41
2018

Reprint of: A three-dimensional in vitro HepG2 cells liver spheroid model for genotoxicity studies.

Shah, Ume-Kulsoom, Mallia, Jefferson de Oliveira, Singh, Neenu, Chapman, Katherine E, Doak, Shareen H, Jenkins, Gareth J S

The liver's role in metabolism of chemicals makes it an appropriate tissue for toxicity testing. Current testing protocols, such as animal testing and two-dimensional liver cell systems, offer limited resemblance to in vivo liver cell behaviour, in terms of gene expression profiles and metabolic competence; thus, they do not always accurately predict human toxicology. In vitro three-dimensional liver cell models offer an attractive alternative. This study reports on the development of a 3D liver model, using HepG2 cells, by a hanging-drop technique, with a focus on evaluating spheroid growth characteristics and suitability for genotoxicity testing. The cytokinesis-blocked micronucleus assay protocol was adapted to enable micronucleus (MN) detection in the 3D spheroid models. This involved evaluating the difference between hanging vs non-hanging drop positions for dosing of the test agents and comparison of automated Metafer scoring with manual scoring for MN detection in HepG2 spheroids. The initial seeding density, used for all experiments, was 5000 cells/20 μl drop hanging spheroids, harvested on day 4, with >75% cell viability. Albumin secretion (7.8 g/l) and both CYP1A1 and CYP1A2 gene expression were highest in the 3D environment at day 4. Exposure to metabolically activated genotoxicants for 24 h resulted in a 6-fold increase in CYP1A1 enzyme activity (3 μM B[a]P) and a 30-fold increase in CYP1A2 enzyme activity (5 μM PhIP) in 3D hanging spheroids. MN inductions in response to B[a]P or PhIP were 2-fold and 3-fold, respectively, and were greater in 3D hanging spheroids than in 2D format, showing that hanging spheroids are more sensitive to genotoxic agents. HepG2 hanging-drop spheroids are an exciting new alternative system for genotoxicity studies, due to their improved structural and physiological properties, relative to 2D cultures.

Digital object identifier (DOI): 10.1016/j.mrgentox.2018.06.020

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

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

Angiogenesis
2018

Improved recovery from limb ischaemia by delivery of an affinity-isolated heparan sulphate.

Poon, Selina, Lu, Xiaohua, Smith, Raymond A A, Ho, Pei, Bhakoo, Kishore, Nurcombe, Victor, Cool, Simon M

Peripheral arterial disease is a major cause of limb loss and its prevalence is increasing worldwide. As most standard-of-care therapies yield only unsatisfactory outcomes, more options are needed. Recent cell- and molecular-based therapies that have aimed to modulate vascular endothelial growth factor-165 (VEGF ) levels have not yet been approved for clinical use due to their uncertain side effects. We have previously reported a heparan sulphate (termed HS7) tuned to avidly bind VEGF . Here, we investigated the ability of HS7 to promote vascular recovery in a murine hindlimb vascular ischaemia model. HS7 stabilised VEGF against thermal and enzyme degradation in vitro, and isolated VEGF from serum via affinity-chromatography. C57BL6 mice subjected to unilateral hindlimb ischaemia injury received daily intramuscular injections of respective treatments (n = 8) and were assessed over 3 weeks by laser Doppler perfusion, magnetic resonance angiography, histology and the regain of function. Mice receiving HS7 showed improved blood reperfusion in the footpad by day 7. In addition, they recovered hindlimb blood volume two- to fourfold faster compared to the saline group; the greatest rate of recovery was observed in the first week. Notably, 17% of HS7-treated animals recovered full hindlimb function by day 7, a number that grew to 58% and 100% by days 14 and 21, respectively. This was in contrast to only 38% in the control animals. These results highlight the potential of purified glycosaminoglycan fractions for clinical use following vascular insult, and confirm the importance of harnessing the activity of endogenous pro-healing factors generated at injury sites.

Digital object identifier (DOI): 10.1007/s10456-018-9622-9

Journal of phycology
2018

Quantitative comparison of taxa and taxon concepts in the diatom genus Fragilariopsis: a case study on using slide scanning, multi-expert image annotation and image analysis in taxonomy.

Beszteri, Bánk, Allen, Claire, Almandoz, Gastón O, Armand, Leanne, Barcena, María Ángeles, Cantzler, Hannelore, Crosta, Xavier, Esper, Oliver, Jordan, Richard W, Kauer, Gerhard, Klaas, Christine, Kloster, Michael, Leventer, Amy, Pike, Jennifer, Rigual Hernández, Andrés S

Semi-automated methods for microscopic image acquisition, image analysis and taxonomic identification have repeatedly received attention in diatom analysis. Less well studied is the question whether and how such methods might prove useful for clarifying the delimitation of species that are difficult to separate for human taxonomists. To try to answer this question, three very similar Fragilariopsis species endemic to the Southern Ocean were targeted in this study: F. obliquecostata, F. ritscheri, and F. sublinearis. A set of 501 extended focus depth specimen images were obtained using a standardized, semi-automated microscopic procedure. Twelve diatomists independently identified these specimen images in order to reconcile taxonomic opinions and agree upon a taxonomic gold standard. Using image analyses, we then extracted morphometric features representing taxonomic characters of the target taxa. The discriminating ability of individual morphometric features was tested visually and statistically, and multivariate classification experiments were performed to test the agreement of the quantitatively-defined taxa assignments with expert consensus opinion. Beyond an updated differential diagnosis of the studied taxa, our study also shows that automated imaging and image analysis procedures for diatoms are coming close to reaching a broad applicability for routine use. This article is protected by copyright. All rights reserved.

Digital object identifier (DOI): 10.1111/jpy.12767

The FEBS journal, 285, 3769--3785
2018

Naphthalene diimide-derivatives G-quadruplex ligands induce cell proliferation inhibition, mild telomeric dysfunction and cell cycle perturbation in U251MG glioma cells.

Muoio, Daniela, Berardinelli, Francesco, Leone, Stefano, Coluzzi, Elisa, di Masi, Alessandra, Doria, Filippo, Freccero, Mauro, Sgura, Antonella, Folini, Marco, Antoccia, Antonio

In the present paper, the biological effects of three different naphthalene diimides (NDIs) G-quadruplex (G4) ligands (H-NDI-Tyr, H-NDI-NMe2, and tetra-NDI-NMe2) were comparatively evaluated to those exerted by RHPS4, a well-characterized telomeric G4-ligand, in an in vitro model of glioblastoma. Data indicated that NDIs were very effective in blocking cell proliferation at nanomolar concentrations, although displaying a lower specificity for telomere targeting compared to RHPS4. In addition, differently from RHPS4, NDIs failed to enhance the effect of ionizing radiation, thus suggesting that additional targets other than telomeres could be involved in the strong NDI-mediated anti-proliferative effects. In order to test telomeric off-target action of NDIs, a panel of genes involved in tumor progression, DNA repair, telomere maintenance, and cell-cycle regulation were evaluated at transcriptional and translational level. Specifically, the compounds were able to cause a marked reduction of TERT and BCL2 amounts as well as to favor the accumulation of proteins involved in cell cycle control. A detailed cytofluorimetric analysis of cell cycle progression by means of bromodeoxyuridine (BrdU) incorporation and staining of phospho-histone H3 indicated that NDIs greatly reduce the progression through S-phase and lead to G1 accumulation of BrdU-positive cells. Taken together, these data indicated that, besides effects on telomeres and oncogenes such as Tert and Bcl2, nanomolar concentrations of NDIs determined a sustained block of cell proliferation by slowing down cell cycle progression during S-phase. In conclusion, our data indicate that NDIs G4-ligands are powerful antiproliferative agents, which act through mechanisms that ultimately lead to altered cell-cycle control.

Digital object identifier (DOI): 10.1111/febs.14628

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

Scientific reports, 8, 1032
2018

The ten-year evolutionary trajectory of a highly recurrent paediatric high grade neuroepithelial tumour with MN1:BEND2 fusion.

Burford, Anna, Mackay, Alan, Popov, Sergey, Vinci, Maria, Carvalho, Diana, Clarke, Matthew, Izquierdo, Elisa, Avery, Aimee, Jacques, Thomas S, Ingram, Wendy J, Moore, Andrew S, Frawley, Kieran, Hassall, Timothy E, Robertson, Thomas, Jones, Chris

Astroblastomas are rare brain tumours which predominate in children and young adults, and have a controversial claim as a distinct entity, with no established WHO grade. Reports suggest a better outcome than high grade gliomas, though they frequently recur. Recently, they have been described to overlap with a newly-discovered group of tumours described as'high grade neuroepithelial tumour with MN1 alteration' (CNS HGNET-MN1), defined by global methylation patterns and strongly associated with gene fusions targeting MN1. We have studied a unique case of astroblastoma arising in a 6 year-old girl, with multiple recurrences over a period of 10 years, with the pathognomonic MN1:BEND2 fusion. Exome sequencing allowed for a phylogenetic reconstruction of tumour evolution, which when integrated with clinical, pathological and radiological data provide for a detailed understanding of disease progression, with initial treatment driving tumour dissemination along four distinct trajectories. Infiltration of distant sites was associated with a later genome doubling, whilst there was evidence of convergent evolution of different lesions acquiring distinct alterations targeting NF-κB. These data represent an unusual opportunity to understand the evolutionary history of a highly recurrent childhood brain tumour, and provide novel therapeutic targets for astroblastoma/CNS HGNET-MN1.

Digital object identifier (DOI): 10.1038/s41598-018-19389-9

eLife, 7
2018

Distinct roles of ATM and ATR in the regulation of ARP8 phosphorylation to prevent chromosome translocations.

Sun, Jiying, Shi, Lin, Kinomura, Aiko, Fukuto, Atsuhiko, Horikoshi, Yasunori, Oma, Yukako, Harata, Masahiko, Ikura, Masae, Ikura, Tsuyoshi, Kanaar, Roland, Tashiro, Satoshi

Chromosomal translocations are hallmarks of various types of cancers and leukemias. However, the molecular mechanisms of chromosome translocations remain largely unknown. The ataxia-telangiectasia mutated (ATM) protein, a DNA damage signaling regulator, facilitates DNA repair to prevent chromosome abnormalities. Previously, we showed that ATM deficiency led to the 11q23 chromosome translocation, the most frequent chromosome abnormalities in secondary leukemia. Here, we show that ARP8, a subunit of the INO80 chromatin remodeling complex, is phosphorylated after etoposide treatment. The etoposide-induced phosphorylation of ARP8 is regulated by ATM and ATR, and attenuates its interaction with INO80. The ATM-regulated phosphorylation of ARP8 reduces the excessive loading of INO80 and RAD51 onto the breakpoint cluster region. These findings suggest that the phosphorylation of ARP8, regulated by ATM, plays an important role in maintaining the fidelity of DNA repair to prevent the etoposide-induced 11q23 abnormalities.

Digital object identifier (DOI): 10.7554/eLife.32222

Pathology, research and practice, 214, 318--324
2018

Osteosarcoma arising in fibrous dysplasia, confirmed by mutational analysis of GNAS gene.

Sugiura, Yoshiya, Kanda, Hiroaki, Motoi, Noriko, Nomura, Kimie, Inamura, Kentaro, Okada, Erina, Matsumoto, Haruna, Shimoji, Takashi, Matsumoto, Seiichi, Nakayama, Jun, Takazawa, Yutaka, Ishikawa, Yuichi, Machinami, Rikuo

Malignancy arising in fibrous dysplasia (FD) is rare. Approximately 100 cases have been reported so far, and osteosarcoma is the most common malignancy. We report a case of osteosarcoma in a 33-year-old Japanese man with monostotic FD of the right proximal femur from the age of 16 years. Histologically, relatively well-differentiated osteosarcoma was found in the FD lesion. Immunohistochemically, the FD was negative for p53 or MDM2, and the MIB-1 index was less than 1%, whereas the osteosarcoma was positive for both p53 and MDM2, and the MIB-1 index was up to 15%. The FD and osteosarcoma were negative for CDK4. Fluorescent in situ hybridization assay showed no amplification of the MDM2 gene, indicating that the osteosarcoma was a conventional osteosarcoma, not an intraosseous well-differentiated type. The original cell of malignancy in FD is unclear. Malignancy can be potentially derived from dysplastic cells in the area of the FD or cells in the adjacent normal tissues. GNAS gene mutation has recently been reported for fibrous dysplasia and the mutation is highly specific to fibrous dysplasia among fibro-osseous lesions including osteosarcoma. In this case, point mutations of GNAS were found in the FD and osteosarcoma but not in the adjacent normal tissues, suggesting that osteosarcoma was derived from the spindle cells of FD. This is the first report to clearly show that osteosarcoma is derived from the spindle cells in fibrous dysplasia (FD).

Digital object identifier (DOI): 10.1016/j.prp.2017.10.018

Cancers, 10
2018

Establishment and Characterization of a Reliable Xenograft Model of Hodgkin Lymphoma Suitable for the Study of Tumor Origin and the Design of New Therapies.

M'kacher, Radhia, Frenzel, Monika, Al Jawhari, Mustafa, Junker, Steffen, Cuceu, Corina, Morat, Luc, Bauchet, Anne-Laure, Stimmer, Lev, Lenain, Aude, Dechamps, Nathalie, Hempel, William M, Pottier, Geraldine, Heidingsfelder, Leonhard, Laplagne, Eric, Borie, Claire, Oudrhiri, Noufissa, Jouni, Dima, Bennaceur-Griscelli, Annelise, Colicchio, Bruno, Dieterlen, Alain, Girinsky, Theodore, Boisgard, Raphael, Bourhis, Jean, Bosq, Jacques, Mehrling, Thomas, Jeandidier, Eric, Carde, Patrice

To identify the cells responsible for the initiation and maintenance of Hodgkin lymphoma (HL) cells, we have characterized a subpopulation of HL cells grown in vitro and in vivo with the aim of establishing a reliable and robust animal model for HL. To validate our model, we challenged the tumor cells in vivo by injecting the alkylating histone-deacetylase inhibitor, EDO-S101, a salvage regimen for HL patients, into xenografted mice. Blood lymphocytes from 50 HL patients and seven HL cell lines were used. Immunohistochemistry, flow cytometry, and cytogenetics analyses were performed. The in vitro and in vivo effects of EDO-S101 were assessed. We have successfully determined conditions for in vitro amplification and characterization of the HL L428-c subline, containing a higher proportion of CD30-/CD15- cells than the parental L428 cell line. This subline displayed excellent clonogenic potential and reliable reproducibility upon xenografting into immunodeficient NOD-SCID-gamma (-/-)(NSG) mice. Using cell sorting, we demonstrate that CD30-/CD15- subpopulations can gain the phenotype of the L428-c cell line in vitro. Moreover, the human cells recovered from the seventh week after injection of L428-c cells into NSG mice were small cells characterized by a high frequency of CD30-/CD15- cells. Cytogenetic analysis demonstrated that they were diploid and showed high telomere instability and telomerase activity. Accordingly, chromosomal instability emerged, as shown by the formation of dicentric chromosomes, ring chromosomes, and breakage/fusion/bridge cycles. Similarly, high telomerase activity and telomere instability were detected in circulating lymphocytes from HL patients. The beneficial effect of the histone-deacetylase inhibitor EDO-S101 as an anti-tumor drug validated our animal model. Our HL animal model requires only 10³ cells and is characterized by a high survival/toxicity ratio and high reproducibility. Moreover, the cells that engraft in mice are characterized by a high frequency of small CD30-/CD15- cells exhibiting high telomerase activity and telomere dysfunction.

Digital object identifier (DOI): 10.3390/cancers10110414

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