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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

Cancers, 10
2018

Independent Mechanisms Lead to Genomic Instability in Hodgkin Lymphoma: Microsatellite or Chromosomal Instability , [email protected], .

Cuceu, Corina, Colicchio, Bruno, Jeandidier, Eric, Junker, Steffen, Plassa, François, Shim, Grace, Mika, Justyna, Frenzel, Monika, Al Jawhari, Mustafa, Hempel, William M, O'Brien, Grainne, Lenain, Aude, Morat, Luc, Girinsky, Theodore, Dieterlen, Alain, Polanska, Joanna, Badie, Christophe, Carde, Patrice, M'Kacher, Radhia

: Microsatellite and chromosomal instability have been investigated in Hodgkin lymphoma (HL). : We studied seven HL cell lines (five Nodular Sclerosis (NS) and two Mixed Cellularity (MC)) and patient peripheral blood lymphocytes (100 NS-HL and 23 MC-HL). Microsatellite instability (MSI) was assessed by PCR. Chromosomal instability and telomere dysfunction were investigated by FISH. DNA repair mechanisms were studied by transcriptomic and molecular approaches. : In the cell lines, we observed high MSI in L428 (4/5), KMH2, and HDLM2 (3/5), low MSI in L540, L591, and SUP-HD1, and none in L1236. NS-HL cell lines showed telomere shortening, associated with alterations of nuclear shape. Small cells were characterized by telomere loss and deletion, leading to chromosomal fusion, large nucleoplasmic bridges, and breakage/fusion/bridge (B/F/B) cycles, leading to chromosomal instability. The MC-HL cell lines showed substantial heterogeneity of telomere length. Intrachromosmal double strand breaks induced dicentric chromosome formation, high levels of micronucleus formation, and small nucleoplasmic bridges. B/F/B cycles induced complex chromosomal rearrangements. We observed a similar pattern in circulating lymphocytes of NS-HL and MC-HL patients. Transcriptome analysis confirmed the differences in the DNA repair pathways between the NS and MC cell lines. In addition, the NS-HL cell lines were radiosensitive and the MC-cell lines resistant to apoptosis after radiation exposure. : In mononuclear NS-HL cells, loss of telomere integrity may present the first step in the ongoing process of chromosomal instability. Here, we identified, MSI as an additional mechanism for genomic instability in HL.

Digital object identifier (DOI): 10.3390/cancers10070233

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

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

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

Revista brasileira de ortopedia, 53, 293--299
2018

Intra-articular viscosupplementation of hyaluronic acids in an experimental osteoarthritis model.

Oliveira, Marcello Zaia, Albano, Mauro Batista, Stirma, Guilherme Augusto, Namba, Mario Massatomo, Vidigal, Leandro, Cunha, Luiz Antonio Munhoz da

To analyze, from the immunohistochemical perspective, the effects of hyaluronic acid of different molecular weights in an experimental model of osteoarthritis in rabbits. Forty-four male California rabbits were randomly assigned to three different groups (PR, S, and P) and submitted to the resection of the anterior cruciate ligament of the right knee. Three weeks after the surgical procedure, three intra-articular weekly injections were carried out with low-molecular-weight native hyaluronic acid (Hyalgan ) to PR group, high molecular weight branched chain hyaluronic acid (Synvisc ) to group S, and saline solution 0.9% to group P. All animals were sacrificed 12 weeks after the surgical procedure, and the tibial plateaus of the infiltrated knees were then dissected. Histological sections of cartilage from the tibial plateau support areas were stained with immunohistochemical markers in order to investigate the amount of metalloproteases (MMPs 3 and 13) and their inhibitors (TIMPs 1 and 3). The staining intensity was quantified on a Zeiss Imager.Z2 Metasystems microscope and analyzed by Metafer4 Msearch software. The chondroprotective effect of the hyaluronic acids used in the study was demonstrated when compared to the control group. However, the comparison between them presented no significant statistical difference regarding chondroprotection. The injection of saline solution demonstrated signs of OA development, while adding native hyaluronic acid of low molecular weight (Hyalgan ) and hyaluronic acid of high molecular weight (Synvisc ) protected the articular cartilage in this model of OA.

Digital object identifier (DOI): 10.1016/j.rboe.2018.03.009

British journal of pharmacology
2018

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

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

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

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

Scientific reports, 8, 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

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 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

Journal of medical entomology, 55, 575--586
2018

Description of Larval Instars To Fill a Gap in Forensic Entomology: The Larvae of Paralucilia pseudolyrcea (Diptera: Calliphoridae).

Da Silva, S M, Vairo, K P, Moura, M O

A fundamental assumption of forensic entomology for estimating the postmortem interval is that insect species are accurately identified, which depends on diagnostic morphological characters. Larvae of the blow fly Paralucilia pseudolyrcea (Mello, 1969) (Diptera: Calliphoridae) were sampled from four corpses in the state of Paraná, Brazil, but despite the forensic importance of this species, morphological data for the identification of its larval instars are lacking, limiting its usefulness in such cases. Thus, the main goal of this study was to describe the larval instars of P. pseudolyrcea. The material was obtained from a colony established by larvae collected from a corpse of a murder case. Overall, the distribution of spines is a key character for identifying this species in the first, second and third instars. Other characteristics, such as the presence of an accessory oral sclerite, the small cirri, the number of lobes of the anterior spiracle and the morphology of posterior spiracles, separates P. pseudolyrcea from other necrophagous blow flies. The detailed morphological description provided here facilitates the identification of larval instars of P. pseudolyrcea and their differentiation from those of other calliphorid species.

Digital object identifier (DOI): 10.1093/jme/tjx257

Scientific Reports, 8(1), 1141
2018

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

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

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

Diagnostics (Basel, Switzerland), 8
2018

Aneuploid CTC and CEC.

Lin, Peter Ping

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

Digital object identifier (DOI): 10.3390/diagnostics8020026

Nature communications, 9, 1048
2018

Integrative genomic profiling of large-cell neuroendocrine carcinomas reveals distinct subtypes of high-grade neuroendocrine lung tumors.

George, Julie, Walter, Vonn, Peifer, Martin, Alexandrov, Ludmil B, Seidel, Danila, Leenders, Frauke, Maas, Lukas, Müller, Christian, Dahmen, Ilona, Delhomme, Tiffany M, Ardin, Maude, Leblay, Noemie, Byrnes, Graham, Sun, Ruping, De Reynies, Aurélien, McLeer-Florin, Anne, Bosco, Graziella, Malchers, Florian, Menon, Roopika, Altmüller, Janine, Becker, Christian, Nürnberg, Peter, Achter, Viktor, Lang, Ulrich, Schneider, Peter M, Bogus, Magdalena, Soloway, Matthew G, Wilkerson, Matthew D, Cun, Yupeng, McKay, James D, Moro-Sibilot, Denis, Brambilla, Christian G, Lantuejoul, Sylvie, Lemaitre, Nicolas, Soltermann, Alex, Weder, Walter, Tischler, Verena, Brustugun, Odd Terje, Lund-Iversen, Marius, Helland, Åslaug, Solberg, Steinar, Ansén, Sascha, Wright, Gavin, Solomon, Benjamin, Roz, Luca, Pastorino, Ugo, Petersen, Iver, Clement, Joachim H, Sänger, Jörg, Wolf, Jürgen, Vingron, Martin, Zander, Thomas, Perner, Sven, Travis, William D, Haas, Stefan A, Olivier, Magali, Foll, Matthieu, Büttner, Reinhard, Hayes, David Neil, Brambilla, Elisabeth, Fernandez-Cuesta, Lynnette, Thomas, Roman K

Pulmonary large-cell neuroendocrine carcinomas (LCNECs) have similarities with other lung cancers, but their precise relationship has remained unclear. Here we perform a comprehensive genomic (n = 60) and transcriptomic (n = 69) analysis of 75 LCNECs and identify two molecular subgroups: "type I LCNECs" with bi-allelic TP53 and STK11/KEAP1 alterations (37%), and "type II LCNECs" enriched for bi-allelic inactivation of TP53 and RB1 (42%). Despite sharing genomic alterations with adenocarcinomas and squamous cell carcinomas, no transcriptional relationship was found; instead LCNECs form distinct transcriptional subgroups with closest similarity to SCLC. While type I LCNECs and SCLCs exhibit a neuroendocrine profile with ASCL1 /DLL3 /NOTCH , type II LCNECs bear TP53 and RB1 alterations and differ from most SCLC tumors with reduced neuroendocrine markers, a pattern of ASCL1 /DLL3 /NOTCH , and an upregulation of immune-related pathways. In conclusion, LCNECs comprise two molecularly defined subgroups, and distinguishing them from SCLC may allow stratified targeted treatment of high-grade neuroendocrine lung tumors.

Digital object identifier (DOI): 10.1038/s41467-018-03099-x