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Journal of nuclear medicine : official publication, Society of Nuclear Medicine, 57, 173–179
February, 2016

DNA Damage in Peripheral Blood Lymphocytes of Thyroid Cancer Patients After Radioiodine Therapy.

Eberlein, Uta, Scherthan, Harry, Bluemel, Christina, Peper, Michel, Lapa, Constantin, Buck, Andreas Konrad, Port, Matthias, Lassmann, Michael

The aim of the study was to investigate DNA double-strand break (DSB) formation and its correlation to the absorbed dose to the blood in patients with surgically treated differentiated thyroid cancer undergoing their first radioiodine therapy for remnant ablation. Twenty patients were included in this study. At least 7 peripheral blood samples were obtained before and between 0.5 and 120 h after administration of radioiodine. From the time-activity curves of the blood and the whole body, residence times for the blood self-irradiation and the irradiation from the whole body were determined. Peripheral blood lymphocytes were isolated, ethanol-fixed, and subjected to immunofluorescence staining for colocalizing γ-H2AX/53BP1 DSB-marking foci. The average number of DSB foci per cell per patient sample was analyzed as a function of the absorbed dose to the blood and compared with an in vitro calibration curve for (131)I and (177)Lu established previously in our institution. The average number of radiation-induced foci (RIF) per cell increased over the first 3 h after radionuclide administration and decreased thereafter. A linear fit from 0 to 2 h as a function of the absorbed dose to the blood agreed with our in vitro calibration curve. At later time points, RIF numbers diminished, along with dropping dose rates, indicating progression of DNA repair. Individual patient data were characterized by a linear dose-dependent increase and a biexponential response function describing a fast and a slow repair component. With the experimental results and model calculations presented in this work, a dose-response relationship is demonstrated, and an analytic function describing the time course of the in vivo damage response after internal irradiation of patients with (131)I is established.

Digital object identifier (DOI): 10.2967/jnumed.115.164814

Mol Med Rep, 13(1), 130–136
January, 2016

Chromosomal radiosensitivity of human immunodeficiency virus positive/negative cervical cancer patients in South Africa.

Herd, Olivia, Francies, Flavia, Kotzen, Jeffrey, Smith, Trudy, Nxumalo, Zwide, Muller, Xanthene, Slabbert, Jacobus, Vral, Anne, Baeyens, Ans

Cervical cancer is the second most common cancer amongst South African women and is the leading cause of cancer-associated mortality in this region. Several international studies on radiation‑induced DNA damage in lymphocytes of cervical cancer patients have remained inconclusive. Despite the high incidence of cervical cancer in South Africa, and the extensive use of radiotherapy to treat it, the chromosomal radiosensitivity of South African cervical cancer patients has not been studied to date. Since a high number of these patients are human immunodeficiency virus (HIV)‑positive, the effect of HIV infection on chromosomal radiosensitivity was also investigated. Blood samples from 35 cervical cancer patients (20 HIV‑negative and 15 HIV‑positive) and 20 healthy controls were exposed to X‑rays at doses of 6 MV of 2 and 4 Gy in vitro. Chromosomal radiosensitivity was assessed using the micronucleus (MN) assay. MN scores were obtained using the Metafer 4 platform, an automated microscopic system. Three scoring methods of the MNScore module of Metafer were applied and compared. Cervical cancer patients had higher MN values than healthy controls, with HIV‑positive patients having the highest MN values. Differences between groups were significant when using a scoring method that corrects for false positive and false negative MN. The present study suggested increased chromosomal radiosensitivity in HIV-positive South African cervical cancer patients.

Digital object identifier (DOI): 10.3892/mmr.2015.4504

Gastroenterol Res Pract, 2016, 6089658

The Role of Chromosomal Instability and Epigenetics in Colorectal Cancers Lacking beta-Catenin/TCF Regulated Transcription.

Abdel-Rahman, Wael M., Lotsari-Salomaa, Johanna E., Kaur, Sippy, Niskakoski, Anni, Knuutila, Sakari, Järvinen, Heikki, Mecklin, Jukka-Pekka, Peltomäki, Päivi

All colorectal cancer cell lines except RKO displayed active β-catenin/TCF regulated transcription. This feature of RKO was noted in familial colon cancers; hence our aim was to dissect its carcinogenic mechanism. MFISH and CGH revealed distinct instability of chromosome structure in RKO. Gene expression microarray of RKO versus 7 colon cancer lines (with active Wnt signaling) and 3 normal specimens revealed 611 differentially expressed genes. The majority of the tested gene loci were susceptible to LOH in primary tumors with various β-catenin localizations as a surrogate marker for β-catenin activation. The immunohistochemistry of selected genes (IFI16, RGS4, MCTP1, DGKI, OBCAM/OPCML, and GLIPR1) confirmed that they were differentially expressed in clinical specimens. Since epigenetic mechanisms can contribute to expression changes, selected target genes were evaluated for promoter methylation in patient specimens from sporadic and hereditary colorectal cancers. CMTM3, DGKI, and OPCML were frequently hypermethylated in both groups, whereas KLK10, EPCAM, and DLC1 displayed subgroup specificity. The overall fraction of hypermethylated genes was higher in tumors with membranous β-catenin. We identified novel genes in colorectal carcinogenesis that might be useful in personalized tumor profiling. Tumors with inactive Wnt signaling are a heterogeneous group displaying interaction of chromosomal instability, Wnt signaling, and epigenetics.

Digital object identifier (DOI): 10.1155/2016/6089658

Sci Rep, 6, 32510

Replication Timing of Human Telomeres is Conserved during Immortalization and Influenced by Respective Subtelomeres.

Piqueret-Stephan, Laure, Ricoul, Michelle, Hempel, William M., Sabatier, Laure

Telomeres are specific structures that protect chromosome ends and act as a biological clock, preventing normal cells from replicating indefinitely. Mammalian telomeres are replicated throughout S-phase in a predetermined order. However, the mechanism of this regulation is still unknown. We wished to investigate this phenomenon under physiological conditions in a changing environment, such as the immortalization process to better understand the mechanism for its control. We thus examined the timing of human telomere replication in normal and SV40 immortalized cells, which are cytogenetically very similar to cancer cells. We found that the timing of telomere replication was globally conserved under different conditions during the immortalization process. The timing of telomere replication was conserved despite changes in telomere length due to endogenous telomerase reactivation, in duplicated homologous chromosomes, and in rearranged chromosomes. Importantly, translocated telomeres, possessing their initial subtelomere, retained the replication timing of their homolog, independently of the proportion of the translocated arm, even when the remaining flanking DNA is restricted to its subtelomere, the closest chromosome-specific sequences (inferior to 500 kb). Our observations support the notion that subtelomere regions strongly influence the replication timing of the associated telomere.

Digital object identifier (DOI): 10.1038/srep32510

Molecular cytogenetics, 9, 90

Inherent variability of cancer-specific aneuploidy generates metastases

Bloomfield, Mathew, Duesberg, Peter

<p>The genetic basis of metastasis is still unclear because metastases carry individual karyotypes and phenotypes, rather than consistent mutations, and are rare compared to conventional mutation. There is however correlative evidence that metastasis depends on cancer-specific aneuploidy, and that metastases are karyotypically related to parental cancers. Accordingly we propose that metastasis is a speciation event. This theory holds that cancer-specific aneuploidy varies the clonal karyotypes of cancers automatically by unbalancing thousands of genes, and that rare variants form new autonomous subspecies with metastatic or other non-parental phenotypes like drug-resistance - similar to conventional subspeciation. To test this theory, we analyzed the karyotypic and morphological relationships between seven cancers and corresponding metastases. We found (1) that the cellular phenotypes of metastases were closely related to those of parental cancers, (2) that metastases shared 29 to 96% of their clonal karyotypic elements or aneusomies with the clonal karyotypes of parental cancers and (3) that, unexpectedly, the karyotypic complexity of metastases was very similar to that of the parental cancer. This suggests that metastases derive cancer-specific autonomy by conserving the overall complexity of the parental karyotype. We deduced from these results that cancers cause metastases by karyotypic variations and selection for rare metastatic subspecies. Further we asked whether metastases with multiple metastasis-specific aneusomies are assembled in one or multiple, sequential steps. Since (1) no stable karyotypic intermediates of metastases were observed in cancers here and previously by others, and (2) the karyotypic complexities of cancers are conserved in metastases, we concluded that metastases are generated from cancers in one step - like subspecies in conventional speciation. We conclude that the risk of cancers to metastasize is proportional to the degree of cancer-specific aneuploidy, because aneuploidy catalyzes the generation of subspecies, including metastases, at aneuploidy-dependent rates. Since speciation by random chromosomal rearrangements and selection is unpredictable, the theory that metastases are karyotypic subspecies of cancers also explains Foulds' rules, which hold that the origins of metastases are "abrupt" and that their phenotypes are "unpredictable."</p>

Digital object identifier (DOI): 10.1186/s13039-016-0297-x

Public Health England. Part of: Radiation: PHE-CRCE report series.

Doses in Radiation Accidents Investigated by Chromosomal Aberration Analysis XXV. Review of cases investigated, 2006-2015.

M Sun, J E Moquet, S Barnard, D C Lloyd, K Rothkamm, E A Ainsbury

<p>During the period 73 people suspected of being overexposed to ionising radiation were referred to Public Health England (and its predecessor the Health Protection Agency) for biological dosimetry. Although the vast majority of cases were suspected occupational overexposures, the most serious case concerned a 2-year-old boy who sustained radiation burns during CT scans performed outside the European Union, which were incorrectly repeated numerous times. The cases included in this summary bring the total number of individuals examined since the laboratory was established in 1968 to 1092. A number of new biological dosimetry techniques have been developed within the last 10 years. These are briefly summarised in this report and represent a large improvement in the laboratory’s ability both to perform accurate routine biological dose estimations and to provide rapid response triage dose estimates following a mass casualty event.</p>

J Med Case Rep, 10, 203

Acute promyelocytic leukemia with the translocation t(15;17)(q22;q21) associated with t(1;2)(q42~43;q11.2~12): a case report.

Wafa, Abdulsamad, Moassass, Faten, Liehr, Thomas, Al-Ablog, Ayman, Al-Achkar, Walid

<p>Acute promyelocytic leukemia is characterized by a typical reciprocal translocation t(15;17)(q22;q21). Additional chromosomal abnormalities are reported in only 23-43 % of cases of acute promyelocytic leukemia.Here we report the case of a 46-year-old Syrian Alawis woman with acute promyelocytic leukemia with the typical t(15;17) translocation, but with a second clone presenting a t(1;2)(q42~43;q11.2~12) translocation as an additional abnormality. To the best of our knowledge, an association between these chromosomal abnormalities has not previously been described in the literature. Our patient started treatment with all-trans retinoic acid 10 days after diagnosis but died the same day of treatment initiation due to hemolysis, intracranial hemorrhage, thrombocytopenia, and disseminated intravascular coagulation.The here reported combination of aberrations in a case of acute promyelocytic leukemia seems to indicate an adverse prognosis, and possibly shows that all-trans retinoic acid treatment may be contraindicated in such cases.</p>

Digital object identifier (DOI): 10.1186/s13256-016-0982-8

Comp Cytogenet, 10(1), 45–59

Sex chromosome diversity in Armenian toad grasshoppers (Orthoptera, Acridoidea, Pamphagidae).

Bugrov, Alexander G., Jetybayev, Ilyas E., Karagyan, Gayane H., Rubtsov, Nicolay B.

Although previous cytogenetic analysis of Pamphagidae grasshoppers pointed to considerable karyotype uniformity among most of the species in the family, our study of species from Armenia has discovered other, previously unknown karyotypes, differing from the standard for Pamphagidae mainly in having unusual sets of sex chromosomes. Asiotmethis turritus (Fischer von Waldheim, 1833), Paranocaracris rubripes (Fischer von Waldheim, 1846), and Nocaracris cyanipes (Fischer von Waldheim, 1846) were found to have the karyotype 2n♂=16+neo-XY and 2n♀=16+neo-XX, the neo-X chromosome being the result of centromeric fusion of an ancient acrocentric X chromosome and a large acrocentric autosome. The karyotype of Paranothrotes opacus (Brunner von Wattenwyl, 1882) was found to be 2n♂=14+X1X2Y and 2n♀=14+X1X1X2X2., the result of an additional chromosome rearrangement involving translocation of the neo-Y and another large autosome. Furthermore, evolution of the sex chromosomes in these species has involved different variants of heterochromatinization and miniaturization of the neo-Y. The karyotype of Eremopeza festiva (Saussure, 1884), in turn, appeared to have the standard sex determination system described earlier for Pamphagidae grasshoppers, 2n♂=18+X0 and 2n♀=18+XX, but all the chromosomes of this species were found to have small second C-positive arms. Using fluorescent in situ hybridization (FISH) with 18S rDNA and telomeric (TTAGG)n DNA repeats to yield new data on the structural organization of chromosomes in the species studied, we found that for most of them, clusters of repeats homologous to 18S rDNA localize on two, three or four pairs of autosomes and on the X. In Eremopeza festiva, however, FISH with labelled 18S rDNA painted C-positive regions of all autosomes and the X chromosome; clusters of telomeric repeats localized primarily on the ends of the chromosome arms. Overall, we conclude that the different stages of neo-Y degradation revealed in the Pamphagidae species studied make the family a very promising and useful model for studying sex chromosome evolution.

Digital object identifier (DOI): 10.3897/CompCytogen.v10i1.6407

Genet Sel Evol, 48, 12

The second highest chromosome count among vertebrates is observed in cultured sturgeon and is associated with genome plasticity.

Havelka, Miloš, Bytyutskyy, Dmytro, Symonová, Radka, Ráb, Petr, Flaj\vshans, Martin

<p>One of the five basal actinopterygian lineages, the Chondrostei, including sturgeon, shovelnose, and paddlefish (Order Acipenseriformes) show extraordinary ploidy diversity associated with three rounds of lineage-specific whole-genome duplication, resulting in three levels of ploidy in sturgeon. Recently, incidence of spontaneous polyploidization has been reported among cultured sturgeon and it could have serious negative implications for the economics of sturgeon farming. We report the occurrence of seven spontaneous heptaploid (7n) Siberian sturgeon Acipenser baerii, which is a functional tetraploid species (4n) with ~245 chromosomes. Our aims were to assess ploidy level and chromosome number of the analysed specimens and to identify the possible mechanism that underlies the occurrence of spontaneous additional chromosome sets in their genome.Among 150 specimens resulting from the mating of a tetraploid (4n) A. baerii (~245 chromosomes) dam with a hexaploid (6n) A. baerii (~368 chromosomes) sire, 143 displayed a relative DNA content that corresponds to pentaploidy (5n) with an absolute DNA content of 8.98 ± 0.03 pg DNA per nucleus and nuclear area of 35.3 ± 4.3 μm(2) and seven specimens exhibited a relative DNA content that corresponds to heptaploidy (7n), with an absolute DNA content of 15.02 ± 0.04 pg DNA per nucleus and nuclear area of 48.4 ± 5.1 μm(2). Chromosome analyses confirmed a modal number of ~437 chromosomes in these heptaploid (7n) individuals. DNA genotyping of eight microsatellite loci followed by parental assignment confirmed spontaneous duplication of the maternal chromosome sets via retention of the second polar body in meiosis II as the mechanism for the formation of this unusual chromosome number and ploidy level in a functional tetraploid A. baerii.We report the second highest chromosome count among vertebrates in cultured sturgeon (~437) after the schizothoracine cyprinid Ptychobarbus dipogon with ~446 chromosomes. The finding also represents the highest documented chromosome count in Acipenseriformes, and the first report of a functional heptaploid (7n) genome composition in sturgeon. To our knowledge, this study provides the first clear evidence of a maternal origin for spontaneous polyploidization in cultured A. baerii. To date, all available data indicate that spontaneous polyploidization occurs frequently among cultured sturgeons.</p>

Digital object identifier (DOI): 10.1186/s12711-016-0194-0

Atom Indonesia, 42(2), 71-77

Comparison of Radiosensitivity of Human Chromosomes 1, 2 and 4 from One Healthy Donor

Ramadhani, Purnami, Yoshida

In general, it was assumed that the chromosome aberration induced by ionizing radiation is proportional to the chromosome size. From this viewpoint, the higher chromosome size, the more resistant to radiation. However, different opinions, in which chromosomes are particularly sensitive or resistant to radiation, are also still followed until now. Here in this research, we compared the chromosome sensitivity between chromosomes number 1, 2, and 4 using the FISH (fluorescence in situ hybridization) technique. From this research, we expect that the information obtained could show clearly whether a longer chromosome is more frequently involved in translocations and also more resistant to radiation than a shorter one. The type of chromosome aberration considered was limited only to translocation and we used one sample donor in order to avoid donor variability. The whole blood from a healthy female was irradiated with γ-rays with doses of 1, 3 and 5 Gy, respectively. Isolated lymphocytes from the whole blood were then cultured for 48 hours. After the culture process was completed, preparations of harvest and metaphase chromosomes were carried out. Chromosomes 1, 2, and 4 were stained with different fluorochromes. The translocation of each chromosome at each dose point was subsequently evaluated from 50 images obtained from an automated metaphase finder and capturing system. An additional analysis was performed to identify which chromosome arm was more frequently involved in translocation. Further analyses were also conducted with the aim of determining which chromosome band had a higher frequency of radiation-induced breakage. The experimental results showed that chromosome number 4 was more frequently involved in translocations compared to chromosomes 1 and 2 at 5 Gy. In contrast, at doses of 1 and 3 Gy translocations involving chromosomes number 1 and 2 were more numerous compared to the ones involving chromosome 4. However, if the number of translocation was accumulated for all the doses applied, the chromosome number 4 was the chromosome most frequently involved in translocations. Breakpoint analysis revealed that in chromosome 1, chromosome 2, and chromosome 4, the highest chromosome bands as break position were in band q32, p13, and q21, respectively. It can be concluded that chromosome 4 is more sensitive to radiation in all doses point, despite having less DNA content than chromosomes 1 and 2. Thus, it was showed that our research cannot support the general assumption about chromosome aberration induced by radiation being proportional to DNA content.

PLoS One, 11(8), e0161369

Tumor Touch Imprints as Source for Whole Genome Analysis of Neuroblastoma Tumors.

Brunner, Clemens, Brunner-Herglotz, Bettina, Ziegler, Andrea, Frech, Christian, Amann, Gabriele, Ladenstein, Ruth, Ambros, Inge M., Ambros, Peter F.

Tumor touch imprints (TTIs) are routinely used for the molecular diagnosis of neuroblastomas by interphase fluorescence in-situ hybridization (I-FISH). However, in order to facilitate a comprehensive, up-to-date molecular diagnosis of neuroblastomas and to identify new markers to refine risk and therapy stratification methods, whole genome approaches are needed. We examined the applicability of an ultra-high density SNP array platform that identifies copy number changes of varying sizes down to a few exons for the detection of genomic changes in tumor DNA extracted from TTIs.DNAs were extracted from TTIs of 46 neuroblastoma and 4 other pediatric tumors. The DNAs were analyzed on the Cytoscan HD SNP array platform to evaluate numerical and structural genomic aberrations. The quality of the data obtained from TTIs was compared to that from randomly chosen fresh or fresh frozen solid tumors (n = 212) and I-FISH validation was performed.SNP array profiles were obtained from 48 (out of 50) TTI DNAs of which 47 showed genomic aberrations. The high marker density allowed for single gene analysis, e.g. loss of nine exons in the ATRX gene and the visualization of chromothripsis. Data quality was comparable to fresh or fresh frozen tumor SNP profiles. SNP array results were confirmed by I-FISH.TTIs are an excellent source for SNP array processing with the advantage of simple handling, distribution and storage of tumor tissue on glass slides. The minimal amount of tumor tissue needed to analyze whole genomes makes TTIs an economic surrogate source in the molecular diagnostic work up of tumor samples.

Digital object identifier (DOI): 10.1371/journal.pone.0161369

Front Microbiol, 7, 739

Backup Expression of the PhaP2 Phasin Compensates for phaP1 Deletion in Herbaspirillum seropedicae, Maintaining Fitness and PHB Accumulation.

Alves, Luis P S., Teixeira, Cícero S., Tirapelle, Evandro F., Donatti, Lucélia, Tadra-Sfeir, Michelle Z., Steffens, Maria B R., de Souza, Emanuel M., de Oliveira Pedrosa, Fabio, Chubatsu, Leda S., Müller-Santos, Marcelo

<p>Phasins are important proteins controlling poly-3-hydroxybutyrate (PHB) granules formation, their number into the cell and stability. The genome sequencing of the endophytic and diazotrophic bacterium Herbaspirillum seropedicae SmR1 revealed two homologous phasin genes. To verify the role of the phasins on PHB accumulation in the parental strain H. seropedicae SmR1, isogenic strains defective in the expression of phaP1, phaP2 or both genes were obtained by gene deletion and characterized in this work. Despite of the high sequence similarity between PhaP1 and PhaP2, PhaP1 is the major phasin in H. seropedicae, since its deletion reduced PHB accumulation by ≈50 % in comparison to the parental and ΔphaP2. Upon deletion of phaP1, the expression of phaP2 was sixfold enhanced in the ΔphaP1 strain. The responsive backup expression of phaP2 partially rescued the ΔphaP1 mutant, maintaining about 50 % of the parental PHB level. The double mutant ΔphaP1.2 did not accumulate PHB in any growth stage and showed a severe reduction of growth when glucose was the carbon source, a clear demonstration of negative impact in the fitness. The co-occurrence of phaP1 and phaP2 homologous in bacteria relatives of H. seropedicae, including other endophytes, indicates that the mechanism of phasin compensation by phaP2 expression may be operating in other organisms, showing that PHB metabolism is a key factor to adaptation and efficiency of endophytic bacteria.</p>

Digital object identifier (DOI): 10.3389/fmicb.2016.00739

Front Neurol, 7, 23

Lesion Size Is Exacerbated in Hypoxic Rats Whereas Hypoxia-Inducible Factor-1 Alpha and Vascular Endothelial Growth Factor Increase in Injured Normoxic Rats: A Prospective Cohort Study of Secondary Hypoxia in Focal Traumatic Brain Injury.

Thelin, Eric Peter, Frostell, Arvid, Mulder, Jan, Mitsios, Nicholas, Damberg, Peter, Aski, Sahar Nikkhou, Risling, M\aarten, Svensson, Mikael, Morganti-Kossmann, Maria Cristina, Bellander, Bo-Michael

Hypoxia following traumatic brain injury (TBI) is a severe insult shown to exacerbate the pathophysiology, resulting in worse outcome. The aim of this study was to investigate the effects of a hypoxic insult in a focal TBI model by monitoring brain edema, lesion volume, serum biomarker levels, immune cell infiltration, as well as the expression of hypoxia-inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF).Female Sprague-Dawley rats (n = 73, including sham and naive) were used. The rats were intubated and mechanically ventilated. A controlled cortical impact device created a 3-mm deep lesion in the right parietal hemisphere. Post-injury, rats inhaled either normoxic (22\% O2) or hypoxic (11\% O2) mixtures for 30 min. The rats were sacrificed at 1, 3, 7, 14, and 28 days post-injury. Serum was collected for S100B measurements using ELISA. Ex vivo magnetic resonance imaging (MRI) was performed to determine lesion size and edema volume. Immunofluorescence was employed to analyze neuronal death, changes in cerebral macrophage- and neutrophil infiltration, microglia proliferation, apoptosis, complement activation (C5b9), IgG extravasation, HIF-1α, and VEGF.The hypoxic group had significantly increased blood levels of lactate and decreased pO2 (p < 0.0001). On MRI post-traumatic hypoxia resulted in larger lesion areas (p = 0.0173), and NeuN staining revealed greater neuronal loss (p = 0.0253). HIF-1α and VEGF expression was significantly increased in normoxic but not in hypoxic animals (p < 0.05). A trend was seen for serum levels of S100B to be higher in the hypoxic group at 1 day after trauma (p = 0.0868). No differences were observed between the groups in cytotoxic and vascular edema, IgG extravasation, neutrophils and macrophage aggregation, microglia proliferation, or C5b-9 expression.Hypoxia following focal TBI exacerbated the lesion size and neuronal loss. Moreover, there was a tendency to higher levels of S100B in the hypoxic group early after injury, indicating a potential validity as a biomarker of injury severity. In the normoxic group, the expression of HIF-1α and VEGF was found elevated, possibly indicative of neuro-protective responses occurring in this less severely injured group. Further studies are warranted to better define the pathophysiology of post-TBI hypoxia.

Digital object identifier (DOI): 10.3389/fneur.2016.00023

Nat Commun, 7, 10529

RAG2 and XLF/Cernunnos interplay reveals a novel role for the RAG complex in DNA repair.

Lescale, Chloé, Abramowski, Vincent, Bedora-Faure, Marie, Murigneux, Valentine, Vera, Gabriella, Roth, David B., Revy, Patrick, de Villartay, Jean-Pierre, Deriano, Ludovic

XRCC4-like factor (XLF) functions in classical non-homologous end-joining (cNHEJ) but is dispensable for the repair of DNA double-strand breaks (DSBs) generated during V(D)J recombination. A long-standing hypothesis proposes that, in addition to its canonical nuclease activity, the RAG1/2 proteins participate in the DNA repair phase of V(D)J recombination. Here we show that in the context of RAG2 lacking the C-terminus domain (Rag2(c/c) mice), XLF deficiency leads to a profound lymphopenia associated with a severe defect in V(D)J recombination and, in the absence of p53, increased genomic instability at V(D)J sites. In addition, Rag2(c/c) XLF(-/-) p53(-/-) mice develop aggressive pro-B cell lymphomas bearing complex chromosomal translocations and gene amplifications involving Igh and c-myc/pvt1 loci. Our results reveal an unanticipated functional interplay between the RAG complex and XLF in repairing RAG-induced DSBs and maintaining genome integrity during antigen receptor gene assembly.

Digital object identifier (DOI): 10.1038/ncomms10529

Sci Rep, 6, 25658

Trafficking of Endogenous Immunoglobulins by Endothelial Cells at the Blood-Brain Barrier.

Villasenor, Roberto, Ozmen, Laurence, Messaddeq, Nadia, Grüninger, Fiona, Loetscher, Hansruedi, Keller, Annika, Betsholtz, Christer, Freskg\aard, Per-Ola, Collin, Ludovic

The Blood-Brain Barrier (BBB) restricts access of large molecules to the brain. The low endocytic activity of brain endothelial cells (BECs) is believed to limit delivery of immunoglobulins (IgG) to the brain parenchyma. Here, we report that endogenous mouse IgG are localized within intracellular vesicles at steady state in BECs in vivo. Using high-resolution quantitative microscopy, we found a fraction of endocytosed IgG in lysosomes. We observed that loss of pericytes (key components of the BBB) in pdgf-b(ret/ret) mice affects the intracellular distribution of endogenous mouse IgG in BECs. In these mice, endogenous IgG was not detected within lysosomes but instead accumulate at the basement membrane and brain parenchyma. Such IgG accumulation could be due to reduced lysosomal clearance and increased sorting to the abluminal membrane of BECs. Our results suggest that, in addition to low uptake from circulation, IgG lysosomal degradation may be a downstream mechanism by which BECs further restrict IgG access to the brain.

Digital object identifier (DOI): 10.1038/srep25658

Mol Cytogenet, 9, 38

Rare case of Killian-Pallister syndrome associated with idiopathic short stature detected with fluorescent in situ hybridization on buccal smear.

Sukarova-Angelovska, Elena, Kocova, Mirjana, Ilieva, Gordana, Angelkova, Natalija, Kochova, Elena

Killian-Pallister syndrome (KPS) is a rare form of chromosomal mosaicism and is defined by the existence of an extra chromosome 12 in some cell lines in one individual. The degree of mosaicism varies among tissues and dictates the clinical presentation of the syndrome. The clinical features of Killian-Pallister syndrome include mental retardation, typical facial dysmorphism and pigmentation defects.We present a rare case of Killian-Pallister syndrome with severe form of the disease associated with isolated growth hormone deficiency and low-rate mosaicism on buccal smear. The absence of a marker chromosome 12p in lymphocyte cultures and the low degree of mosaicism lead to frequent misdiagnosis of this condition.The selection of tissue sampling is crucial in establishing the diagnosis of Killian-Pallister syndrome. Fluorescent in situ hybridisation on buccal smear remains the golden standard as a screening method if a suspicion of the syndrome exists.

Digital object identifier (DOI): 10.1186/s13039-016-0239-7

Public Health England. Part of: Radiation: PHE-CRCE report series.

Doses in Radiation Accidents Investigated by Chromosomal Aberration Analysis XXV. Review of cases investigated, 2006–2015.

M Sun, J E Moquet, S Barnard, D C Lloyd, K Rothkamm, E A Ainsbury

During the period 73 people suspected of being overexposed to ionising radiation were referred to Public Health England (and its predecessor the Health Protection Agency) for biological dosimetry. Although the vast majority of cases were suspected occupational overexposures, the most serious case concerned a 2-year-old boy who sustained radiation burns during CT scans performed outside the European Union, which were incorrectly repeated numerous times. The cases included in this summary bring the total number of individuals examined since the laboratory was established in 1968 to 1092. A number of new biological dosimetry techniques have been developed within the last 10 years. These are briefly summarised in this report and represent a large improvement in the laboratory’s ability both to perform accurate routine biological dose estimations and to provide rapid response triage dose estimates following a mass casualty event.

Dental materials : official publication of the Academy of Dental Materials, 31, 1335–1344
November, 2015

Dental composite components induce DNA-damage and altered nuclear morphology in gingiva fibroblasts.

Styllou, Marianthi, Reichl, Franz-Xaver, Styllou, Panorea, Urcan, Ebru, Rothmund, Lena, Hickel, Reinhard, Högg, Christof, Scherthan, Harry

<p>Released dental composite components can damage human gingival fibroblasts (HGFs) and their DNA. The cytotoxicity, chromatin condensation and the induction of DNA double strand breaks (DSBs) by different compounds of dental composites was investigated using an improved γ-H2AX focus assay. HGFs were incubated with the monomers: bisphenol-A-ethoxylate-dimethacrylate (Bis-DMA), bisphenol-A-glycerolate-dimethacrylate (BisGMA), ethyltriethylen glycol methacrylate (ETEGMA), glycidyl methacrylate (GMA), 1,6-hexandiol-dimethycrylate (HDDMA), trimethylolpropane ethoxylate triacrylate (TMPTA), and acrylamide (ACR). DSBs were determined by enumerating γ-H2AX and 53BP1 foci colocalized at DSBs. A concentration-dependent induction of DSBs was found in the order: GMA&gt;BisGMA&gt;ACR&gt;Bis-DMA&gt;HDDMA&gt;TMPTA&gt;ETEGMA. HGFs exposure to GMA (0.3mM) and to BisGMA (0.09mM) induced the highest rate of DSB foci, i.e. 12-fold and 8-fold, respectively, relative to control (0.33 DSB foci/cell). At the highest concentrations (EC50) prominent changes in the chromatin morphology of HGF cell nuclei, i.e. compaction of nuclear chromatin and reduction of the area covered by the ovoid fibroblast nuclei, were observed. Nuclear condensation was significantly induced by GMA (1.7-fold at 0.3mM) and BisGMA (1.6-fold at 0.09mM), which correlated with the highest numbers of induced DSB foci (GMA, BisGMA, 3.9 and 2.6 foci/cell, respectively). The improved γ-H2AX/53BP1 focus assay revealed a concentration-dependent increase in DSBs for all tested substances. Furthermore, concentration-dependent changes in HGF cell nucleus morphology was noted, demonstrating genotoxic effects of the substances tested.</p>

Digital object identifier (DOI): 10.1016/

J Neurosci Methods, 247, 41–49
May, 2015

Assessing fibrinogen extravasation into Alzheimer's disease brain using high-content screening of brain tissue microarrays.

Narayan, Pritika J., Kim, Sue-Ling, Lill, Claire, Feng, Sheryl, Faull, Richard L M., Curtis, Maurice A., Dragunow, Michael

Tissue microarrays are commonly used to evaluate disease pathology however methods to automate and quantify pathological changes are limited.This article demonstrates the utility of the VSlide scanner (MetaSystems) for automated image acquisition from immunolabelled tissue microarray slides, and subsequent automated image analysis with MetaXpress (Molecular Devices) software to obtain objective, efficient and reproducible data from immunolabelled tissue microarray sections.Significant increases in fibrinogen immunolabelling were observed in 29 Alzheimer's disease cases compared to 28 control cases analysed from a single tissue microarray slide. Western blot analysis also demonstrated significant increases in fibrinogen immunolabelling in 6 Alzheimer's cases compared to 6 control cases. The observed changes were also validated with gold standard blinded manual H-scoring.VSlide Metafer software offers a 'tissue microarray acquisition' plugin for easy mapping of tissue cores with their original position on the tissue microarray map. High resolution VSlide images are compatible with MetaXpress image analysis software. This article details the coupling of these two technologies to accurately and reproducibly analyse immunolabelled tissue microarrays within minutes, compared to the gold standard method of manual counting using H-scores which is significantly slower and prone to inter-observer variation.Here, we couple brain tissue microarray technology with high-content screening and automated image analysis as a powerful way to address bottle necks in data generation and improve throughput, as well as sensitivity to study biological/pathological changes in brain disease.

Digital object identifier (DOI): 10.1016/j.jneumeth.2015.03.017

April, 2015

Sesamol attenuates genotoxicity in bone marrow cells of whole-body γ-irradiated mice

Arun Kumar, Tamizh G. Selvan, Akanchha M. Tripathi, Sandeep Choudhary, Shahanshah Khan, Jawahar S. Adhikari, Nabo K. Chaudhury

<p>Ionising radiation causes free radical-mediated damage in cellular DNA. This damage is manifested as chromosomal aberrations and micronuclei (MN) in proliferating cells. Sesamol, present in sesame seeds, has the potential to scavenge free radicals; therefore, it can reduce radiation-induced cytogenetic damage in cells. The aim of this study was to investigate the radioprotective potential of sesamol in bone marrow cells of mice and related haematopoietic system against radiation-induced genotoxicity. A comparative study with melatonin was designed for assessing the radioprotective potential of sesamol. C57BL/6 mice were administered intraperitoneally with either sesamol or melatonin (10 and 20mg/kg body weight) 30min prior to 2-Gy whole-body irradiation (WBI) and sacrificed after 24h. Total chromosomal aberrations (TCA), MN and cell cycle analyses were performed using bone marrow cells. The comet assay was performed on bone marrow cells, splenocytes and lymphocytes. Blood was drawn to study haematological parameters. Prophylactic doses of sesamol (10 and 20mg/kg) in irradiated mice reduced TCA and micronucleated polychromatic erythrocyte frequency in bone marrow cells by 57 % and 50 %, respectively, in comparison with radiation-only groups. Sesamol-reduced radiation-induced apoptosis and facilitated cell proliferation. In the comet assay, sesamol (20mg/kg) treatment reduced radiation-induced comets (% DNA in tail) compared with radiation only (P &lt; 0.05). Sesamol also increased granulocyte populations in peripheral blood similar to melatonin. Overall, the radioprotective efficacy of sesamol was found to be similar to that of melatonin. Sesamol treatment also showed recovery of relative spleen weight at 24h of WBI. The results strongly suggest the radioprotective efficacy of sesamol in the haematopoietic system of mice.</p>