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Molecular cell
2017

DNA Double-Strand Break Resection Occurs during Non-homologous End Joining in G1 but Is Distinct from Resection during Homologous Recombination.

Biehs, Ronja, Steinlage, Monika, Barton, Olivia, Juhász, Szilvia, Künzel, Julia, Spies, Julian, Shibata, Atsushi, Jeggo, Penny A, Löbrich, Markus

Canonical non-homologous end joining (c-NHEJ) repairs DNA double-strand breaks (DSBs) in G1 cells with biphasic kinetics. We show that DSBs repaired with slow kinetics, including those localizing to heterochromatic regions or harboring additional lesions at the DSB site, undergo resection prior to repair by c-NHEJ and not alt-NHEJ. Resection-dependent c-NHEJ represents an inducible process during which Plk3 phosphorylates CtIP, mediating its interaction with Brca1 and promoting the initiation of resection. Mre11 exonuclease, EXD2, and Exo1 execute resection, and Artemis endonuclease functions to complete the process. If resection does not commence, then repair can ensue by c-NHEJ, but when executed, Artemis is essential to complete resection-dependent c-NHEJ. Additionally, Mre11 endonuclease activity is dispensable for resection in G1. Thus, resection in G1 differs from the process in G2 that leads to homologous recombination. Resection-dependent c-NHEJ significantly contributes to the formation of deletions and translocations in G1, which represent important initiating events in carcinogenesis.

Digital object identifier (DOI): 10.1016/j.molcel.2016.12.016

Endocr Relat Cancer, 23(8), 635–650
August, 2016

Effect of low doses of estradiol and tamoxifen on breast cancer cell karyotypes.

Rondón-Lagos, Milena, Rangel, Nelson, Di Cantogno, Ludovica Verdun, Annaratone, Laura, Castellano, Isabella, Russo, Rosalia, Manetta, Tilde, Marchiò, Caterina, Sapino, Anna

Evidence supports a role of 17&-estradiol (E2) in carcinogenesis and the large majority of breast carcinomas are dependent on estrogen. The anti-estrogen tamoxifen (TAM) is widely used for both treatment and prevention of breast cancer; however, it is also carcinogenic in human uterus and rat liver, highlighting the profound complexity of its actions. The nature of E2- or TAM-induced chromosomal damage has been explored using relatively high concentrations of these agents, and only some numerical aberrations and chromosomal breaks have been analyzed. This study aimed to determine the effects of low doses of E2 and TAM (10(&8 )mol L(&1) and 10(&6 )mol L(&1) respectively) on karyotypes of MCF7, T47D, BT474, and SKBR3 breast cancer cells by comparing the results of conventional karyotyping and multi-FISH painting with cell proliferation. Estrogen receptor (ER)-positive (+) cells showed an increase in cell proliferation after E2 treatment (MCF7, T47D, and BT474) and a decrease after TAM treatment (MCF7 and T47D), whereas in ER& cells (SKBR3), no alterations in cell proliferation were observed, except for a small increase at 96 h. Karyotypes of both ER+ and ER& breast cancer cells increased in complexity after treatments with E2 and TAM leading to specific chromosomal abnormalities, some of which were consistent throughout the treatment duration. This genotoxic effect was higher in HER2+ cells. The ER&/HER2+ SKBR3 cells were found to be sensitive to TAM, exhibiting an increase in chromosomal aberrations. These in vitro results provide insights into the potential role of low doses of E2 and TAM in inducing chromosomal rearrangements in breast cancer cells.

Digital object identifier (DOI): 10.1530/ERC-16-0078

Mol Med Rep, 14(1), 103–110
July, 2016

A semi‑automated FISH‑based micronucleus‑centromere assay for biomonitoring of hospital workers exposed to low doses of ionizing radiation.

Vral, Anne, Decorte, Veerle, Depuydt, Julie, Wambersie, André, Thierens, Hubert

The aim of the present study was to perform cytogenetic analysis by means of a semi‑automated micronucleus‑centromere assay in lymphocytes from medical radiation workers. Two groups of workers receiving the highest occupational doses were selected: 10 nuclear medicine technicians and 10 interventional radiologists/cardiologists. Centromere‑negative micronucleus (MNCM‑) data, obtained from these two groups of medical radiation workers were compared with those obtained in matched controls. The blood samples of the matched controls were additionally used to construct a 'low‑dose' (0‑100 mGy) MNCM‑ dose‑response curve to evaluate the sensitivity and suitability of the micronucleus‑centromere assay as an 'effect' biomarker in medical surveillance programs. The physical dosimetry data of the 3 years preceding the blood sampling, based on single or double dosimetry practices, were collected for the interpretation of the micronucleus data. The in vitro radiation results showed that for small sized groups, semi‑automated scoring of MNCM‑ enables the detection of a dose of 50 mGy. The comparison of MNCM‑ yields in medical radiation workers and control individuals showed enhanced MNCM‑ scores in the medical radiation workers group (P=0.15). The highest MNCM‑ scores were obtained in the interventional radiologists/cardiologists group, and these scores were significantly higher compared with those obtained from the matched control group (P=0.05). The higher MNCM‑ scores observed in interventional radiologists/cardiologists compared with nuclear medicine technicians were not in agreement with the personal dosimetry records in both groups, which may point to the limitation of 'double dosimetry' procedures used in interventional radiology/cardiology. In conclusion, the data obtained in the present study supports the importance of cytogenetic analysis, in addition to physical dosimetry, as a routine biomonitoring method in medical radiation workers receiving the highest occupational radiation burdens.

Digital object identifier (DOI): 10.3892/mmr.2016.5265

Radiat Prot Dosimetry
July, 2016

A New Cytogenetic Biodosimetry Image Repository for the Dicentric Assay.

Romm, Horst, Beinke, Christina, Garcia, Omar, Di Giorgio, Marina, Gregoire, Eric, Livingston, Gordon, Lloyd, David, Martinez-Lopez, Wilner, Moquet, Jayne E., Sugarman, Stephen L., Wilkins, Ruth C., Ainsbury, Elizabeth A.

The BioDoseNet was founded by the World Health Organization as a global network of biodosimetry laboratories for building biodosimetry laboratory capacities in countries. The newly established BioDoseNet image repository is a databank of ~25 000 electronically captured images of metaphases from the dicentric assay, which have been previously analysed by international experts. The detailed scoring results and dose estimations have, in most cases, already been published. The compilation of these images into one image repository provides a valuable tool for training and research purposes in biological dosimetry. No special software is needed to view and score the image galleries. For those new to the dicentric assay, the BioDoseNet Image Repository provides an introduction to and training for the dicentric assay. It is an excellent instrument for intra-laboratory training purposes or inter-comparisons between laboratories, as recommended by the International Organization for Standardisation standards. In the event of a radiation accident, the repository can also increase the surge capacity and reduce the turnaround time for dose estimations. Finally, it provides a mechanism for the discussion of scoring discrepancies in difficult cases.

Digital object identifier (DOI): 10.1093/rpd/ncw158

Indian J Hematol Blood Transfus, 32(2), 154–161
June, 2016

Evaluation of ETV6/RUNX1 Fusion and Additional Abnormalities Involving ETV6 and/or RUNX1 Genes Using FISH Technique in Patients with Childhood Acute Lymphoblastic Leukemia.

Aydin, Cigdem, Cetin, Zafer, Manguoglu, Ayse Esra, Tayfun, Funda, Clark, Ozden Altiok, Kupesiz, Alphan, Akkaya, Bahar, Karauzum, Sibel Berker

Childhood acute lymphoblastic leukemia (ALL) is the most common type of childhood leukemia. Specifically, ALL is a malignant disorder of the lymphoid progenitor cells, with a peak incidence among children aged 2-5 years. The t(12;21)(p13;q22) translocation occurs in 25 \% of childhood B cell precursor ALL. In this study, bone marrow samples were obtained from 165 patients with childhood ALL. We analyzed the t(12;21) translocation and other related abnormalities using the fluorescent in situ hybridization (FISH) technique with the ETV6(TEL)/RUNX1(AML1) ES dual color translocation probe. Conventional cytogenetic analyses were also performed. ETV6 and RUNX1 related chromosomal abnormalities were found in 42 (25.5 \%) of the 165 patients with childhood ALL. Among these 42 patients, structural changes were detected in 33 (78.6 \%) and numerical abnormalities in 9 (21.4 \%). The frequency of FISH abnormalities in pediatric ALL cases were as follows: 8.5 \% for t(12;21)(p13;q22) ETV6/RUNX1 fusion, 6.0 \% for RUNX1 amplification, 3.0 \% for tetrasomy/trisomy 21, 1.8 \% for ETV6 deletion, 1.21 \% for ETV6 deletion with RUNX1 amplification, 1.21 \% for ETV6 amplification with RUNX1 amplification, 0.6 \% for polyploidy, 0.6 \% for RUNX1 deletion, and 0.6 \% for diminished ETV6 signal. The most common structural abnormality was the t(12;21) translocation, followed by RUNX1 amplification and ETV6 deletion, while the most commonly observed numerical abnormality was trisomy 21.

Digital object identifier (DOI): 10.1007/s12288-015-0557-7

Nucleic Acids Res
June, 2016

Chromosome thripsis by DNA double strand break clusters causes enhanced cell lethality, chromosomal translocations and 53BP1-recruitment.

Schipler, Agnes, Mladenova, Veronika, Soni, Aashish, Nikolov, Vladimir, Saha, Janapriya, Mladenov, Emil, Iliakis, George

Chromosome translocations are hallmark of cancer and of radiation-induced cell killing, reflecting joining of incongruent DNA-ends that alter the genome. Translocation-formation requires DNA end-joining mechanisms and incompletely characterized, permissive chromatin conditions. We show that chromatin destabilization by clusters of DNA double-strand-breaks (DSBs) generated by the I-SceI meganuclease at multiple, appropriately engineered genomic sites, compromises c-NHEJ and markedly increases cell killing and translocation-formation compared to single-DSBs. Translocation-formation from DSB-clusters utilizes Parp1 activity, implicating alt-EJ in their formation. Immunofluorescence experiments show that single-DSBs and DSB-clusters uniformly provoke the formation of single γ-H2AX foci, suggesting similar activation of early DNA damage response (DDR). Live-cell imaging also shows similar single-focus recruitment of the early-response protein MDC1, to single-DSBs and DSB-clusters. Notably, the late DDR protein, 53BP1 shows in live-cell imaging strikingly stronger recruitment to DSB-clusters as compared to single-DSBs. This is the first report that chromatin thripsis, in the form of engineered DSB-clusters, compromises first-line DSB-repair pathways, allowing alt-EJ to function as rescuing-backup. DSB-cluster-formation is indirectly linked to the increased biological effectiveness of high ionization-density radiations, such as the alpha-particles emitted by radon gas or the heavy-ions utilized in cancer therapy. Our observations provide the first direct mechanistic explanation for this long-known effect.

Digital object identifier (DOI): 10.1093/nar/gkw487

J Radiat Res, 57(3), 220–226
June, 2016

Analysis of chromosome translocation frequency after a single CT scan in adults.

Abe, Yu, Miura, Tomisato, Yoshida, Mitsuaki A., Ujiie, Risa, Kurosu, Yumiko, Kato, Nagisa, Katafuchi, Atsushi, Tsuyama, Naohiro, Kawamura, Fumihiko, Ohba, Takashi, Inamasu, Tomoko, Shishido, Fumio, Noji, Hideyoshi, Ogawa, Kazuei, Yokouchi, Hiroshi, Kanazawa, Kenya, Ishida, Takashi, Muto, Satoshi, Ohsugi, Jun, Suzuki, Hiroyuki, Ishikawa, Tetsuo, Kamiya, Kenji, Sakai, Akira

We recently reported an increase in dicentric chromosome (DIC) formation after a single computed tomography (CT) scan (5.78-60.27 mSv: mean 24.24 mSv) and we recommended analysis of 2000 metaphase cells stained with Giemsa and centromere-FISH for dicentric chromosome assay (DCA) in cases of low-dose radiation exposure. In the present study, we analyzed the frequency of chromosome translocations using stored Carnoy's-fixed lymphocyte specimens from the previous study; these specimens were from 12 patients who were subject to chromosome painting of Chromosomes 1, 2 and 4. Chromosomes 1, 2 and 4 were analyzed in ∼5000 cells, which is equivalent to the whole-genome analysis of almost 2000 cells. The frequency of chromosome translocation was higher than the number of DICs formed, both before and after CT scanning. The frequency of chromosome translocations tended to be higher, but not significantly higher, in patients with a treatment history compared with patients without such a history. However, in contrast to the results for DIC formation, the frequency of translocations detected before and after the CT scan did not differ significantly. Therefore, analysis of chromosome translocation may not be a suitable assay for detecting chromosome aberrations in cases of low-dose radiation exposure from a CT scan. A significant increase in the frequency of chromosome translocations was not likely to be detected due to the high baseline before the CT scan; the high and variable frequency of translocations was probably due to multiple confounding factors in adults.

Digital object identifier (DOI): 10.1093/jrr/rrv090

Chromosome Res
May, 2016

Karyotype diversity suggests that Laonastes aenigmamus (Laotian rock rat) (Rodentia, Diatomyidae) is a multi-specific genus.

Richard, Florence, Gerbault-Seureau, Michèle, Douangboupha, Bounneuang, Keovichit, Kham, Hugot, Jean-Pierre, Dutrillaux, Bernard

Laonastes aenigmamus (Khanyou) is a recently described rodent species living in geographically separated limestone formations of the Khammuan Province in Lao PDR. Chromosomes of 21 specimens of L. aenigmamus were studied using chromosome banding as well as fluorescent in situ hybridization (FISH) techniques using human painting, telomere repeats, and 28S rDNA probes. Four different karyotypes were established. Study with human chromosome paints and FISH revealed that four large chromosomes were formed by multiple common tandem fusions, with persistence of some interstitial telomeres. The rearrangements separating the different karyotypes (I to IV) were also reconstructed. Various combinations of Robertsonian translocations or tandem fusions involving the same chromosomes differentiate these karyotypes. These rearrangements create a strong gametic barrier, which isolates specimens with karyotype II from the others. C-banding and FISH with telomere repeats also exhibit large and systematized differences between karyotype II and others. These data indicate an ancient reproductive separation and suggest that Laonastes is not a mono-specific genus.

Digital object identifier (DOI): 10.1007/s10577-016-9527-7

Radiat Environ Biophys
March, 2016

Chromosome aberrations in Japanese fishermen exposed to fallout radiation 420-1200~km distant from the nuclear explosion test site at Bikini Atoll: report 60~years after the incident.

Tanaka, Kimio, Ohtaki, Megu, Hoshi, Masaharu

During the period from March to May, 1954, the USA conducted six nuclear weapon tests at the "Bravo" detonation sites at the Bikini and Enewetak Atolls, Marshall Islands. At that time, the crew of tuna fishing boats and cargo ships that were operating approximately 150-1200 km away from the test sites were exposed to radioactive fallout. The crew of the fishing boats and those on cargo ships except the "5th Fukuryu-maru" did not undergo any health examinations at the time of the incident. In the present study, chromosome aberrations in peripheral blood lymphocytes were examined in detail by the G-banding method in 17 crew members from 8 fishing boats and 2 from one cargo ship, 60 years after the tests. None of the subjects examined had suffered from cancer. The percentages of both stable-type aberrations such as translocation, inversion and deletion, and unstable-type aberrations such as dicentric and centric ring in the study group were significantly higher (1.4- and 2.3-fold, respectively) than those in nine age-matched controls. In the exposed and control groups, the percentages of stable-type aberrations were 3.35 \% and 2.45 \%, respectively, and the numbers of dicentric and centric ring chromosomes per 100 cells were 0.35 and 0.15, respectively. Small clones were observed in three members of the exposed group. These results suggest that the crews were exposed to slightly higher levels of fallout than had hitherto been assumed.

Digital object identifier (DOI): 10.1007/s00411-016-0648-3

Tumour Biol, 37(3), 4041–4052
March, 2016

Establishment and characterization of a human intrahepatic cholangiocarcinoma cell line derived from an Italian patient.

Cavalloni, Giuliana, Peraldo-Neia, Caterina, Varamo, Chiara, Casorzo, Laura, Dell'Aglio, Carmine, Bernabei, Paola, Chiorino, Giovanna, Aglietta, Massimo, Leone, Francesco

Biliary tract carcinoma is a rare malignancy with multiple causes, which underlie the different genetic and molecular profiles. Cancer cell lines are affordable models, reflecting the characteristics of the tumor of origin. They represent useful tools to identify molecular targets for treatment. Here, we established and characterized from biological, molecular, and genetic point of view, an Italian intrahepatic cholangiocarcinoma cell line (ICC), the MT-CHC01. MT-CHC01 cells were isolated from a tumor-derived xenograft. Immunophenotypical characterization was evaluated both at early and after stabilization passages. In vitro biological, genetic, and molecular features were also investigated. In vivo tumorigenicity was assessed in NOD/SCID mice. MT-CHC01cells retain epithelial cell markers, EPCAM, CK7, and CK19, and some stemness and pluripotency markers, i.e., SOX2, Nanog, CD49f/integrin-α6, CD24, PDX1, FOXA2, and CD133. They grow as a monolayer, with a population double time of about 40 h; they show a low migration and invasion potential. In low attachment conditions, they are able to form spheres and to growth in anchorage-independent manner. After subcutaneous injection, they retain in vivo tumorigenicity; the expression of biliary markers as CA19-9 and CEA were maintained from primary tumor. The karyotype is highly complex, with a hypotriploid to hypertriploid modal number (3n+/-) (52 to 77 chromosomes); low level of HER2 gene amplification, TP53 deletion, gain of AURKA were identified; K-RAS G12D mutation were maintained from primary tumor to MT-CHC01 cells. We established the first ICC cell line derived from an Italian patient. It will help to study either the biology of this tumor or to test drugs both in vitro and in vivo.

Digital object identifier (DOI): 10.1007/s13277-015-4215-3

Oncotarget, 7(9), 10182–10192
March, 2016

Chromothripsis-like chromosomal rearrangements induced by ionizing radiation using proton microbeam irradiation system.

Morishita, Maki, Muramatsu, Tomoki, Suto, Yumiko, Hirai, Momoki, Konishi, Teruaki, Hayashi, Shin, Shigemizu, Daichi, Tsunoda, Tatsuhiko, Moriyama, Keiji, Inazawa, Johji

Chromothripsis is the massive but highly localized chromosomal rearrangement in response to a one-step catastrophic event, rather than an accumulation of a series of subsequent and random alterations. Chromothripsis occurs commonly in various human cancers and is thought to be associated with increased malignancy and carcinogenesis. However, the causes and consequences of chromothripsis remain unclear. Therefore, to identify the mechanism underlying the generation of chromothripsis, we investigated whether chromothripsis could be artificially induced by ionizing radiation. We first elicited DNA double-strand breaks in an oral squamous cell carcinoma cell line HOC313-P and its highly metastatic subline HOC313-LM, using Single Particle Irradiation system to Cell (SPICE), a focused vertical microbeam system designed to irradiate a spot within the nuclei of adhesive cells, and then established irradiated monoclonal sublines from them, respectively. SNP array analysis detected a number of chromosomal copy number alterations (CNAs) in these sublines, and one HOC313-LM-derived monoclonal subline irradiated with 200 protons by the microbeam displayed multiple CNAs involved locally in chromosome 7. Multi-color FISH showed a complex translocation of chromosome 7 involving chromosomes 11 and 12. Furthermore, whole genome sequencing analysis revealed multiple de novo complex chromosomal rearrangements localized in chromosomes 2, 5, 7, and 20, resembling chromothripsis. These findings suggested that localized ionizing irradiation within the nucleus may induce chromothripsis-like complex chromosomal alterations via local DNA damage in the nucleus.

Digital object identifier (DOI): 10.18632/oncotarget.7186

Reprod Domest Anim, 51(1), 171–174
February, 2016

A Non-Reciprocal Autosomal Translocation 64,XX, t(4;10)(q21;p15) in an Arabian Mare with Repeated Early Embryonic Loss.

Ghosh, S., Das, P. J., Avila, F., Thwaits, B. K., Chowdhary, B. P., Raudsepp, T.

Balanced autosomal translocations are a known cause for repeated early embryonic loss (REEL) in horses. In most cases, carriers of such translocations are phenotypically normal, but the chromosomal aberration negatively affects gametogenesis giving rise to both genetically balanced and unbalanced gametes. The latter, if involved in fertilization, result in REEL, whereas gametes with the balanced form of translocation will pass the defect into next generation. Therefore, in order to reduce the incidence of REEL, identification of translocation carriers is critical. Here, we report about a phenotypically normal 3-year-old Arabian mare that had repeated resorption of conceptuses prior to day 45 of gestation and was diagnosed with REEL. Conventional and molecular cytogenetic analyses revealed that the mare had normal chromosome number 64,XX but carried a non-mosaic and non-reciprocal autosomal translocation t(4;10)(q21;p15). This is a novel translocation described in horses with REEL and the first such report in Arabians. Previous cases of REEL due to autosomal translocations have exclusively involved Thoroughbreds. The findings underscore the importance of routine cytogenetic screening of breeding animals.

Digital object identifier (DOI): 10.1111/rda.12636

Am J Hematol, 91(2), 233–237
February, 2016

Classic and extracavitary primary effusion lymphoma in 51 HIV-infected patients from a single institution.

Guillet, Stéphanie, Gérard, Laurence, Meignin, Véronique, Agbalika, Felix, Cuccini, Wendy, Denis, Blandine, Katlama, Christine, Galicier, Lionel, Oksenhendler, Eric

Human immunodeficiency virus (HIV)-associated primary effusion lymphoma (PEL) is a rare B-cell non-Hodgkin lymphoma with poor prognosis. Lymphoma cells are always infected with human herpesvirus-8 (HHV-8) and in most cases coinfected with Epstein-Barr virus. In classic presentation, PEL is characterized by body cavity effusions with or without mass lesions. A variant with only extracavitary localization has also been described. We report on a large single-center series of patients with PEL in the era of combined antiretroviral therapy (cART). The main objective was to compare the characteristics and the outcome of patients with classic (n = 34) and extracavitary (n = 17) variant PEL. At PEL diagnosis, no major difference was observed between the two groups in terms of demographic and HIV characteristics. Extracavitary localizations were exclusively nodal in six patients and involved various organs in 11 patients. Another HHV-8-associated disease was observed in 31 patients, Kaposi sarcoma in 25, and multicentric Castleman disease in 18 patients, without difference between the two groups. Thirty-two patients were treated with CHOP associated with high-dose methotrexate, 13 were treated with CHOP-derived regimen alone, and six patients received low-dose/no chemotherapy. Complete remission was achieved in 21 (62\%) and seven (41\%) patients of the classic and extracavitary groups, respectively. The median overall survival (OS) was 10.2 months. Despite a higher disease-free survival in the extracavitary group, there was no difference in OS between the two variants. Based on this series, characteristics of classic and extracavitary variants were very close. Although prognosis of PEL remains very severe in cART era, the median survival compares favorably with earlier series.

Digital object identifier (DOI): 10.1002/ajh.24251

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

Front Microbiol, 7, 739
2016

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

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.

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

Front Neurol, 7, 23
2016

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
2016

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

Journal of applied toxicology : JAT
2016

Genotoxic risk of ethyl-paraben could be related to telomere shortening.

Finot, F, Kaddour, A, Morat, L, Mouche, I, Zaguia, N, Cuceu, C, Souverville, D, Négrault, S, Cariou, O, Essahli, A, Prigent, N, Saul, J, Paillard, F, Heidingsfelder, L, Lafouge, P, Al Jawhari, M, Hempel, W M, El May, M, Colicchio, B, Dieterlen, A, Jeandidier, E, Sabatier, L, Clements, J, M'Kacher, R

The ability of parabens to promote the appearance of multiple cancer hallmarks in breast epithelium cells provides grounds for regulatory review of the implication of the presence of parabens in human breast tissue. It is well documented that telomere dysfunction plays a significant role in the initiation of genomic instability during carcinogenesis in human breast cancer. In the present study, we evaluated the genotoxic effect of ethyl 4-hydroxybenzoate (ethyl-paraben), with and without metabolic activation (S9), in studies following OECD guidelines. We observed a significant increase in genotoxic damage using the Mouse Lymphoma Assay and in vitro micronucleus (MN) tests in the L5178Y cell line in the presence of S9 only after a short exposure. A high frequency of MN was observed in the TK6 cells after a short exposure (3 h) in the presence of S9 and a long exposure (26 h) without S9. We found significant increases in the MN frequency and induced chromosomal aberrations in the lymphocytes of only one donor after ethyl-paraben exposure in the presence of S9 after a short exposure. Cytogenetic characterization of the paraben-treated cells demonstrated telomere shortening associated with telomere loss and telomere deletions in L5178Y and TK6 cells and lymphocytes of the paraben sensitive-donor. In a control cohort of 68 human lymphocytes, telomere length and telomere aberrations were age-dependent and showed high inter-individual variation. This study is the first to link telomere shortening and the genotoxic effect of ethyl paraben in the presence of S9 and raises the possibility that telomere shortening may be a proxy for underlying inter-individual sensitivity to ethyl-paraben. Copyright © 2016 John Wiley & Sons, Ltd.

Digital object identifier (DOI): 10.1002/jat.3425

Sci Rep, 6, 25658
2016

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