XLF and APLF bind Ku80 at two remote sites to ensure DNA repair by non-homologous end joining

International audienceThe Ku70-Ku80 (Ku) heterodimer binds rapidly and tightly to the ends of DNA double-strand breaks and recruits factors of the non-homologous end-joining (NHEJ) repair pathway through molecular interactions that remain unclear. We have determined crystal structures of the Ku-binding motifs (KBM) of the NHEJ proteins APLF (A-KBM) and XLF (X-KBM) bound to a Ku-DNA complex. The two KBM motifs bind remote sites of the Ku80 alpha/beta domain. The X-KBM occupies an internal pocket formed by an unprecedented large outward rotation of the Ku80 alpha/beta domain. We observe independent recruitment of the APLF-interacting protein XRCC4 and of XLF to laser-irradiated sites via binding of A- and X-KBMs, respectively, to Ku80. Finally, we show that mutation of the X-KBM and A-KBM binding sites in Ku80 compromises both the efficiency and accuracy of end joining and cellular radiosensitivity. A- and X-KBMs may represent two initial anchor points to build the intricate interaction network required for NHEJ

Editing the genome of chicken primordial germ cells to introduce alleles and study gene function

With continuing advances in genome sequencing technology, the chicken genome assembly is now better annotated with improved accuracy to the level of single nucleotide polymorphisms. Additionally, the genomes of other birds such as the duck, turkey and zebra finch have now been sequenced. A great opportunity exists in avian biology to use genome editing technology to introduce small and defined sequence changes to create specific haplotypes in chicken to investigate gene regulatory function, and also perform rapid and seamless transfer of specific alleles between chicken breeds. The methods for performing such precise genome editing are well established for mammalian species but are not readily applicable in birds due to evolutionary differences in reproductive biology. A significant leap forward to address this challenge in avian biology was the development of long-term culture methods for chicken primordial germ cells (PGCs). PGCs present a cell line in which to perform targeted genetic manipulations that will be heritable. Chicken PGCs have been successfully targeted to generate genetically modified chickens. However, genome editing to introduce small and defined sequence changes has not been demonstrated in any avian species. To address this deficit, the application of CRISPR/Cas9 and short oligonucleotide donors in chicken PGCs for performing small and defined sequence changes was investigated in this thesis. Specifically, homology-directed DNA repair (HDR) using oligonucleotide donors along with wild-type CRISPR/Cas9 (SpCas9-WT) or high fidelity CRISPR/Cas9 (SpCas9-HF1) was investigated in cultured chicken PGCs. The results obtained showed that small sequences changes ranging from a single to a few nucleotides could be precisely edited in many loci in chicken PGCs. In comparison to SpCas9-WT, SpCas9-HF1 increased the frequency of biallelic and single allele editing to generate specific homozygous and heterozygous genotypes. This finding demonstrates the utility of high fidelity CRISPR/Cas9 variants for performing sequence editing with high efficiency in PGCs. Since PGCs can be converted into pluripotent stem cells that can potentially differentiate into many cell types from the three germ layers, genome editing of PGCs can, therefore, be used to generate PGC-derived avian cell types with defined genetic alterations to investigate the host-pathogen interactions of infectious avian diseases. To investigate this possibility, the chicken ANP32A gene was investigated as a target for genetic resistance to avian influenza virus in PGC-derived chicken cell lines. Targeted modification of ANP32A was performed to generate clonal lines of genome-edited PGCs. Avian influenza minigenome replication assays were subsequently performed in the ANP32A-mutant PGC-derived cell lines. The results verified that ANP32A function is crucial for the function of both avian virus polymerase and human-adapted virus polymerase in chicken cells. Importantly, an asparagine to isoleucine mutation at position 129 (N129I) in chicken ANP32A failed to support avian influenza polymerase function. This genetic change can be introduced into chickens and validated in virological studies. Importantly, the results of my investigation demonstrate the potential to use genome editing of PGCs as an approach to generate many types of unique cell models for the study of avian biology. Genome editing of PGCs may also be applied to unravel the genes that control the development of the avian germ cell lineage. In the mouse, gene targeting has been extensively applied to generate loss-of-function mouse models to use the reverse genetics approach to identify key genes that regulate the migration of specified PGCs to the genital ridges. Avian PGCs express similar cytokine receptors as their mammalian counterparts. However, the factors guiding the migration of avian PGCs are largely unknown. To address this, CRISPR/Cas9 was used in this thesis to generate clonal lines of chicken PGCs with loss-of-function deletions in the CXCR4 and c-Kit genes which have been implicated in controlling mouse PGC migration. The results showed that CXCR4-deficient PGCs are absent from the gonads whereas c-Kit-deficient PGCs colonise the developing gonads in reduced numbers and are significantly reduced or absent from older stages. This finding shows a conserved role for CXCR4 and c-Kit signalling in chicken PGC development. Importantly, other genes suspected to be involved in controlling the development of avian germ cells can be investigated using this approach to increase our understanding of avian reproductive biology. Finally, the methods developed in this thesis for editing of the chicken genome may be applied in other avian species once culture methods for the PGCs from these species are develope

A two-dimensional mixed-metal phosphate templated by ethylene diamine, [enH(2)][CoIn(PO4)(2)H(OH2)(2)F-2]

A fluorinated mixed-metal phosphate containing indium and cobalt in 1:1 ratio, [enH(2)][CoIn(PO4)(2)H(OH2)(2)F-2], 1, has been synthesized by hydrothermal methods from a gel-composition of 1.0 Co(OAc)(2): 1.0 In(OAc)(3): 2.0 H3PO4: 2.0 en: 2.0 HF: 240 H2O. Large pale pink-purple single crystals (200 pm) were formed in 70\% yield based on cobalt, with only minor amorphous impurity phases. Compound 1 is triclinic, space group P (1) over bar, a 5.217(1), b=7.596(2), c=8.904(2) Angstrom, alpha=109.11(1), beta=97.46(1), gamma=109.95(1), V=301.6(1) Angstrom(3). The magnetic moment is 5.02 BM, indicating a d(7) high-spin Co2+ center. Fluoride is crucial to formation of the mixed-metal phase since the In and Co metals are connected to each other by bent F- bridges, as well as hydrogen phosphate groups, which connect the (Co-F-In) chains into a 2-D sheet structure. (C) 1999 Academic Press

Zirconium phosphates of variable dimension templated by ethylene diamine: Crystal structures of 1-D [enH(2)][Zr(HPO4)(3)] and 2-D [enH(2)](0.5)[Zr(PO4)(HPO4)]

Hydrothermal reaction of Zr(OPr)(4) with H3PO4 in the presence of ethylene diamine and HE yields three crystalline phases; the 1-D [enH(2)][Zr(HPO4)(3)], 1, which can grow as needles over 1 mm in length, the 2-D [enH(2)](0.5)[Zr(PO4)(HPO4)], 2, which is found as thin plates, and the previously reported fluorinated 3-D open-framework compound [enH(2)](0.5)[Zr-2(PO4)(2)(HPO4)F]. H2O, 3. The 1-D phase is the first of its type, with each Zr linked to neighboring metal atoms in the chain by three bridging phosphate groups via Zr-O-P-O-Zr linkages. Its crystals are monoclinic, space group C2/c, a = 8.996(3), b = 15.373(5), c = 9.582(4) Angstrom, beta = 102.97(2)degrees, V = 1287(1) Angstrom(3). Compound 2 is orthorhombic, space group Pnnm, a = 24.087(4), b = 5.381(1), c = 6.660(1) Angstrom, V = 863.0(4) Angstrom(3). It is related to the `double layer' sheet compound gamma-ZrP and its ion exchanged analogues. (C) 1998 Academic Press

Cytotoxic diterpenoids from the roots of Euphorbia ebracteolata

Three new diterpenoids, yuexiandajisu D (1), E (2) and F were isolated from the roots of Euphorbia ebracteolata, along with eight known diterpenoids, jolkinolide B (4), jolkinolide A, ent-11 alpha-hydroxyabieta-8(14),13(15)-dien-16,12 alpha-olide (6), ent-(13S)hydroxyatis-16-ene-3,14-dione, ent-3 beta,(13S)-diliydroxyatis-16-en-14-one, ent-3-oxokaurane-16 alpha,17-diol, ent-16 alpha,17-dihydroxyatisan-3-one and ent-atisane-3 beta,16 alpha,17-triol. The structures of all compounds were deduced using spectroscopic methods and confirmed for 1 and 2 by single-crystal X-ray diffraction. A biogenetic pathway for the formation of 1 and 2 is proposed briefly. Cytotoxic activities were evaluated against ANA-1, B 16 and Jurkat tumor cells. Jolkinolide B (4) displayed modest activity on ANA-1, B 16 and Jurkat tumor cells with IC50 values 4.46 x 10(-2), 4.48 x 10(-2), 6.47 x 10(-2) mu M, and ent-11 alpha-hydroxyabieta-8(14), 13(15)-dien-16,12 alpha-olide (6) showed significant activity against ANA-1 and Jurkat cells with IC50 values 7.12 x 10(-3) and 1.79 x 10(-2) mu M. Compound 1 was found to be slightly active against ANA-1 cells with an IC50 value 2.88 x 10(-1) mu M. Structure-activity relationships of isolated compounds are also discussed

Adjuvant therapy for the reduction of postoperative intra-abdominal adhesion formation

BACKGROUND: To review currently available evidence on the use of adjuvant therapy to reduce the formation of postoperative intra-abdominal adhesions. METHODS: A search on Pubmed and the Cochrane library was undertaken using the keywords "abdominal", "adhesion", "postoperative", "prevention" and "reduction". Only randomised controlled trials, prospective non-randomised controlled studies and review articles published in the English language between 1990 and 2006 were included. RESULTS: Two prospective non-randomised controlled studies and 18 randomised controlled trials were included in this review. Adjuvant therapies reviewed included pharmacological agents (streptokinase, recombinant tissue plasminogen activator, vitamin E antioxidant molecules), and mechanical barriers (hyaluronic acid barriers, oxidised regenerated cellulose barriers, nanofibrous barriers and collagen foils). Hyaluronate/carboxymethylcellulose-based bioresorbable membrane (Seprafilm) appeared to be the most efficacious in reducing adhesion formation as well as decreasing the incidence of adhesion obstruction requiring reoperation in clinical studies. Drawbacks to the use of Seprafilm include high cost and complications such as haemorrhage and poor wound healing. CONCLUSIONS: Only a limited number of adjuvant treatment methods are currently available for the reduction of postoperative adhesions. Seprafilm has been proven to be the efficacious method to reduce adhesions. Investigations into the novel therapies are showing promising results in experimental studies and clinical studies before their wider application. [Asian J Surg 2009;32(3): 180-6] © 2009 Elsevier. All rights reserved.link_to_subscribed_fulltex