Ataxia Telangiectasia Mutated and Rad3 Related (ATR) Protein Kinase Inhibition Is Synthetically Lethal in XRCC1 Deficient Ovarian Cancer Cells

Introduction

Ataxia telangiectasia mutated and Rad3 Related (ATR) protein kinase is a key sensor of single-stranded DNA associated with stalled replication forks and repair intermediates generated during DNA repair. XRCC1 is a critical enzyme in single strand break repair and base excision repair. XRCC1-LIG3 complex is also an important contributor to the ligation step of the nucleotide excision repair response.

Methods

In the current study, we investigated synthetic lethality in XRCC1 deficient and XRCC1 proficient Chinese Hamster ovary (CHO) and human ovarian cancer cells using ATR inhibitors (NU6027). In addition, we also investigated the ability of ATR inhibitors to potentiate cisplatin cytotoxicity in XRCC1 deficient and XRCC1 proficient CHO and human cancer cells. Clonogenic assays, alkaline COMET assays, γH2AX immunocytochemistry, FACS for cell cycle as well as FITC-annexin V flow cytometric analysis were performed.

Results

ATR inhibition is synthetically lethal in XRCC1 deficient cells as evidenced by increased cytotoxicity, accumulation of double strand DNA breaks, G2/M cell cycle arrest and increased apoptosis. Compared to cisplatin alone, combination of cisplatin and ATR inhibitor results in enhanced cytotoxicity in XRCC1 deficient cells compared to XRCC1 proficient cells.

Conclusions

Our data provides evidence that ATR inhibition is suitable for synthetic lethality application and cisplatin chemopotentiation in XRCC1 deficient ovarian cancer cells.     

A robust CRISPR–Cas9-based fluorescent reporter assay for the detection and quantification of DNA double-strand break repair

DNA double-strand breaks (DSBs) are highly cytotoxic lesions that can lead to chromosome rearrangements, genomic instability and cell death. Consequently, cells have evolved multiple mechanisms to efficiently repair DSBs to preserve genomic integrity. We have developed a DSB repair assay system, designated CDDR (CRISPR–Cas9-based Dual-fluorescent DSB Repair), that enables the detection and quantification of DSB repair outcomes in mammalian cells with high precision. CDDR is based on the introduction and subsequent resolution of one or two DSB(s) in an intrachromosomal fluorescent reporter following the expression of Cas9 and sgRNAs targeting the reporter. CDDR can discriminate between high-fidelity (HF) and error-prone non-homologous end-joining (NHEJ), as well as between proximal and distal NHEJ repair. Furthermore, CDDR can detect homology-directed repair (HDR) with high sensitivity. Using CDDR, we found HF-NHEJ to be strictly dependent on DNA Ligase IV, XRCC4 and XLF, members of the canonical branch of NHEJ pathway (c-NHEJ). Loss of these genes also stimulated HDR, and promoted error-prone distal end-joining. Deletion of the DNA repair kinase ATM, on the other hand, stimulated HF-NHEJ and suppressed HDR. These findings demonstrate the utility of CDDR in characterizing the effect of repair factors and in elucidating the balance between competing DSB repair pathways.     

Comparative immunohistochemical study of tissue integration of macroporous and laminar surgical meshes

Intraperitoneal surgical mesh implantation is required for laparoscopic ventral hernia repair. Composite meshes are well known in animal models and human practice. The aim of our study is to compare the biological behaviour of two different textured silicone-covered polypropylene meshes. Transmural abdominal wall defect was created in 40 rabbits and treated as follows: In 20 animals a polypropylene mesh with a laminar silicone covering (LSPP) and in the rest a macroporous textured mesh knitted of silicone-impregnated polypropylene filaments (MSPP) was applied. One and three weeks after implantation we evaluated the intraperitoneal adhesion formation of the mesh macroscopically, histologically and immunohistochemically to detect the reactive cells, especially inflammatory, endothelial and mesothelial cells, as well as their proliferative activity, and with Scanning Electron microscopy to visualize the surface of the meshes. The adhesion formation caused by the composites showed no statistical difference after one week although in the three weeks old samples the LSPP adhesion was significantly weaker than that of MSPP. As complications, serome formation in both groups, fistulas, abscesses, and sc. haematoma in the LSPP group were found. Only in MSPP containing tissues was the decrease of Ki-67 positive proliferating cells significant. A significant increase in VEGF expressing cells was observed only in MSPP containing three week old samples, suggesting better regulation of vascular growth in tissues surrounding the implants. In one week old specimens we observed an irregular proliferation of cytokeratin containing mesothelial cells in both group. The intraperitoneal surface of MSPP mesh was covered with neoperitoneum, while it was not regularly seen on LSPP mesh after three week.     

Distribution, Movements and Growth of Young Sandbar Sharks, Carcharhinus Plumbeus, in the Nursery Grounds of Delaware Bay

During the spring and summer months of 1995, 1996 and 1997, gillnet and longline surveys were conducted in conjunction with tag and recapture experiments to outline spatial and seasonal distribution of young sandbar sharks, Carcharhinus plumbeus, in Delaware Bay for essential fish habitat mapping, to assess abundance of young sandbar sharks, and to quantify growth during the summer nursery season. Sandbar sharks (n = 943) ranging from 40 to 120cm fork length (48 to 130cm total length) were captured; yearly totals were 199, 314 and 430 in 1995, 1996 and 1997, respectively. Individuals were captured between June and October in water temperatures ranging from 15.4° to 28.5°C and salinities ranging from 22.8 to 30.3 ppt. Presence of neonates and catch per unit effort data indicate that pupping begins in late June near the southwestern coast of the Bay. Juveniles were present from early June through September and their spatial distribution in the Bay appeared uniform. Of 782 sandbar sharks tagged and released during the three years, 50 were recaptured. Mean distance from tag to recapture location and mean days-at-liberty of sandbar sharks recaptured in Delaware Bay during the year of tagging were 10km and 18 days, respectively. Some sharks were recaptured as far as 957km from the release location. Length distributions show young-of-the-year sandbar sharks grow about 2–3cm in length during the nursery season.     

Environmental performance of three innovative calcium carbonate-based consolidants used in the field of built cultural heritage

Purpose

In this study, the environmental performance of three newly developed materials for the conservation of built Cultural Heritage is preliminary evaluated by means of life cycle assessment (LCA). Although LCA has been adopted since many years in many fields, including the construction industry, still it is barely used the field of the Cultural Heritage conservation. Therefore, this paper aims to put a step forward to a wider use of the method for a more sustainable conservation of built heritage.

Methods

Cradle-to-gate approach was applied, which includes activities beginning with production of constituent and ancillary materials and concluding with the manufacturing of the product and its packaging. The functional unit was the production of 1 kg of the consolidant stored in a canister and ready to use. Ecoinvent database was used for the life cycle inventory of chemicals. This database was also taken into account to evaluate impacts related with energy (electricity) needs during manufacturing of the consolidants and production of polyethylene canisters (packaging material). The IMPACT 2002+ method was used to select the impact categories.

Results and discussion

The consolidants under investigation are calcium acetoacetate and two calcium alkoxides (calcium ethoxide and calcium isopropoxide). In the case of calcium acetoacetate consolidant, the highest environmental impact is related to the use of acetone dicarboxylic acid as raw material. Manufacturing process of the final product is not an energy-demanding process; hence, it is regarded as relatively clean from the environmental point of view. In the case of calcium ethoxide and calcium isopropoxide nanosuspensions, the innovative materials just recently developed for conservation purpose, manufacturing has been performed and optimized at a kg/lab scale; therefore, the results must be considered provisional. The two calcium alkoxides nanosuspensions show a relatively high environmental footprint that reflect the high consumption of ancillary materials, i.e. solid CO₂ used to cool down the reaction and liquid ammonia as catalyst, currently released in the atmosphere.

Conclusions

The environmental footprint of calcium acetoacetate is relatively low showing that 1 kg of the consolidant is associated to 0.32 kg of CO₂ equivalent emissions. In the case of two calcium alkoxides nanosuspensions, the global warming impact amounts to 198 kg (calcium ethoxide, 1 kg) and 132 kg (calcium isopropoxide, 1 kg) of CO₂ equivalent emissions. Given the planned optimization of the industrial production currently under investigation, the environmental footprint of the two calcium alkoxides nanosuspensions is expected to reduce between 60 and 85%.

     

Synthesis and evaluation of AKR1C inhibitory properties of A-ring halogenated oestrone derivatives

Enzymes AKR1C regulate the action of oestrogens, androgens, and progesterone at the pre-receptor level and are also associated with chemo-resistance. The activities of these oestrone halides were investigated on recombinant AKR1C enzymes. The oestrone halides with halogen atoms at both C-2 and C-4 positions (13β-, 13α-methyl-17-keto halogen derivatives) were the most potent inhibitors of AKR1C1. The lowest IC₅₀ values were for the 13α-epimers 2_2I,4Br and 2_2I,4Cl (IC₅₀, 0.7 μM, 0.8 μM, respectively), both of which selectively inhibited the AKR1C1 isoform. The 13α-methyl-17-keto halogen derivatives 2_2Br and 2_4Cl were the most potent inhibitors of AKR1C2 (IC₅₀, 1.5 μM, 1.8 μM, respectively), with high selectivity for the AKR1C2 isoform. Compound 1_2Cl,4Cl showed the best AKR1C3 inhibition, and it also inhibited AKR1C1 (Ki: AKR1C1, 0.69 μM; AKR1C3, 1.43 μM). These data show that halogenated derivatives of oestrone represent a new class of potent and selective AKR1C inhibitors as lead compounds for further optimisations.