Imaging the cellular response to an antigen tagged interstrand crosslinking agent.

Immunofluorescence imaging is a standard experimental tool for monitoring the response of cellular factors to DNA damage. Visualizing the recruitment of DNA Damage Response (DDR) components requires high affinity antibodies, which are generally available. In contrast, reagents for the display of the lesions that induce the response are far more limited. Consequently, DDR factor accumulation often serves as a surrogate for damage, without reporting the actual inducing structure. This limitation has practical implications given the importance of the response to DNA reactive drugs such as those used in cancer therapy. These include interstrand crosslink (ICL) forming compounds which are frequently employed clinically. Among them are the psoralens, natural products that form ICLs upon photoactivation and applied therapeutically since antiquity. However, despite multiple attempts, antibodies against psoralen ICLs have not been developed. To overcome this limitation, we developed a psoralen tagged with an antigen for which there are commercial antibodies. In this report we describe our application of the tagged psoralen in imaging experiments, and the unexpected discoveries they revealed.

Treatment of Acetabular Bone Defect in Revision of Total Hip Arthroplasty Using 3D Printed Tantalum Acetabular Augment.

OBJECTIVE: The treatment of acetabular defects is one of the most difficult challenges of revision of total hip arthroplasty (RTHA), and tantalum is regarded as a promising bone substitute material. This study aims to investigate the effectiveness of 3D printed acetabular augment used in RTHA for the treatment of acetabular bone defect. METHODS: A retrospective analysis of the clinical data of seven patients who had undergone RTHA was carried out using 3D printed acetabular augment from January 2017 to December 2018. The CT data of the patients were exported to Mimics 21.0 software (Materialise, Leuven, Belgium), and the acetabular bone defect augment were designed, printed and then implanted during operation. The postoperative Harris score, visual analogue scale (VAS) score and prosthesis position were observed to evaluate the clinical outcome. A I-test was used for preoperative and postoperative comparison of the paired-design dataset. RESULTS: A firm attachment of the bone augment to the acetabulum during operation without any complications was found during the follow-up time 2.8-4.3 years. The VAS score of all patients was found 6.9 ± 1.4 before operation and was 0.7 ± 0.7 at the last follow-up (P ≤ 0.001), and the Harris hip scores, were 31.9 ± 10.3 and 73.3 ± 12.8 before operation, and at the last follow-up (P ≤ 0.001), respectively. Moreover, no loosening sign between the bone defect augment and the acetabulum was observed during the entire implantation period. CONCLUSION: 3D printed acetabular augment is effective in reconstructing the acetabulum following an acetabular bone defect revision, which enhances the hip joint function and eventually makes a satisfactory stable prosthetic.

Modulation of Osteogenesis and Angiogenesis Activities Based on Ionic Release from Zn-Mg Alloys.

The enhancement of osteogenesis and angiogenesis remains a great challenge for the successful regeneration of engineered tissue. Biodegradable Mg and Zn alloys have received increasing interest as potential biodegradable metallic materials, partially due to the biological functions of Mg2+ and Zn2+ with regard to osteogenesis and angiogenesis, respectively. In the present study, novel biodegradable Zn-xMg (x = 0.2, 0.5, 1.0 wt.%) alloys were designed and fabricated, and the effects of adding different amounts of Mg to the Zn matrix were investigated. The osteogenesis and angiogenesis beneficial effects of Zn2+ and Mg2+ release during the biodegradation were characterized, demonstrating coordination with the bone regeneration process in a dose-dependent manner. The results show that increased Mg content leads to a higher amount of released Mg2+ while decreasing the Zn2+ concentration in the extract. The osteogenesis of pre-osteoblasts was promoted in Zn-0.5Mg and Zn-1Mg due to the higher concentration of Mg2+. Moreover, pure Zn extract presented the highest activity in angiogenesis, owing to the highest concentration of Zn2+ release (6.415 µg/mL); the proliferation of osteoblast cells was, however, inhibited under such a high Zn2+ concentration. Although the concentration of Zn ion was decreased in Zn-0.5Mg and Zn-1Mg compared with pure Zn, the angiogenesis was not influenced when the concentration of Mg in the extract was sufficiently increased. Hence, Mg2+ and Zn2+ in Zn-Mg alloys show a dual modulation effect. The Zn-0.5Mg alloy was indicated to be a promising implant candidate due to demonstrating the appropriate activity in regulating osteogenesis and angiogenesis. The present work evaluates the effect of the Mg content in Zn-based alloys on biological activities, and the results provide guidance regarding the Zn-Mg composition in designs for orthopedic application.

Visualizing replication fork encounters with DNA interstrand crosslinks.

Replication forks encounter numerous challenges as they move through eu- and hetero-chromatin during S phase in mammalian cells. These include a variety of impediments to the unwinding of DNA by the replicative helicase such as alternate DNA structures, transcription complexes and R-loops, DNA-protein complexes, and DNA chemical adducts. Much of our knowledge of these events is based on analysis of markers of the replication stress and DNA Damage Response that follow stalling of replisomes. To examine consequences for the replisomes more directly, we developed an approach for imaging collisions of replication forks with the potent block presented by an interstrand crosslink (ICL). The strategy is based on the visualization on DNA fibers of the encounter of replication tracts and an antigen tagged ICL. Our studies revealed an unexpected restart of DNA synthesis past an intact ICL. In addition, and also unexpected, we found two distinct versions of the replisome, one biased toward euchromatin and the other more prominent in heterochromatin. Here, we present details of our experimental procedures that led to these observations.

Replication of the Mammalian Genome by Replisomes Specific for Euchromatin and Heterochromatin.

Replisomes follow a schedule in which replication of DNA in euchromatin is early in S phase while sequences in heterochromatin replicate late. Impediments to DNA replication, referred to as replication stress, can stall replication forks triggering activation of the ATR kinase and downstream pathways. While there is substantial literature on the local consequences of replisome stalling-double strand breaks, reversed forks, or genomic rearrangements-there is limited understanding of the determinants of replisome stalling vs. continued progression. Although many proteins are recruited to stalled replisomes, current models assume a single species of "stressed" replisome, independent of genomic location. Here we describe our approach to visualizing replication fork encounters with the potent block imposed by a DNA interstrand crosslink (ICL) and our discovery of an unexpected pathway of replication restart (traverse) past an intact ICL. Additionally, we found two biochemically distinct replisomes distinguished by activity in different stages of S phase and chromatin environment. Each contains different proteins that contribute to ICL traverse.

Visualization of Replisome Encounters with an Antigen Tagged Blocking Lesion.

Considerable insight is present into the cellular response to double strand breaks (DSBs), induced by nucleases, radiation, and other DNA breakers. In part, this reflects the availability of methods for the identification of break sites, and characterization of factors recruited to DSBs at those sequences. However, DSBs also appear as intermediates during the processing of DNA adducts formed by compounds that do not directly cause breaks, and do not react at specific sequence sites. Consequently, for most of these agents, technologies that permit the analysis of binding interactions with response factors and repair proteins are unknown. For example, DNA interstrand crosslinks (ICLs) can provoke breaks following replication fork encounters. Although formed by drugs widely used as cancer chemotherapeutics, there has been no methodology for monitoring their interactions with replication proteins. Here, we describe our strategy for following the cellular response to fork collisions with these challenging adducts. We linked a steroid antigen to psoralen, which forms photoactivation dependent ICLs in nuclei of living cells. The ICLs were visualized by immunofluorescence against the antigen tag. The tag can also be a partner in the Proximity Ligation Assay (PLA) which reports the close association of two antigens. The PLA was exploited to distinguish proteins that were closely associated with the tagged ICLs from those that were not. It was possible to define replisome proteins that were retained after encounters with ICLs and identify others that were lost. This approach is applicable to any structure or DNA adduct that can be detected immunologically.

DONSON and FANCM associate with different replisomes distinguished by replication timing and chromatin domain.

Duplication of mammalian genomes requires replisomes to overcome numerous impediments during passage through open (eu) and condensed (hetero) chromatin. Typically, studies of replication stress characterize mixed populations of challenged and unchallenged replication forks, averaged across S phase, and model a single species of "stressed" replisome. Here, in cells containing potent obstacles to replication, we find two different lesion proximal replisomes. One is bound by the DONSON protein and is more frequent in early S phase, in regions marked by euchromatin. The other interacts with the FANCM DNA translocase, is more prominent in late S phase, and favors heterochromatin. The two forms can also be detected in unstressed cells. ChIP-seq of DNA associated with DONSON or FANCM confirms the bias of the former towards regions that replicate early and the skew of the latter towards regions that replicate late.

Remodeling of Interstrand Crosslink Proximal Replisomes Is Dependent on ATR, FANCM, and FANCD2.

Eukaryotic replisomes are driven by the mini chromosome maintenance (MCM [M]) helicase complex, an offset ring locked around the template for leading strand synthesis by CDC45 (C) and GINS (G) proteins. Although the CDC45 MCM GINS (CMG) structure implies that interstrand crosslinks (ICLs) are absolute blocks to replisomes, recent studies indicate that cells can restart DNA synthesis on the side of the ICL distal to the initial encounter. Here, we report that restart requires ATR and is promoted by FANCD2 and phosphorylated FANCM. Following introduction of genomic ICLs and dependent on ATR and FANCD2 but not on the Fanconi anemia core proteins or FAAP24, FANCM binds the replisome complex, with concomitant release of the GINS proteins. In situ analysis of replisomes proximal to ICLs confirms the ATR-dependent release of GINS proteins while CDC45 is retained on the remodeled replisome. The results demonstrate the plasticity of CMG composition in response to replication stress.

Imaging cellular responses to antigen tagged DNA damage.

Repair pathways of covalent DNA damage are understood in considerable detail due to decades of brilliant biochemical studies by many investigators. An important feature of these experiments is the defined adduct location on oligonucleotide or plasmid substrates that are incubated with purified proteins or cell free extracts. With some exceptions, this certainty is lost when the inquiry shifts to the response of living mammalian cells to the same adducts in genomic DNA. This reflects the limitation of assays, such as those based on immunofluorescence, that are widely used to follow responding proteins in cells exposed to a DNA reactive compound. The lack of effective reagents for adduct detection means that the proximity between responding proteins and an adduct must be assumed. Since these assumptions can be incorrect, models based on in vitro systems may fail to account for observations made in vivo. Here we discuss the use of a detection tag to address the problem of lesion location, as illustrated by our recent work on replication dependent and independent responses to interstrand crosslinks.

Single Molecule Analysis of Laser Localized Psoralen Adducts.

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live cells. The DDR to helix distorting covalent DNA modifications, including interstrand DNA crosslinks (ICLs), is not as well defined. We have studied the DDR stimulated by ICLs, localized by laser photoactivation of immunotagged psoralens, in the nuclei of live cells. In order to address fundamental questions about adduct distribution and replication fork encounters, we combined laser localization with two other technologies. DNA fibers are often used to display the progress of replication forks by immunofluorescence of nucleoside analogues incorporated during short pulses. Immunoquantum dots have been widely employed for single molecule imaging. In the new approach, DNA fibers from cells carrying laser localized ICLs are spread onto microscope slides. The tagged ICLs are displayed with immunoquantum dots and the inter-lesion distances determined. Replication fork collisions with ICLs can be visualized and different encounter patterns identified and quantitated.

Peptide and peptide nucleic acid syntheses using a DNA/RNA synthesizer.

The use of an ABI 394 DNA/RNA synthesizer for peptide and peptide nucleic acid (PNA) syntheses is described. No additional physical part or software is needed for the application. A commercially available large DNA synthesis column was used, and only about half of its volume was filled with resin when the resin was fully swollen. With additional space in the top portion of the column, agitation of reaction mixture was achieved by bubbling argon from the bottom without losing solution. Removing solutions from column was achieved by flushing argon from top to bottom. Two peptide and two PNA sequences were synthesized. Good yields were obtained in all the cases. The method is easy to follow by researchers who are familiar with DNA/RNA synthesizer.

Purification of synthetic peptides using a catching full-length sequence by polymerization approach.

During automated solid-phase peptide synthesis, failure sequences were capped with acetic anhydride. After synthesis, a polymerizable methacrylamide tag was attached to the full-length sequences. Peptide purification was then achieved by polymerizing the full-length sequences, washing away impurities, and cleaving the peptide product from the polymer.