Hierarchical Ceramic Coating Reduces Adherence of Cells, Blood, Bacteria, and Tissue on Titanium Microsurgical Instruments.

BACKGROUND: Progress in the field of microsurgery allows more detailed reconstructions of the smallest tissue structures. The applied instruments are left with biological residues after coming into contact with body fluids or tissue, leading to compromised surgical precision. Designing of residue-free innovative instruments would reduce the necessity of subsidiary practices and would improve the surgical precision. METHODS: We designed a ceramic coating (Lotus ceramic coating system 26-LCC-26) that exhibits self-cleaning surface properties on coated titanium specimens. A titanium surface was modified by blasting technology and electropolishing, followed by applying a high-performance ceramic and sol-gel finish layer. The physical surface characterization was performed by scanning electron microscopy and measuring the contact angle. The cell-repellent properties and cytotoxicity were investigated using live-dead staining, BrdU, and lactate dehydrogenase assay. Furthermore, bacterial and fluid-adhesion tests were performed. Finally, blood compatibility was analyzed according to DIN ISO 10993. RESULTS: The composite system LCC-26 increased the hydrophobic character of the titanium surface (the water contact angle of 74.9 degrees was compared with 62.7 degrees of the uncoated native titanium; p < 0.01) and led to the fluid and cell-repellent properties shown by the reduction in fibroblast adherence by ∼50.7% (p < 0.05), the reduction in Staphylococcus aureus pathogen colonization by 74.1% (p < 0.001), and the decrease in erythrocyte adherence by 62.9% (p < 0.01). Furthermore, the LCC-26 coated titanium microforceps dipped in human whole blood exhibited blood-repellent character (reduction in blood adherence by 46.1%; p < 0.05). Additionally, cyto- and hemocompatibility was guaranteed in direct and indirect tests. CONCLUSION: Titanium surface modification on surgical instruments exhibits cell, bacteria, and blood-repellent properties with a full guarantee of cyto- and hemocompatibility. Thus, innovatively coated instruments could contribute to increased precision during microsurgical interventions and optimized medical operation routines in the future.

Therapeutic melanoma inhibition by local micelle-mediated cyclic nucleotide repression.

The acidic tumor microenvironment in melanoma drives immune evasion by up-regulating cyclic adenosine monophosphate (cAMP) in tumor-infiltrating monocytes. Here we show that the release of non-toxic concentrations of an adenylate cyclase (AC) inhibitor from poly(sarcosine)-block-poly(L-glutamic acid γ-benzyl ester) (polypept(o)id) copolymer micelles restores antitumor immunity. In combination with selective, non-therapeutic regulatory T cell depletion, AC inhibitor micelles achieve a complete remission of established B16-F10-OVA tumors. Single-cell sequencing of melanoma-infiltrating immune cells shows that AC inhibitor micelles reduce the number of anti-inflammatory myeloid cells and checkpoint receptor expression on T cells. AC inhibitor micelles thus represent an immunotherapeutic measure to counteract melanoma immune escape.

Inflammatory processes and elevated osteoclast activity chaperon atrophic non-union establishment in a murine model.

BACKGROUND: Delayed bone healing, especially in long bones poses one of the biggest problems in orthopeadic and reconstructive surgery and causes tremendous costs every year. There is a need for exploring the causes in order to find an adequate therapy. Earlier investigations of human scaphoid non-union revealed an elevated osteoclast activity, accompanied by upregulated levels of TGF-beta and RANKL. Interestingly, scaphoid non-union seemed to be well vascularized. METHODS: In the current study, we used a murine femur-defect model to study atrophic non unions over a time-course of 10 weeks. Different time points were chosen, to gather insights into the dynamic processes of non-union establishment. RESULTS: Histological analyses as well as western blots and qRT-PCR indicated enhanced osteoclast activity throughout the observation period, paralleled by elevated levels of TGF-beta, TNF-alpha, MMP9, MMP13 and RANKL, especially during the early phases of non-union establishment. Interestingly, elevated levels of these mediators decreased markedly over a period of 10 weeks, as inflammatory reaction during non-union establishment seemed to wear out. To our surprise, osteoblastogenesis seemed to be unaffected during early stages of non-union establishment. CONCLUSION: Taken together, we gained first insights into the establishment process of atrophic non unions, in which inflammatory processes accompanied by highly elevated osteoclast activity seem to play a leading role.

A spiked human proteomic dataset from human osteogenic differentiated BMSCs and ASCs for use as a spectral library, for modelling pathways as well as protein mapping.

This article describes a mass spectrometry data set generated from osteogenic differentiated bone marrow stromal cells (BMSCs) and adipose tissue derived stromal cells (ASCs) of a 24-year old healthy donor. Before osteogenic differentiation and performing mass spectrometric measurements cells have been characterized as mesenchymal stromal cells via FACS-analysis positive for CD90 and CD105 and negative for CD14, CD34, CD45 and CD11b and tri-lineage differentiation. After osteogenic differentiation, both cell types were homogenized and then fractionated by SDS gel electrophoresis, resulting in 12 fractions. The proteins underwent an in-gel digestion, spiked with iRT peptides and analysed by nanoHPLC-ESI-MS/MS, resulting in 24 data files. The data files generated from the described workflow are hosted in the public repository ProteomeXchange with identifier PXD015026. The presented data set can be used as a spectral library for analysis of key proteins in the context of osteogenic differentiation of mesenchymal stromal cells for regenerative applications. Moreover, these data can be used to perform comparative proteomic analysis of different mesenchymal stromal cells or stem cells upon osteogenic differentiation. In addition, these data can also be used to determine the optimal settings for measuring proteins and peptides of interest.

Bone allografts combined with adipose-derived stem cells in an optimized cell/volume ratio showed enhanced osteogenesis and angiogenesis in a murine femur defect model.

Critical sized defects, especially in long bones, pose one of the biggest problems in orthopedic surgery. By definition, these defects do not heal without further treatment. Different therapeutic options range from autologous bone grafts, for example, free vascularized bone grafts, to commercially available bone allografts. Disadvantages of these bone allografts are related to reduced osteogenesis, since they are solely composed of cell-free bone matrix. The purpose of this study was to investigate the cell seeding efficiency of human adipose-derived stem cells (hASCs) on human bone allografts in vitro and furthermore analyze these optimized seeded allografts in a critical sized defect model in vivo. Cancellous human bone allografts were colonized with human ASCs in vitro. Cell seeding efficiency was evaluated by Cell Counting Kit-8 assay. Thereafter, optimized hASC-seeded bone scaffolds were examined in a murine femur defect model, stabilized with the MouseExFix system. Subsequently, x-ray analysis and histology were performed. Examination of cell seeding efficiency revealed an optimum starting population of 84,600 cells per 100 mm3 scaffold. In addition, scaffolds seeded with hASCs showed increased osteogenesis compared with controls. Histological analysis revealed increased remodeling and elevated new bone formation within hASC-seeded scaffolds. Moreover, immunohistochemical stainings revealed increased proliferation, osteogenesis, and angiogenesis. In this study, we systemically optimized cell/volume ratio of two promising components of tissue engineering: hASCs and human bone allografts. These findings may serve as a basis for future translational studies. KEY MESSAGES: Bone tissue engineering. Mesenchymal stem cells derived from human adipose tissue (hASCs). Optimal cell/volume ratio of cell-seeded scaffolds. Increased osteogenesis and angiogenesis in vivo.

Adipose-Derived Stromal Cells Are Capable of Restoring Bone Regeneration After Post-Traumatic Osteomyelitis and Modulate B-Cell Response.

Bone infections are a frequent cause for large bony defects with a reduced healing capacity. In previous findings, we could already show diminished healing capacity after bone infections, despite the absence of the causing agent, Staphylococcus aureus. Moreover, these bony defects showed reduced osteoblastogenesis and increased osteoclastogenesis, meaning elevated bone resorption ongoing with an elevated B-cell activity. To overcome the negative effects of this postinfectious inflammatory state, we tried to use the regenerative capacity of mesenchymal stem cells derived from adipose tissue (adipose-derived stem cells [ASCs]) to improve bone regeneration and moreover were curious about immunomodulation of applicated stem cells in this setting. Therefore, we used our established murine animal model and applicated ASCs locally after sufficient debridement of infected bones. Bone regeneration and resorption as well as immunological markers were investigated via histology, immunohistochemistry, Western blot, and fluorescence-activated cell scanning (FACS) analysis and µ-computed tomography (CT) analysis. Interestingly, ASCs were able to restore bone healing via elevation of osteoblastogenesis and downregulation of osteoclasts. Surprisingly, stem cells showed an impact on the innate immune system, downregulating B-cell population. In summary, these data provide a fascinating new and innovative approach, supporting bone healing after bacterial infections and moreover gain insights into the complex ceremony of stem cell interaction in terms of bone infection and regeneration. Stem Cells Translational Medicine 2019;8:1084-1091.

P2000 - A high-nitrogen austenitic steel for application in bone surgery.

Optimal treatment of bone fractures with minimal complications requires implant alloys that combine high strength with high ductility. Today, TiAl6V4 titanium and 316L steel are the most applied alloys in bone surgery, whereas both share advantages and disadvantages. The nickel-free, high-nitrogen austenitic steel X13CrMnMoN18-14-3 (1.4452, brand name: P2000) exhibits high strength in combination with superior ductility. In order to compare suitable alloys for bone implants, we investigated titanium, 316L steel, CoCrMo and P2000 for their biocompatibility and hemocompatibility (according to DIN ISO 10993-5 and 10993-4), cell metabolism, mineralization of osteoblasts, electrochemical and mechanical properties. P2000 exhibited good biocompatibility of fibroblasts and osteoblasts without impairment in vitality or changing of cell morphology. Furthermore, investigation of the osteoblasts function by ALP activity and protein levels of the key transcription factor RUNX2 revealed 2x increased ALP activity and more than 4x increased RUNX2 protein levels for P2000 compared to titanium or 316 steel, respectively. Additionally, analyses of osteoblast biomineralization by Alizarin Red S staining exhibited more than 6x increased significant mineralization of osteoblasts grown on P2000 as compared to titanium. Further, P2000 showed no hemolytic effect and no significant influence on hemocompatibility. Nanoindentation hardness tests of Titanium and 316L specimens exposed an indentation hardness (HIT) of about 4 GPa, whereas CoCrMo and P2000 revealed HIT of 7.5 and 5.6 GPa, respectively. Moreover, an improved corrosion resistance of P2000 compared to 316L steel was observed. In summary, we could demonstrate that the nickel-free high-nitrogen steel P2000 appears to be a promising alternative candidate for applications in bone surgery. As to nearly all aspects like biocompatibility and hemocompatibility, cell metabolism, mineralization of osteoblasts and mechanical properties, P2000 was similar to or revealed advantages against titanium, 316L or CoCrMo.

Activin Receptor 2 Antagonization Impairs Adipogenic and Enhances Osteogenic Differentiation in Mouse Adipose-Derived Stem Cells and Mouse Bone Marrow-Derived Stem Cells In Vitro and In Vivo.

Tumors, traumata, burn injuries or surgeries can lead to critical-sized bony defects which need to be reconstructed. Mesenchymal stem cells (MSCs) have the ability to differentiate into multiple cell lineages and thus present a promising alternative for use in tissue engineering and reconstruction. However, there is an ongoing debate whether all MSCs are equivalent in their differentiation and proliferation ability. The goal of this study was to assess osteogenic and adipogenic characteristic changes of adipose-derived stem cells (ASCs) and bone marrow-derived stem cells (BMSCs) upon Myostatin inhibition with Follistatin in vitro and in vivo. We harvested ASCs from mice inguinal fat pads and BMSCs from tibiae of mice. By means of histology, real-time cell analysis, immunohistochemistry, and PCR osteogenic and adipogenic proliferation and differentiation in the presence or absence of Follistatin were analyzed. In vivo, osteogenic capacity was investigated in a tibial defect model of wild-type (WT) mice treated with mASCs and mBMSCs of Myo-/- and WT origin. In vitro, we were able to show that inhibition of Myostatin leads to markedly reduced proliferative capacity in mBMSCs and mASCs in adipogenic differentiation and reduced proliferation in osteogenic differentiation in mASCs, whereas proliferation in mBMSCs in osteogenic differentiation was increased. Adipogenic differentiation was inhibited in mASCs and mBMSCs upon Follistatin treatment, whereas osteogenic differentiation was increased in both cell lineages. In vivo, we could demonstrate increased osteoid formation in WT mice treated with mASCs and mBMSCs of Myo-/- origin and enhanced osteogenic differentiation and proliferation of mASCs of Myo-/- origin. We could demonstrate that the osteogenic potential of mASCs could be raised to a level comparable to mBMSCs upon inhibition of Myostatin. Moreover, Follistatin treatment led to inhibition of adipogenesis in both lineages.

Interaction with the GDF8/11 pathway reveals treatment options for adenocarcinoma of the breast.

Breast adenocarcinoma continues to be the most frequently diagnosed tumor entity. Despite established therapy options, mortality for breast cancer remains to be as high as 40,000 patients in the US annually. Thus, a need to develop a patient-oriented, targeted therapy exists. In this study, we investigated the interaction of breast adenocarcinoma with the ubiquitously present protein Follistatin and subsequently the GDF8/11 pathway. We analyzed primary histological samples from adenocarcinoma patients for expression of Follistatin and GDF8/11. Furthermore, expression levels of Follistatin and GDF8/11 in MCF7 were compared with MCF10a cells. From the resulting data, GDF8 and Follistatin were used as chemotherapeutic agents in MCF7 cells and their migratory, proliferative behavior and viability were measured. From the experiments, we were able to detect a significantly increased expression of Follistatin and GDF8/11 in the low malignant breast adenocarcinoma (G1) as compared to benign breast fibroadenoma. Interestingly, a decrease was demonstrated in higher grade malignancies. These findings were accompanied by the clinical observation that increased expression of Follistatin and GDF8 is associated with a higher overall survival rate of breasts cancer patients. Substitution of GDF8 and Follistatin reduces the viability of the MCF7 cells and disrupts the migrative and proliferative potential. In summary, MCF7 cells show high chemosensitivity to Follistatin and especially GDF8 and both proteins might serve as targets to improve systemic treatment in breast cancer. In contrast to most established chemotherapy regimens Follistatin and GDF8 show no cytotoxicity to other organs.

Inhibition of GDF8 (Myostatin) accelerates bone regeneration in diabetes mellitus type 2.

Metabolic diseases like diabetes mellitus cause bone healing deficiencies. We found significant impairment of bone regeneration, osteogenic differentiation and proliferation in diabetic bone. Moreover recent studies suggest a highly underestimated importance of GDF8 (Myostatin) in bone metabolism. Our goal was to analyze the role of GDF8 as a regulator of osteogenic differentiation, proliferation and bone regeneration. We used a murine tibial defect model in diabetic (Leprdb-/-) mice. Myostatin-Inhibitor Follistatin was administered in tibial bony defects of diabetic mice. By means of histology, immunohistochemistry and QRT-PC osteogenesis, differentiation and proliferation were analyzed. Application of Myostatin-inhibitor showed a significant improvement in diabetic bone regeneration compared to the control group (6.5 fold, p < 0.001). Immunohistochemistry revealed a significantly higher proliferation (7.7 fold, p = 0.009), osteogenic differentiation (Runx-2: 3.7 fold, p = 0.011, ALP: 9.3 fold, p < 0.001) and calcification (4.9 fold, p = 0.024) in Follistatin treated diabetic animals. Therapeutical application of Follistatin, known for the importance in muscle diseases, plays an important role in bone metabolism. Diabetic bone revealed an overexpression of the catabolic protein Myostatin. Antagonization of Myostatin in diabetic animals leads to a restoration of the impaired bone regeneration and represents a promising therapeutic option.

Age-dependent alterations in osteoblast and osteoclast activity in human cancellous bone.

It is assumed that the activity of osteoblasts and osteoclasts is decreased in bone tissue of aged individuals. However, detailed investigation of the molecular signature of human bone from young compared to aged individuals confirming this assumption is lacking. In this study, quantitative expression analysis of genes related to osteogenesis and osteoclastogenesis of human cancellous bone derived from the distal radius of young and aged individuals was performed. Furthermore, we additionally performed immunohistochemical stainings. The young group included 24 individuals with an average age of 23.2 years, which was compared to cancellous bone derived from 11 body donators with an average age of 81.0 years. In cancellous bone of young individuals, the osteogenesis-related genes RUNX-2, OSTERIX, OSTEOPONTIN and OSTEOCALCIN were significantly up-regulated compared to aged individuals. In addition, RANKL and NFATc1, both markers for osteoclastogenesis, were significantly induced in cancellous bone of young individuals, as well as the WNT gene family member WNT5a and the matrix metalloproteinases MMP-9. However, quantitative RT-PCR analysis of BMP-2, ALP, FGF-2, CYCLIN-D1, MMP-13, RANK, OSTEOPROTEGERIN and TGFb1 revealed no significant difference. Furthermore, Tartrate-resistant acid phosphatase (TRAP) staining was performed which indicated an increased osteoclast activity in cancellous bone of young individuals. In addition, pentachrome stainings revealed significantly less mineralized bone matrix, more osteoid and an increased bone density in young individuals. In summary, markers related to osteogenesis as well as osteoclastogenesis were significantly decreased in the aged individuals. Thus, the present data extends the knowledge about reduced bone regeneration and healing capacity observed in aged individuals.

Diminished bone regeneration after debridement of posttraumatic osteomyelitis is accompanied by altered cytokine levels, elevated B cell activity, and increased osteoclast activity.

Osteomyelitis is a frequent consequence of open fractures thus representing a common bone infection with subsequent alteration of bone regeneration. Impaired bone homeostasis provokes serious variations in the bone remodeling process, thereby involving multiple inflammatory cytokines to activate bone healing. Our previously established mouse model of posttraumatic osteomyelitis provides the chance to study regulation of selected cytokines after surgical debridement of osteomyelitis thus illustrating the course of initial infectious recovery. An inflammatory cytokine array revealed specifically upregulated cytokines in debrided animals after bone infection, that were verified by Western blot analysis, identifying increased levels of CCL2, CCL3, and CXCL2. Increased osteoclastogenesis after debridement of osteomyelitis was demonstrated by Calcitonin-receptor and RANKL detection via immunohistochemical and -fluorescence stainings. The substantial protein analysis was complemented by uncovering diminished osteogenesis and proliferation in debrided group, tracking Osteocalcin, RUNX2, and PCNA expression. Interestingly TNF-α expression seemed to have no effect on altered bone regeneration after bone infection. Additional flow cytometry analysis proved elevated B cell activity, subsequently increased osteoclast activity and accelerated bone resorption. Based on the variety of severely altered cytokines, we propose a RANKL-dependent osteoclastogenesis after debridement of osteomyelitis coinciding with elevated B cells and simultaneously decreased osteogenesis. A comprehensive understanding of these mechanisms provides new therapeutic options of osteomyelitis cure and is of great importance in prospective medical treatment. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2425-2434, 2017.

Label-free in situ imaging of oil body dynamics and chemistry in germination.

Plant oleosomes are uniquely emulsified lipid reservoirs that serve as the primary energy source during seed germination. These oil bodies undergo significant changes regarding their size, composition and structure during normal seedling development; however, a detailed characterization of these oil body dynamics, which critically affect oil body extractability and nutritional value, has remained challenging because of a limited ability to monitor oil body location and composition during germination in situ Here, we demonstrate via in situ, label-free imaging that oil bodies are highly dynamic intracellular organelles that are morphologically and biochemically remodelled extensively during germination. Label-free, coherent Raman microscopy (CRM) combined with bulk biochemical measurements revealed the temporal and spatial regulation of oil bodies in native soya bean cotyledons during the first eight days of germination. Oil bodies undergo a cycle of growth and shrinkage that is paralleled by lipid and protein compositional changes. Specifically, the total protein concentration associated with oil bodies increases in the first phase of germination and subsequently decreases. Lipids contained within the oil bodies change in saturation and chain length during germination. Our results show that CRM is a well-suited platform to monitor in situ lipid dynamics and local chemistry and that oil bodies are actively remodelled during germination. This underscores the dynamic role of lipid reservoirs in plant development.

Conformational transitions in the glycine-bound GluN1 NMDA receptor LBD via single-molecule FRET.

The N-methyl-D-aspartate receptor (NMDAR) is a member of the glutamate receptor family of proteins and is responsible for excitatory transmission. Activation of the receptor is thought to be controlled by conformational changes in the ligand binding domain (LBD); however, glutamate receptor LBDs can occupy multiple conformations even in the activated form. This work probes equilibrium transitions among NMDAR LBD conformations by monitoring the distance across the glycine-bound LBD cleft using single-molecule Förster resonance energy transfer (smFRET). Recent improvements in photoprotection solutions allowed us to monitor transitions among the multiple conformations. Also, we applied a recently developed model-free algorithm called "step transition and state identification" to identify the number of states, their smFRET efficiencies, and their interstate kinetics. Reversible interstate conversions, corresponding to transitions among a wide range of cleft widths, were identified in the glycine-bound LBD, on much longer timescales compared to channel opening. These transitions were confirmed to be equilibrium in nature by shifting the distribution reversibly via denaturant. We found that the NMDAR LBD proceeds primarily from one adjacent smFRET state to the next under equilibrium conditions, consistent with a cleft-opening/closing mechanism. Overall, by analyzing the state-to-state transition dynamics and distributions, we achieve insight into specifics of long-lived LBD equilibrium structural dynamics, as well as obtain a more general description of equilibrium folding/unfolding in a conformationally dynamic protein. The relationship between such long-lived LBD dynamics and channel function in the full receptor remains an open and interesting question.

Structural dynamics of the glycine-binding domain of the N-methyl-D-aspartate receptor.

N-Methyl-D-aspartate receptors mediate the slow component of excitatory neurotransmission in the central nervous system. These receptors are obligate heteromers containing glycine- and glutamate-binding subunits. The ligands bind to a bilobed agonist-binding domain of the receptor. Previous x-ray structures of the glycine-binding domain of NMDA receptors showed no significant changes between the partial and full agonist-bound structures. Here we have used single molecule fluorescence resonance energy transfer (smFRET) to investigate the cleft closure conformational states that the glycine-binding domain of the receptor adopts in the presence of the antagonist 5,7-dichlorokynurenic acid (DCKA), the partial agonists 1-amino-1-cyclobutanecarboxylic acid (ACBC) and L-alanine, and full agonists glycine and D-serine. For these studies, we have incorporated the unnatural amino acid p-acetyl-L-phenylalanine for specific labeling of the protein with hydrazide derivatives of fluorophores. The single molecule fluorescence resonance energy transfer data show that the agonist-binding domain can adopt a wide range of cleft closure states with significant overlap in the states occupied by ligands of varying efficacy. The difference lies in the fraction of the protein in a more closed-cleft form, with full agonists having a larger fraction in the closed-cleft form, suggesting that the ability of ligands to select for these states could dictate the extent of activation.

Cytolethal distending toxin (CDT) is a radiomimetic agent and induces persistent levels of DNA double-strand breaks in human fibroblasts.

Cytolethal distending toxin (CDT) is a unique genotoxin produced by several pathogenic bacteria. The tripartite protein toxin is internalized into mammalian cells via endocytosis followed by retrograde transport to the ER. Upon translocation into the nucleus, CDT catalyzes the formation of DNA double-strand breaks (DSBs) due to its intrinsic endonuclease activity. In the present study, we compared the DNA damage response (DDR) in human fibroblasts triggered by recombinant CDT to that of ionizing radiation (IR), a well-known DSB inducer. Furthermore, we dissected the pathways involved in the detection and repair of CDT-induced DNA lesions. qRT-PCR array-based mRNA and western blot analyses showed a partial overlap in the DDR pattern elicited by CDT and IR, with strong activation of both the ATM-Chk2 and the ATR-Chk1 axis. In line with its in vitro DNase I-like activity on plasmid DNA, neutral and alkaline Comet assay revealed predominant induction of DSBs in CDT-treated fibroblasts, whereas irradiation of cells generated higher amounts of SSBs and alkali-labile sites. Using confocal microscopy, the dynamics of the DSB surrogate marker γ-H2AX was monitored after pulse treatment with CDT or IR. In contrast to the fast induction and disappearance of γ-H2AX-foci observed in irradiated cells, the number of γ-H2AX-foci induced by CDT were formed with a delay and persisted. 53BP1 foci were also generated following CDT treatment and co-localized with γ-H2AX foci. We further demonstrated that ATM-deficient cells are very sensitive to CDT-induced DNA damage as reflected by increased cell death rates with concomitant cleavage of caspase-3 and PARP-1. Finally, we provided novel evidence that both homologous recombination (HR) and non-homologous end joining (NHEJ) protect against CDT-elicited DSBs. In conclusion, the findings suggest that CDT functions as a radiomimetic agent and, therefore, is an attractive tool for selectively inducing persistent levels of DSBs and unveiling the associated cellular responses.