The present study investigates the effect of using lignin at nanoscale as new flame-retardant additive for polylactide (PLA). Lignin nanoparticles (LNP) were prepared from Kraft lignin microparticles (LMP) through a dissolution-precipitation process. Both micro and nano lignins were functionalized using diethyl chlorophosphate  and diethyl (2-(triethoxysilyl)ethyl) phosphonate  to enhance their flame-retardant effect in PLA. From the use of inductively coupled plasma (ICP) spectrometry, it can be considered that a large amount of phosphorus has been grafted onto the nanoparticles. It has been previously shown that blending lignin with PLA induces degradation of the polymer matrix. However, phosphorylated lignin nanoparticles seem to limit PLA degradation during melt processing and the nanocomposites were shown to be relatively thermally stable. Cone calorimeter tests revealed that the incorporation of untreated lignin, whatever its particle size, induced an increase in pHRR. Using phosphorylated lignin nanoparticles, especially those treated with diethyl (2-(triethoxysilyl)ethyl) phosphonate allows this negative effect to be overcome. Moreover, the pHRR is significantly reduced, even when only 5 wt% LNP-SiP is used. Biobased polymers have expanded significantly during the last decade. Many scientific studies have shown the ability to develop polymers from renewable resources  in order to propose alternatives to fossil-based polymers, notably polylactic acid (PLA) ,2. FolloStudies about the development of fire retardants from biobased materials have been reviewed and widely discussed ,4. For eAt the same time, nano-technologies have allowed the improvement of the fire behavior of polymers owing to several processes such as the modification of the degradation pathway of the polymeric matrix as well as the modification of the rheological behavior. The thermal stability and flammability of poly(methyl methacrylate) (PMMA) containing organo-modified montmorillonite (OMMT) and metal oxide nanoparticles or both have been examined . ThermalOnly a few studies have associated both bio and nano-technologies for the development of flame retardants additives. The utility of this approach has been demonstrated especially when nanoparticles are associated with phosphorous compounds in the case of cellulose nanocrystals . Lignin In this study, lignin nanoparticles (LNP) were prepared from Kraft lignin microparticles (LMP) by dissolution-precipitation. LMP and LNP were functionalized using diethyl chlorophosphate and diethyl (2-(triethoxysilyl)ethyl) phosphonate. All these lignins were incorporated into polylactic acid by melt blending. Thermal properties and fire behavior of the blends were determined using thermogravimetric analysis (TGA) and cone calorimeter experiments.PLA resin (3052D) was purchased from NatureWorks . Kraft water soluble lignin with a low sulfonate content, diethyl chlorophosphate, and diethyl (2-(triethoxysilyl)ethyl) phosphonate were purchased from Sigma-Aldrich . Diethyl (2-(triethoxysilyl)ethyl) phosphonate was purchased from Specific Polymers . Absolute ethanol, ethylene glycol, and acetone were purchased from PanReac AppliChem . Hydrochloric acid solution (35%) was purchased from Fisher Scientific .Lignin nanoparticles (LNP) were prepared from Kraft water-soluble lignin microparticles (LMP), by precipitation from ethylene glycol solution. A 4 wt% solution of lignin in ethylene glycol was prepared at 30 °C under stirring for 2 h. A 0.25M aqueous HCl solution was added at the rate of 2 drops per minute. After addition, the solution was stirred for another hour. Then, the solution was dialyzed against ethanol over 4 days in order to provoke non-solvent precipitation of lignin.With diethyl chlorophosphate (diEtP) a: 30 g oWith diethyl (2-(triethoxysilyl) ethyl) phosphonate (SiP) b: a mixt3 were compression molded from pellets using a Darragon thermopress at 170 °C. First pellets were brought into contact with the heated platens for 4 min, then a 50 bar pressure was applied for 2 min and finally a 100 bar pressure was applied for 2 more min. Plates were cooled in ambient atmosphere.PLA 3052D containing 5 wt% and 10 wt% of the untreated and treated lignin micro and nano-particles were prepared in a Rheomix 3000 internal mixer from Thermo Fisher Scientific. PLA was first ground in 2 mm diameter powder and a suspension of lignin particles in acetone was added to the powder. Acetone was evaporated under a ventilated hood, then the powder mixture was dried overnight in a ventilated oven at 70 °C. Powders were blended in the internal mixer at 170 °C, first at 30 rpm during 3 min and then at 70 rpm during 7 min. The obtained composite was ground into 6 mm diameter pellets. Composite plates of 100 × 100 × 3 mma- Particle Size AnalysisLaser particle size analyzer LS 13 320 from Beckman-Coulter Company was used for determining particle size distribution of untreated and treated lignin nanoparticles in acetone. Particle size distribution was obtained by scattering of monochromatic light with a wavelength of 780 nm diffracted and transmitted through the suspension. Each analysis was at least reproduced 3 times and the maximum relative standard deviation for the median size was 10%. Quanta 200 FEG Scanning Electron Microscope (SEM) from FEI Company  was also used for investigating the lignin particles shape under high vacuum at a voltage of 12.5 kV and a working distance between 8.2 mm and 10.6 mm.b- Phosphorus Content3 and H2SO4 solutions, followed by microwaves irradiation using a Milestone 1200-Mega from Gemini BV . A calibration curve was used to determine the exact amount of phosphorus in the samples.Phosphorus content in functionalized lignins was determined using an Activa M Inductively Coupled Plasma (ICP) spectrometer from Horiba Jobin Yvon . In a first step, the organic matrix was decomposed by a mineralization process using HNOc- Thermal Degradation5%, T10%, and T50%) and char yield at 650 °C were determined.Thermal degradation of lignins and composites was studied by thermogravimetric analysis (TGA). Dried lignins samples and composites were submitted to a temperature ramp from 70 °C to 700 °C at a heating rate of 10 °C/min. TGA were performed under both air and nitrogen flow of 100 mL/min using a Setsys Evolution device from Setaram Instrumentation. 5%, 10%, and 50% weight loss temperatures  was measured as a function of time and Time To Ignition (TTI) and peak of Heat Release Rate (pHRR) were determined. For each sample, at least 2 tests were performed.The fire behavior of the different composites was studied using a cone calorimeter from Fire Testing Technology. Samples of 100 × 100 × 3 mme- Degradation of PLA During Melt Processing−1) and filtered through a 0.2 μm filter. The molecular weight distributions were determined in CHCl3 at 23 °C using an Agilent size exclusion chromatograph equipped with a Knauer 2320 refractometer index detector and two PLGel columns (MIXED-D and 103 A). 20 μL of the sample solutions were injected into the columns using a flow rate of 1 mL/min. Monodisperse polystyrene standards  were used for the primary calibration. The number average molecular weight (Mn) was determined.PLA thermal degradation during melt processing was investigated using a Steric Exclusion Chromatography (SEC) analysis. Samples were dissolved in chloroform  were performed under both air and nitrogen to evaluate the effect of lignin particle size on its thermal stability . The theThe phosphorylation process is mainly used to increase the char stability during lignin aerobic thermal degradation and thus enhances the flame-retardant effect of lignin. First of all, the amount of grafted phosphorus with both diethyl chlorophosphate and diethyl (2-(triethoxysilyl) ethyl) phosphonate has been determined. ICP analyses  highligh50) and 150 nm (d90) for LNP to 545 nm (d50) and 10 µm (d90) for LNP-SiP. The increase of lignin particle size observed in the case of LNP treated with SiP results from self-condensation of SiP as well as from the coupling of different nanoparticles due to the multifunctional reactivity of SiP. Such behavior has also been observed in the literature and resulted in an increase of lignin molecular weight [However, grafting SiP induces some changes on the lignin particles structural and thermal properties. Firstly, in contrast to diEtP that does not affect lignin particle size, using SiP induces a significant increase in lignin particle size. Indeed, r weight  and lignr weight . SEM obsAll these observations enable us to propose the following schematic representation of LNP-SiP  that expThe effect of lignin phosphorylation on its thermal stability has been also evaluated using TGA analysis . The phoOn the contrary, under air, the presence of phosphorus induces important changes in TGA curves  and the It has been proven that lignin could be advantageously used to improve the char formation during the combustion of polymeric matrices ,14,17. HFirst, we investigated the effect of lignin particle size on PLA degradation during melt processing, thermal stability by TGA and fire properties using cone calorimeter test. Both lignin micro and nano-particles were incorporated in PLA by melt mixing at different contents (5 and 10 wt%). SEM observation in It has been reported in a previous work  that theThus, the effect of the incorporation of both lignins (nanoparticles and microparticles) on the thermal degradation of PLA during melt processing was first studied to evaluate whether the incorporation of very small lignin particles, which are supposed to exhibit higher specific surface area , induces higher degradation with respect to lignin microparticles. Increasing lignin content, whatever the particle size, also induces an important reduction of PLA thermal stability during TGA analysis under nitrogen . Using lThe incorporation of untreated lignins entails some modifications of the fire behavior of PLA assessed using cone calorimeter test at 35 kW/m². Moreover, the incorporation of lignin particles, whatever their size, does not induce any significant reduction of pHRR except the composition containing 20 wt% LMP that only presents however a slight reduction of about 10%. In addition, using reduced lignin content does not present any pHRR reduction. Using such a low lignin content (5 wt%) is not enough to promote the formation of a continuous protective layer and only an ‘islands-in-the-sea’ structure is generated.The amount of the final char left after cone calorimeter test is mainly affected by lignin particle size . Using lFire testing results clearly indicate that no superior effect was obtained using untreated lignin nanoparticles. Performances remain similar to those obtained with microparticles.The results obtained with untreated lignin nanoparticles did not highlight any superior effect of LNP with respect to LMP. The other way for taking advantage of lignin nanoparticles is to modify their surface by phosphorous based compound in order to limit their degradant effect, enhancing the amount of the char as well as its thermal stability for developing nanocomposites presenting high molecular weight and improved fire performance. With this view, untreated and phosphorylated lignin micro and nano particles were incorporated into PLA at reduced content (5 and 10 wt%). Their effect on PLA degradation, its thermal stability and fire behavior were studied.Phosphorylation of lignin nanoparticles doesn’t enable significant increase of PLA thermal stability during melt processing. In fact, PLA molecular weights remain similar to those obtained with untreated LMP and LNP. Some changes are observed but do not demonstrate a significant effect and the Mn remains lower to 55,000 g/mol, while PLA containing 10 wt% untreated lignin microparticles and nanoparticles present Mn around 40,000 g/mol. The effect resulting from the presence of LNP-SiP is a little bit disappointing regarding the presence of a significant amount of non-degradant SiP groups at the surface of lignin. The number of average molecular weight of PLA composites containing LNP-SiP and LMP-SiP are very similar to those obtained with untreated LNP and LMP, respectively. The presence of SiP seems to not affect PLA thermal degradation during melt processing.5%), 10% weight loss (T10%), 50% weight loss (T50%), as well as the value of the final char at 650 °C, were determined. TGA curves are presented in SEC analysis enables determining the degradant effect of additives on PLA during melt processing at 170 °C while TGA analysis gives information on composites thermal stability at higher temperatures. TGA tests were run on dried samples, with a ramp of temperature from 100 °C to 700 °C. From these analyses, temperatures corresponding to 5% weight loss  was left at the end of the test. At similar incorporation content, LMP enabled obtaining higher char yield than LNP. This could be due to the volatilization of a part of lignin nanoparticles with the degradation product. Moreover, under nitrogen, experimental char yields of the composites filled with LMP were higher than theoretical ones. This result evidences the protective effect of lignin that, owing to its char forming ability, avoids total PLA degradation, leading to a higher residue. With respect to untreated lignins, the incorporation of chemically treated lignin particles into PLA entailed different changes on composites thermal stability. While the presence of phosphorylated microparticles induced the early degradation of PLA under nitrogen, the incorporation of LNP-SiP nanoparticles enabled maintaining the thermal stability of PLA since the nanocomposite TGA curve was very close to that of pristine PLA. These results clearly indicate the interest of using phosphorylated nanoparticles instead of microparticles and especially those treated by SiP. The superior effect of LNP-SiP nanoparticles is related to their higher phosphorus content.The addition of untreated lignin, whatever its particle size, induced an important reduction of PLA thermal stability for all compositions. Tterature . As prevterature . It meanThe effect of the incorporation of phosphorylated lignin nanoparticles on the composites fire behavior was investigated using cone calorimeter tests. As can be seen in However, using SiP as grafting agent enables obtaining higher performances with treated lignin nanoparticles . Hence, Interestingly enough, the flame retardant effect of LNP-SiP remains effective even at more reduced content. Indeed, the incorporation of only 5 wt% LNP-SiP  still alLignin nanoparticles were synthesized from Kraft lignin. Both micro and nano lignins were functionalized with diethyl chlorophosphate and diethyl (2-(triethoxysilyl) ethyl) phosphonate. Using diethyl (2-(triethoxysilyl) ethyl) phosphonate enables grafting higher P content. Moreover, highest P content was obtained with lignin nanoparticles (3.2 wt%). While both untreated lignin micro and nano-particles induce dramatic reduction of PLA thermal stability during TGA analysis, grafting SiP enables limiting this negative effect and enhancing the composites thermal stability that becomes very close to that of unfilled PLA. While the other lignin particles do not show any flame-retardant effect into PLA, the incorporation of LNP-SiP content at relatively low content (10 wt%) into PLA enables important increase of time to ignition as well as a reduction of pHRR. Remarkably enough, this effect is obtained even with the lowest LNP-SiP content (5 wt%). This study reports, for the first time, the interest of using lignin nanoparticles as flame-retardant agent in polymers. Results showed that grafting significant P content at the surface of lignin nanoparticles enables their use as flame-retardant additives, effective even at low incorporation content (5 wt%).


Ambystoma mexicanum) embryo tail regeneration assay. A relatively large number of compounds (N = 55) inhibited tail regeneration, including 18 histone deacetylase inhibitors (HDACi). In particular, romidepsin, an FDA-approved anticancer drug, potently inhibited tail regeneration when embryos were treated continuously for 7 days. Additional experiments revealed that romidepsin acted within a very narrow, post-injury window. Romidepsin treatment for only 1-minute post amputation inhibited regeneration through the first 7 days, however after this time, regeneration commenced with variable outgrowth of tailfin tissue and abnormal patterning. Microarray analysis showed that romidepsin altered early, transcriptional responses at 3 and 6-hour post-amputation, especially targeting genes that are implicated in tumor cell death, as well as genes that function in the regulation of transcription, cell differentiation, cell proliferation, pattern specification, and tissue morphogenesis. Our results show that HDAC activity is required at the time of tail amputation to regulate the initial transcriptional response to injury and regeneration.Tissue regeneration is associated with complex changes in gene expression and post-translational modifications of proteins, including transcription factors and histones that comprise chromatin. We tested 172 compounds designed to target epigenetic mechanisms in an axolotl ( In contrast to mammals, highly regenerative salamanders repair damaged tissues without scarring and are capable of regenerating appendages and organs2. While many aspects of wound healing are conserved among organisms, critical differences in early wound healing processes may explain scar-free healing and competence for regeneration3. Open wounds in salamanders are rapidly closed by epidermal cells that migrate from the basal layer of the epidermis4. Macrophages infiltrate the injury site5 and presumably release molecules that prevent infection, including reactive oxygen species (ROS) and antibacterial proteins. These cells clear the injury site of debris, and dying and senescent cells6, change the nature of the extracellular matrix, and release cytokines that induce cellular and molecular changes among infiltrating and surviving cells, including changes in gene expression. The diversity of the early transcriptional response suggests that regeneration in salamanders requires simultaneous expression of wound-healing and developmental genes8.Wound healing processes are rapidly activated after injury to prevent infection and repair damaged tissues9], injury cues likely act through epigenetic mechanisms that alter chromatin structure and properties of proteins that function in transcriptional regulation and cell signaling10. These epigenetic mechanisms include DNA methylation and various chemical modifications of histones, including acetylation-deacetylation [reviewed by11]. Regions of the genome with high levels of DNA methylation and/or low levels of histone acetylation are characterized by compacted chromatin and transcriptional repression, while low levels of DNA methylation and/or high levels of DNA acetylation are characterized by open chromatin and transcriptional activation. Because genomic data have been lacking until recently13, few studies have examined epigenetic mechanisms during salamander regeneration. Yakushiji et al.14 found that sonic hedgehog (shh), an essential limb patterning gene, is expressed during limb regeneration in regeneration competent Xenopus tadpoles and axolotls, but not during stages of development when Xenopus are refractory to regeneration. Expression of shh was correlated with a lower methylation status of the shh promoter region and histone modifications15, thus implicating epigenetic regulation. Building on this work, Hayashi et al.16 showed that histone modifications are important for regulating genes that maintain intrinsic limb-cell identities during Xenopus limb bud regeneration. Aguilar and Gardiner17 examined transcriptional changes of dnmt3 during axolotl limb regeneration, a methyltransferase that mediates de novo methylation of cytosine residues in DNA. They found that nerve signaling transiently downregulated dnmt3 in basal keratinocytes of the early wound epidermis, suggesting a decrease in methylation status and an associated de-repression of genes (e.g. sp9) necessary for the formation of a specialized wound epidermis that is required for blastema formation. Taylor and Beck18 used valproic acid (VPA) to reduce histone deacetylase (HDAC) activity and inhibit tail regeneration in Xenopus and axolotl embryos. Interestingly, they found that pre-treatment of Xenopus embryos with VPA prior to tail amputation did not inhibit regeneration, nor did VPA treatment administered after 6-hours post amputation (6 hpa). Their results suggested that HDAC activity is required sometime within the first 6 hours of injury to trigger the re-expression of developmental genes, although no transcriptional data were presented to support this hypothesis. Tseng et al.19 also used VPA and trichostatin A to inhibit Xenopus tadpole tail regeneration. They observed spatially aberrant expression of developmental genes (at 24 hpa) that are required for tail regeneration , consistent with the idea that HDAC activity is required during regeneration to regulate gene expression. While these few studies show that DNA methylation and HDAC activity are essential for amphibian appendage regeneration, much remains to be learned about these and other mechanisms of epigenetic regulation.Although there have been few studies to date , were dysregulated in romidepsin-treated embryos; and homeotic genes  that define medial spatial positions of the developing vertebrate body axis were upregulated. These expression patterns suggest that HDACi activated proximal patterning genes in the distal tip of the regenerating tail, as this is where tissue was collected for gene expression analyses.In addition to genes that are typically dysregulated in cancer cells after HDACi treatment, we observed changes in axolotl genes that regulate transcription and developmental signaling pathways  and BMP  pathway genes and gene targets that were dysregulated by romidepsin. Overall, many transcription factors, chromatin remodeling genes, and regulators of signaling pathways  were differentially expressed at 3 hpa in romidepsin-treated embryos. HDAC activity at the time of tissue injury is critical for regulating an initial transcriptional response to injury that leads to a successful regenerative outcome.The temporal regulation of transcription factor complexes at the time of injury is probably essential for regeneration. Presumably, the genes that were activated at 3 and 6 hpa in romidepsin-treated embryos are normally repressed by HDAC activity after injury. Consistent with this inference, 37]. Metamorphosis in amphibians is initiated when thyroid hormone (TH) reaches critical levels within cells and thus transcriptional output is dose-dependent. The binding of TH to TRs de-represses HDAC corepressor complexes and activates transcription. It is possible that regeneration-specific, signaling mechanisms may act to de-repress HDAC corepressor complexes during regeneration to regulate transcription temporally and spatially. In support of this hypothesis, we note that many of the genes that were expressed differently in response to romidepsin treatment were also identified in an experiment that used a chemical inhibitor to block WNT-ligand secretion23. Further temporal and spatial analyses of gene expression and regulation will be needed to rigorously test this hypothesis.HDAC-containing corepressor complexes are known to regulate the timing of transcription-mediated developmental events in anuran amphibians. For example, HDACs interact with thyroid hormone receptors (TRs) to regulate the proper timing of metamorphic transcriptional programs among different tissues and organs [reviewed byThe use of pre-feeding stage axolotls does not require a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at University of Kentucky, however embryos used in this study were treated according to the same ethical standards that apply to feeding axolotls. Some of the axolotl embryos were reared to feeding stages (>12 dph at 17–18 C) to image larval tail anatomy. These individuals were fed brine shrimp and cared for using standard axolotl husbandry protocols approved under IACUC protocol 2017–2580. Embryos (RRID:AGSC_100E) were obtained from the Ambystoma Genetic Stock Center (RRID:SCR_006372).23. Briefly, developmental stage 4238 axolotl embryos were manually hatched by removing the egg jelly and membrane, anesthetized in 0.02% benzocaine, and administered tail amputations with a razor blade to remove 2 mm (~20% of the body length) of the distal tail tip. Axolotl embryos were then distributed into microtiter plates containing epigenetic targeting compounds from two libraries: (1) the Structural Genomics Consortium epigenetic chemical panel (N = 22); (2) the Selleckchem epigenetics compound library (N = 151). Chemicals were dissolved in DMSO and diluted to a stock concentration of 10 mM (0.1% DMSO). The initial screen tested 4 replicate embryos per chemical at 10 μM. All chemicals that inhibited tail regeneration were tested again at 10 μM and chemicals were considered hits if both tests showed inhibition. Some chemicals that were not inhibitory at 10 μm were re-tested and found to be inhibitory at 20 μM. Embryos were imaged at the time of amputation and 7 dpa. Tail and spinal cord measurements were tested statistically using Student’s t-test. Embryo survival and distal tail shape were used to classify chemicals as toxic, inhibitory, or having no effect on tail regeneration.The amphibian tail amputation assay was described previouslyThree microarray experiments were performed. In the first experiment, 336 embryos were administered tail amputations  and placed into microtiter plates containing rearing water (40% modified Holtfreter’s Solution) and 0.1% DMSO (controls) or rearing water with 10 μm romidepsin or 10 μm belinostat. Exactly 1 mm of the distal tail tip was removed from 24 embryos immediately after tail amputation to obtain Day 0 samples. Tissues from 8 embryos were pooled into a 1.5 ml tubes with 0.5 ml of RNA-later (Qiagen) to obtain three replicate pools. This tissue sampling and pooling procedure was used to create replicate pools of tissue for 12 hpa, 24 hpa, 48 hpa, and 72 hpa. Four replicates were processed for all but 12 hpa control (3 replicates) and 12 hpa belinostat (2 replicates). In the second microarray experiment, the same tissue sampling design was followed to generate 3 replicates for 0–3 hr romidepsin-treated and control embryos for each of 2 time points (3 and 6 hpa). In the third experiment, the same tissue sampling design was followed to generate 3 replicates for 0–1 mpa romidepsin-treated and control embryos at 3 hpa.39 was performed by the University of Kentucky Microarray Core Facility. The raw microarray data (.CEL files) were deposited in the GEO database (accession number GSE118515). GeneChips were normalized using the affy R package40 to accomplish robust multichip averaging (RMA)41. Differential expression analysis was conducted using the limma R package42. RMA normalized signal intensity values were fit to a linear model and empirical Bayes smoothing applied to standard errors. Moderated t-tests were performed separately for each time point to identify probe sets that yielded significantly different average expression values as a function of treatment. These lists were further filtered using a false discovery rate of α = 0.05 and by requiring a 1.5-fold difference between treatment and control means. Hierarchical clustering of significant genes was performed using Pearson correlation as the distance metric. Optimal k parameters were selected by plotting the within-cluster sum of squares by k, varying from 1 through 10; the k aligning with the observed bend in the resulting plot was chosen as the k parameter. Heatmaps were constructed and visualized using ComplexHeatmap43. Graphs of probe signal intensities were visualized using ggplot244. Correlation plots were constructed with CorrPlot45 and ggplot2 using Pearson correlation.The tissue samples were maintained at 4 C in RNA later prior to RNA isolation using first the Trizol method and then a Qiagen minikit with on-the-column DNAse treatment of DNA. Microarray hybridization using an Ambystoma Affymetrix arrayAxolotl embryos were administered tail amputations and placed into microtiter wells containing either rearing water and 0.1% DMSO, belinostat (10 μM) and 0.1% DMSO, or romidepsin (10 μM) and 0.1% DMSO. Embryos were initially treated for 7-days however shorter exposure times were also tested. For example, the briefest exposure time was for 1 mpa; after exposure, embryos were rinsed in 1 liter of rearing water before being placed into microtiter plates with 2 ml of rearing water. Embryos were imaged at 7 dpa and in some cases at later post-amputation times.et al.46. All EdU labeled cells within 200 μ of the amputation plane were counted as well as cells staining positive for DAPI, after normalizing for tissue area. Student’s t-test was used to assess statistical significance and t-statistics with P < 0.05 were considered statistically significant.Embryos at stage 42 were anesthetized and 0.5 ul of 8 mM EdU (EdU click-iT) was microinjected into the intraperitoneal cavity. Then, 2 mm of the distal tail tip was amputated and embryos were treated with either 10 μM romidepsin (n = 6) or 0.04% DMSO (n = 6). Subsequent steps of the EdU staining methodology followed the protocol reported in Baddar SUPPLEMENTARY INFORMATIONSupplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Supplementary Table 5Supplementary Table 6


MICA rs2596542 and DEPDC5 rs1012068 were identified as being associated with the development of HCV-induced hepatocellular carcinoma (HCC) in Japanese patients. The role of these variants on hepatic inflammation and fibrosis that are closely associated with HCC development is not known, nor are the biological mechanisms underlying their impact on the liver. Here, we demonstrate in 1689 patients with chronic hepatitis C (CHC) , that the MICA (T) allele, despite not being associated with HCC susceptibility, is associated with increased fibrosis stage  and fibrosis progression rate . The DEPDC5 variant was not associated with any of these phenotypes. MICA expression was down-regulated in advanced fibrosis stages. Further, (T) allele carriage was associated with lower MICA expression in liver and serum. Transforming growth factor-β1 (TGF-β1) expression suppresses MICA expression in hepatic stellate cells. Our findings suggest a novel mechanism linking susceptibility to advanced fibrosis and subsequently indirectly to HCC, to the level of MICA expression through TGF-β1-dependent mechanisms.Hepatocarcinogenesis is tightly linked to liver fibrosis. Recently, two GWAS variants,  Advanced liver fibrosis or cirrhosis represents the major risk factor for developing liver-related complications and mortality, but currently there are no approved anti-fibrotic therapies2.Chronic hepatitis C virus (HCV) infection is a leading cause of cirrhosis, hepatocellular carcinoma (HCC) and liver transplantation, with an estimated 339,000 people dying annually from complications4. This risk is likely polygenic and dependent on multiple genetic and epigenetic factors since variations in single loci are usually of modest effect size and explain only a small fraction of the phenotype5. This has led in the recent past to a shift towards the discovery of novel variants with limited effects which ultimately could guide the development of polygenic scores with high predictive value.The rate of liver fibrosis progression varies greatly according to disease etiology and also between individuals; the latter is at least partially attributable to genetic factors. In chronic HCV infection, host genetics play a pivotal role in shaping the immune response, virus-host interactions and ultimately the predilection to and progress of liver fibrosisMICA) on 6p21.33 (rs2596542) to be strongly associated with HCC and the progression from CHC to HCC6. MICA is a ligand for natural-killer group 2 member D (NKG2D), a highly conserved C-type lectin-like membrane glycoprotein and one of the major activating receptors on NK cells7. The second GWAS identified a susceptibility locus near DEPDC5 (rs1012068)8. The function of DEPDC5 is not well understood, but has been linked with hereditary forms of epilepsy9, bladder cancer10 and malignant glioblastomas11.Two genome wide association studies (GWAS) have investigated the risk of HCV-related HCC in Japanese patients. The first identified a locus in the 5′ flanking region of the MHC class I-related chain A , which has some difference from the Japanese population.The clinical, demographic and biochemical characteristic of the patients in the cohort with chronic HCV infection n = 1501) are presented in Supplementary Table 501 are pMICA rs2596542 or DEPDC5 rs1012068 genotype, we examined the association of the genotypes with baseline clinical variables; the results are presented in Supplementary Tables To explore if baseline clinical variables differ between chronic HCV patients according to MICA rs2596542 and DEPDC5 rs1012068 and liver damage (hepatic inflammation and fibrosis). The distribution of rs2596542 and rs1012068 genotypes according to histological features is depicted in Fig. We next assessed the association between MICA rs2596542 was significantly associated with stage of fibrosis  (Supplementary Table MICA rs2596542 (T) allele was associated consistently with a significant increased risk of advanced fibrosis, independent of age, gender, BMI, HCV genotype and alcohol intake   in a multivariate model that included age, gender, BMI and HCV genotype . In contrast, DEPDC5 rs1012068 was not associated with FPR   expression in liver tissue from patients with chronic HCV infection is not known as all available current data are based on the soluble form (sMICA) present in serum16. As shown in Fig. Whether the MICA rs2596542 CC genotype, sMICA levels were significantly reduced in those with the risk T allele  by ELISA in serum samples from a sub-cohort of 214 chronic HCV infection patients; their characteristics are summarized in Supplementary Table ele Fig. . We deteele Fig. .In total, MICA expression is upregulated in HCV infection in an rs2596542 genotype dependent manner with individuals carrying rs2596542 (T) expressing lower levels of mMICA and sMICA. This suggests that differences in expression levels of mMICA according to genotype are not a consequence of differing shedding rates of MICA.The overall contribution of the MICA/NKG2D pathway to fibrosis is unclear. Interestingly and consistent with the genetic data, hepatic MICA expression was significantly lower in subjects with significant hepatic fibrosis (F2–F4) compared to those with no or mild hepatic fibrosis (F0/1) (n = 94) Fig. . MICA ex17 and as we have shown, also have the highest expression of MICA. Killing of activated HSCs by NK cells to ameliorate liver fibrosis is in part by a MICA/NKG2D-dependent manner18. Thus, we investigated the regulation of MICA expression on HSCs in a fibrotic context.We next thought to explore how differential expression of hepatic MICA could be linked to fibrosis progression. Hepatic stellate cells (HSC) are responsible for the deposition of extracellular matrix that is evident histologically as fibrosis19, an established growth factor mediating liver injury and fibrosis12. Hence, we characterized TGF-β1 mediated regulation of MICA expression on HSCs. We first assessed expression of TGF-β1 mRNA after HCV infection in vitro using qRT-PCR. This revealed a significant increase in TGF-β1 in HCV-infected JFH-1/Huh7 cells compared to non-infected cells . We observed a marked repression of MICA mRNA transcripts in TGF-β1 stimulated LX2 cells Fig. , as well20, we asked whether shedding of sMICA was enhanced in TGF-β1 stimulated cells, thus contributing to the attenuation of surface expression on these cells. To this end, we measured sMICA in cell culture supernatant and showed that the level of sMICA released by TGF-β1 stimulated cells was not different compared with control cells suggesting that cleavage of MICA is not mediated by TGF-β1-dependent proteases  to non-HCC from the entire chronic HCV infection cohort described above . Again, no difference in MICA rs2596542 distribution was observed between subjects with HCC and those with cirrhosis (p = 0.77). Results were not changed after adjustment for age and sex. These findings imply that MICA rs2596542 polymorphism is unlikely to be directly associated with the occurrence of HCC. As MICA originally identified by GWAS as a risk variant for susceptibility to HCV-associated HCC in Japanese patients6 associates with fibrosis severity and progression, but not with HCC. Functionally our data suggest that the association with HCC is plausibly mediated by indirect effects through modulation of fibrosis risk. Following the data for effects on fibrosis progression, we show MICA genotype-dependent reductions in hepatic MICA gene expression and soluble MICA protein levels. Tying these findings to fibrosis, TGF-β1 was increased with advancement of fibrosis and there was a corresponding reduction in MICA expression. In vitro, TGF-β1 treatment resulted in MICA transcriptional and surface protein repression on HSC cells. These effects we propose leads to an increased likelihood of fibrosis in those with the risk genotype. Our findings therefore suggest a molecular explanation for the association of rs2596542 with liver fibrosis  are associated with liver fibrosis in schistosomiasis21. It is noteworthy that in the original GWAS report of MICA in HCV-related HCC, Kumar et al., did not use HCV-related cirrhosis without HCC as their control cohort, masking any potential confounding by fibrosis6. This and the failure to find an association with MICA in a similar Japanese study published 2 months later8, as well as the functional data we provide, suggest that the reported association with HCC actually relates to liver fibrosis risk.Consistent with the present findings in the setting of chronic HCV infection, a recent report suggests that 15. Consistently, we demonstrated increases in MICA expression during the early stages of HCV infection, while it decreases with increasing fibrosis in an rs2596542 genotype dependent manner. Furthermore, the (T) risk allele correlated with lower mMICA in liver. Consistent with other groups16, we demonstrated decreased expression levels of sMICA in the serum of patients with the (T) rs2596542 allele.MICA is a stress inducible protein present at very low levels in healthy tissues, but can be induced by cellular stresses such as viral infectionMICA genotypes with fibrosis risk have not been previously elucidated. MICA is a ligand for the activating NK cell receptor NKG2D and this interaction shapes the anti-fibrotic capacity of NK-cells via elimination of activated HSCs22 We verified that HCV infection is associated with an increase in TGF-β1 expression in liver23 and the latter induces marked repression of MICA expression in HSC. This suggests that MICA genotype might be associated with alterations in TGF-β1 (through as yet undefined mechanisms) and may underlie the accelerated fibrosis we observed.The mechanisms that link DEPDC5 rs1012068, the other variant identified by GWAS as a risk variant for HCC8, with either fibrosis or HCC. A large Japanese study of 2,266 HCV patients who had eradicated HCV and with long term follow up to 20 years, was also unable to find any association between DEPDC5 SNPs at rs1012068 and the cumulative risk of HCC24. Some studies have reported an association of DEPDC5 with fibrosis but with opposite effects of the risk allele26. A recent smaller study conducted in Europeans suggested an association between DEPDC5 rs1012068 but not MICA rs2596542 and risk of cirrhosis in chronic HCV infection13. The reasons for the discrepancy between our study and this work are unclear, but may relate to differences in baseline characteristics and that the association analyses for fibrosis were performed differently, as those authors compared F0-F1 vs. F4. Interestingly, in that study, MICA rs2596542 showed a tendency for significance with cirrhosis (p = 0.07) in the discovery cohort (n = 477) but was not investigated in the validation cohort. The function of DEPDC5 in liver needs to be defined.We did not observe any association between MICA rs2596542 is the functional polymorphism or if there are other variants in linkage disequilibrium and the detailed functional mechanisms for the effects are still not fully understood.Our study has some limitations, including the fact that the sample size of the PCR and ELISA sub-cohorts was relatively modest. In addition, whether MICA rs2596542 is associated with liver fibrosis progression, likely mediated and amplified in part through TGFβ-1 dependent mechanisms.In conclusion, we have demonstrated that 29. Briefly, all subjects who had a liver biopsy with scoring for fibrosis stage and disease activity before anti-viral treatment, and genomic DNA were included. For those with CHC, those with HCV RNA serum positivity of Self-reported Caucasian descent were included. Patients were excluded if they had evidence of co-infection with other hepatitis viruses, other liver diseases by standard tests or current or previous hepatic decompensation. Diagnosis of HCC was based on the EASL–EORTC Clinical Practice Guidelines30.The study comprised 1689 consecutive patients  from the International Liver Disease Genetics Consortium (ILDGC) database. Details of the cohort and inclusion criteria have been reportedThe study protocol was conformed to the ethical guidelines of the 1975 Declaration of Helsinki. Ethics approval was obtained from the Human Research Ethics Committees of the Western Sydney Local Health District and the University of Sydney. All other sites had ethics approval from their respective ethics committees and study was performed according to the recommendations of the centers involved. Written informed consent for genetic testing was obtained from all participants.2).The following data were collected at time of liver biopsy from all patients: gender, age, ethnicity, recruitment center, alcohol intake (g/day), body mass index (BMI) and routine laboratory tests. BMI was calculated as weight divided by the square of the height . 1051 samples were genotyped using the Sequenom MassARRAY system and iPLEX Gold chemistry while 638 samples were genotyped using the TaqMan SNP genotyping allelic discrimination method . All genotyping was blinded to clinical variables.Genotyping for 33. Fibrosis was staged from F0 (no fibrosis) to F4 (cirrhosis). Necroinflammation (A) was graded as A0 (absent), A1 (mild), A2 (moderate) or A3 (severe). The inter-observer agreement between pathologists was studied previously and was good (κ = 77.5) for METAVIR staging using κ statistics34.Liver histopathology was scored according to METAVIRMethods for cell culture, real-time PCR, ELISA and flow Cytometry are described in Materials and Methods of the Supporting InformationStatistical analyses were performed using the statistical software package SPSS for Windows, version 21 . All tests were two-tailed and p values < 0.05 were considered significant. Details of statistical analyses are described in the Materials and Methods of the Supporting Information.Supplementary file


Heterotopic Ossification (HO) is a potential long-term complication in orthopaedic surgery. It is commonly classified according to the Brooker classification, which is based on radiological findings. To our knowledge the correlation of histological features to the Brooker grade is unknown as is the association between HO and the indication for revision. The aim of this paper is to analyze the ossification grade of HO tissue in patients undergoing revision hip and knee arthroplasty and to propose a histologically based classification system for HO. We also assess the relationship between the grade of HO and the indication for revision (septic and aseptic revision). From January to May 2019 we collected 50 human HO samples from hip and knee revision arthroplasty cases. These tissue samples were double-blinded and sent for histopathological diagnostic. Based on these results, we developed a classification system for the progression of HO. The grade of ossification was based on three characteristics: Grade of heterotopic ossification (Grade 1–3), presence of necrosis (N0 or N1) and the presence of osteomyelitis (HOES-Score Type 1 to 5). Demographic data as well as surgical details and indication for surgery was prospectively collected from clinical records. Fifty tissue samples were harvested from 44 hips and 6 knee joints. Of these 33 exhibited Grade I ossifications (66%), followed by 11 Grade II (22%) and one Grade III (2%). Necrosis was noted in two tissue samples (4%) and 2 more had osteomyelitis findings according to HOES-Score. Six samples (12%) with radiologically suggestive of HO turned out to be wear-induced synovitis, SLIM Type 1. Of these cases 16 were septic (32%) and 34 aseptic (68%) revisions. Most of the HO tissue samples were classified as a low-grade. High-grade ossification-Score is rare. Higher grades of ossification seem to be associated with septic revision cases. Wear-induced synovitis potentially influences HO development. A histological scoring system for ossification grading can be derived from the data presented in this study. It is more commonly noted in revision arthroplasty cases and its etiology is poorly understood and thought to be multifactorial3 Fig. . The inc12. These progenitor cells transform into osteoblasts under the influence of morphogenes2. The concentrations of morphogene can be elevated in muscle and soft tissue after any kind of trauma, so that an enchondromal ossification leads to formation and maturation of lamellar bone2. This is the proposed mechanism by which HO occurs following major procedures such as revision total joint arthroplasty, particularly cases with significant surgical dissection and surgical trauma such as septic cases13.It has been proposed that mesenchymal progenitor cells are responsible for the development of HO16. The Brooker classification is the most popular of these and has been used since 1973. It is based on observations from a cohort of 100 patients17. In this study, patients were evaluated 6 months postoperatively after THA. The Harris Hip Score and radiographic incidence of HO were noted. HO was found in 21% of the patients. Since this original description, HO has been described in different joints and muscle groups. The Brooker classification was not described for joints apart from the hip and existing classifications do not account for HO in other regions24.Several classification systems for HO have been proposed25.The aim of this study is to propose a histopathological Ossification Score that allows to express ossification grades in any human joint. Furthermore, these characters involve not only the ossification grade but also necrosis and osteomyelitis findings as well as the SLIM-classification (LIT) and the particle algorithm (LIT) in case of peri-implant tissue and the identification of particles depositsThis study was approved by the Institutional Review Board of Hamburg University College of Medicine . All study procedures followed the tenets of the Declaration of Helsinki and written informed consent was obtained from all participants, no patients under an age of 18 years were included. The study used an experimental design, experimental methods were carried out in accordance with the ethical guidelines and regulations of the medical council Hamburg.This study was performed between January and May 2019. Fifty human tissue samples were harvested from 44 hip (88%) and 6 knee (12%) joints. All tissue samples were taken from patients undergoing revision hip and knee arthroplasty. These patients underwent revision surgery either due to periprosthetic joint infection, implant loosening (septic or aseptic) or excision periprosthetic heterotopic bone. All tissue samples were double-blinded and sent for histopathological processing.We also prospectively collected the following patients’ data from available medical records as well as our electronic database: age, sex, body mass index (BMI), length of hospital stay, postoperative intensive care unit (ICU) stay, preoperative symptoms of systemic inflammatory reaction including night sweats, fever, shivers, and/or unintended weight loss, preoperative C-reactive protein (CRP) and white blood cell count (WBC). Patients’ characteristics are presented in Table Histopathological processing of the tissue samples: The macroscopic, gross examination, tissue sampling, the processing of the samples including tissue sample paraffin-processing, decalcifying procedures, histochemical methods diagnostic histopathological classification had been performed in automatic and semi-automatic systems under a certified and accredited framework .Decalcification: The decalcification was carried out by means of acid . The ratio of decalcification liquid volume to tissue sample volume was about 1:20. The reaction temperature was room temperature with an incubation time of about 1 to 3 days. The consistency of the samples had been checked every 12–24 hours carrying out a very small, superficial cut at the periphery of the samples using a scalpel in order to improve the applicability for further cutting procedure in the microtome.25. Only in cases of large tissue samples (more than 10 mm in diameter) osseous tissue had been separated in principle from adhering soft tissue and embedded separately, so that a soft tissue fraction (1.1) and an osseous tissue fraction (1.2) were generated which guaranties a complete histopathologic evaluation of osseous tissue as well as soft tissue. Tissues samples with a size up to 20 × 20 × 10 mm where completely embedded, large tissue samples where representatively embedded, including at least 50% of the tissue mass26. All these methods follow the published recommendations which are part of the DGORh, DGOOC and IAP (German section) recommendations for bone tissue processing in non-neoplastic orthopedic pathology26.Soft tissue fraction and osseous tissue fraction: After fixing in buffered formalin (4%) for at least 24 hours, tissue samples were cut representatively according to previously described recommendations which guaranties a complete and correct histopathologic diagnosisHistopathology and tissue staining: The microtomised tissue sections with a section thickness of 1–3 µm were stained with haematoxylin and eosin (H&E) in a fully automated system (Leica- PELORIS® or SACURA-VIP-6 -AI®) with a barcode-tracking system, Roche, VANTAGE workflow solution®). These systems are closed and pressure-vacuum based systems with very low emissions ensuring additionally high quality and high consistency as well reproducibility of the staining results. In some cases, a periodic-acid-Schiff (PAS) as well Preussian-blue-reaction staining was additionally performed. These techniques were carried out after evaluating the HE stained slides especially in cases with intense inflammatory infiltration and intracellular, granular deposits allowing the discrimination and specification of neutrophilic segmented granulocytes and intracellular haemosiderin deposits, and by this way ruling out intracytoplasmic particles from prosthetic materials .27. The HOES allows a histopathological stratification in following 5 types: I- Signs of an acute osteomyelitis, II- Signs of a chronically florid (that is to say active) osteomyelitis, III- Signs of a chronic osteomyelitis, IV- Signs of a subsided  osteomyelitis and V- No signs of osteomyelitis. If peri-implant synovial tissue  was included in the tissue sample typing of peri-implant tissue and the identification of particles deposits was evaluated according to the SLIM-consensus classification (LIT) which defines 7 different types of the periimplant synovial membrane : SLIM Typ 1 particle induced, Type 2 infection induced, the combination of particle induced and infection, SLIM Typ 3, SLIM Typ 4, neither particle induced nor infection, SLIM Type 5, endoprosthetic-associated arthrofibrosis, Type 6 particle-induced immunological, inflammatory and toxic mechanisms (adverse reactions). In cases of particulate deposits, the particle algorithm had been used28 which allows a descriptive particle identification of endogenous and exogenous particulate materials and is the basis for particle diagnosis in peri-implant tissue. Both published scoring and typing systems are well accepted scoring systems in diagnostic histopathology30.General considerations: The ossification-score (OS) is a semiquantitative score evaluating three different pathogenetic tissue characteristis. These are heterotopic ossification, necrosis and osteomyelitis. The first two characteristics (ossification and necrosis) comprise the newly defined score whereas the last quality (osteomyelitis) is scored according to the HOES  which had been already accepted in routine diagnostic histopathology for typing of infectious osteomyelitis29. For the detection of score features in conventionally stained HE-slides the objective-magnification of 20x was used (area size about: 1.3 mm2). Polarization analysis is necessary for bone tissue typing (lamellar and non-lamellar bone) and for particle definition. The Quantitative definitions concerning the bone formation and numbers of cells per HPF had been defined after subsequent evaluation of all tissue samples and defining retrospectively a quantitative three-step modality. PAS-staining as well as Prussian-blue-reaction may be used for analysis of inflammatory infiltrate and particle-deposit identification.Scoring principles: The proposed scoring principles of the Ossification-Score are the evaluation of heterotopic ossification and the evaluation of necrosis. Ossification is evaluated on a three-step evaluation, whereas necrosis on a two-step modality. Heterotopic ossification is defined by the detection of osseous tissue (non-osteon and osteon bone) embedded in the peri-implant fibrous tissue compartment. Semiquantitative and graduated scoring  is a general principle of diagnostic histopathologyGrade 1: Low bone formation, low bone destruction, low inflammatory infiltration.Grade 2: Moderate bone formation, moderate bone destruction, moderate inflammatory infiltration.Grade 3: High bone formation, high bone destruction, high inflammatory infiltrationBone formation (heterotopic ossification):Low (=1): Area of bone formation is lower than one third of the evaluated area.Moderate (=2): Area of bone formation is between one and two third of the evaluated areHigh (=3): Area of bone formation is more than two third of the evaluated area.Low (=1): low quantity of osteoclast and osteoblast (fewer than 5/HPF)Moderate (=2): moderate quantity of osteoclast and osteoblast (5 to 10/HPF)High (=3): high quantity of osteoclast and osteoblast (more than 10/HPF)Low (=1): low Inflammatory infiltration (fewer than 5 leucocytes/HPF)Moderate (=2): moderate inflammatory infiltration (5 to 10 leucocytes/HPF)High (=3): high inflammatory infiltration (more 10 leucocytes/HPF)Non-existent=0This finding is generally possible but had not been included in this study since all tissue samples had been exclusively taken from regions with pathologic tissue alterations.Ossification: Heterotopic ossification is defined by non-lamellar fibrous bone tissue embedded in fibrous tissue characterized morphologically by irregular distributed osteocytes, irregular fibrils which are visualized by polarization optic analysis.Destruction: Destruction is defined by on a cellular level the detection of osteoclasts, whereas osteoblasts are more the cellular substrate of bone turnover which however accompanies osteoclastic cells.Inflammatory infiltration: Inflammatory infiltration is defined by the inflammatory infiltration of leucocytic including the whole spectrum of infiltrating leucocytes ranging from polymorphic segmented neutrophilic granulocytes to mononuclear lymphocytes, macrophages and histiocytic cells.Qualitative definition: Necrosis is defined under HE-conditions as eosinophilic areas showing no vital cells respectively cells in state of apoptosis.N-0: No Necrosis: No necrotic tissue is detected, very focal areas below 5% are scored as no necrosis.N-1: Necrosis: Necrotic tissue is detected in more than 5% of the tissue area. 5% had been defined as the cut off since a very low content of necrosis in the sense of very small areas of necrosis may be found in nearly every form of inflammatory infiltration.27. The features of the HOES are described in the text. According to this definition, the HOES is an independent evaluation to the above described ossification score (OS) which should give further information in respect to a bacterial infection of the bone tissue. Figure Evaluation of Osteomyelitis had been carried out according to the HOESOssification-Score (Grade 1 to Grade 3), Necrosis  and HOES (I to V). If peri-implant tissue or synovial tissue is presented the SLIM types are listed (type I to type VI) and if particles are detectable the particle-identities are described according to the particle algorithm.This score is reported in a simple formula: The distributions of the continuous variables are presented by count (N), mean, standard deviation (SD), extrema , quartiles  and median (median). The distribution of categorical data is described by absolute and relative frequencies.The distributions of a continuous variable of two independent groups were compared using the Mann–Whitney-U test due to small groups. For the comparison of categorical variables Fisher’s exact test was performed. All tests were two-tailed. The analysis has an explorative character. The p values are interpreted in a descriptive manner accordingly. All data were processed using statistical analysis software .Most of the heterotopic ossifications were Grade I results , followed by 11 Grade II 22%) and 1 Grade III (2%) results. Furthermore, we had 2 tissue samples with necrosis 4%) and 2 showing osteomyelitis results according to HOES-Score, one Type II (2%) and one Type III (2%). Under the 50 tissue samples there were 6 samples with preoperative radiological suspect of HO which turned out to be wear-induced synovitis, SLIM Type 1 (12%). Looking at the Brooker Score, we had six Grade I, fifteen Grade II, five Grade III and two Grade IV patients. Another five samples were taken out of knee joints, which are not scorable in the currently used Brooker score. Detailed information can be found in Table 2% and 1 % and 2 ssample 1: low-grade ossification, sample 2: high-grade ossification and sample 3: wear-induced synovitis, SLIM Type 1.We now present three exemplary cases of histological findings of intraoperatively taken tissue samples using the presented scoring system: Figure 1.1Bone-adherent fibrinous tissue with slightly increased cellularity.1.2Fokal heterotopic ossification with intramedullary PMMA depositions (zirconium dioxide), few osteoclasts, low inflammatory infiltration with intramedullary edema and fibrosis.Figure 1.1Bone-adherent fibrinous tissue.15 × 12 × 5 mm sized tissue sample with minimal adherent fibrinous tissue. H&E staining, original magnification approximately 200x.1.2Irregulary contoured cartilage tissue.1.3Moderate bone formation with woven bone tissue (non-osteon bone) with edema, medullary fibrosis and moderate osteoblasts/intramedullary histiocyte infiltrates.40 × 15 × 11 mm sized predominantly bony tissue with adherent fibrinous tissue. H&E staining, original magnification approximately 200x.Figure 1.1Collagenous, tense organized connective tissue with PMMA depositions (zirconium dioxide).1.2Periprosthetic Membrane of wear-induced type, SLIM-type 1.11 × 16 × 3 mm size, predominantly fibrinous tissue showing initial indurations, no adherent osseous tissue. H&E staining, original magnification approximately 200x.31. The majority of cases with ossification grades II & III were associated with septic revision cases . This can be potentially explained by the fact that infections cause more significant inflammation and therefore are observed to have a higher incidence and grade of heterotopic ossifications. Manrique et al. have reported on more severe HO in septic revision cases13. The authors assumed that not only patient-related data such as an increased age, male gender or increased BMI may be causal for severe ossification, but that also surgery-related factors such as a higher tissue injury consisting of more aggressive and extensive soft tissue debridement, higher number of surgical procedures within short period of time (either due to treatment strategies with multiple procedures such as two-stage arthroplasty or because of persistent or recurrent PJI) and the lengthier surgical procedures lead to higher ossification grades. Rosteius et al. recently confirmed this suspicion and were able to demonstrate that chronic infections and multiple surgical interventions present significant risk factors for high-grade ossification32.We encountered 66% Grade I ossifications with neither necrosis nor osteomyelitis. These findings might be explained by a generally higher awareness for HO in orthopaedic and trauma surgery and therefore focused use of NSAIDs which are often prescribed not only for postoperative pain but also for an extended postoperative period for anti-inflammatory effectsIn the current study all tissue samples with findings of necrosis were associated with septic revision cases and positive intraoperative cultures. Though we could not demonstrate a correlation between positive intraoperative cultures and higher ossification grades, these two aspects of high-grade ossification on the one hand and necrosis findings on the other hand, suggest that extensive inflammation is caused by bacterial colonization and therefore more intense soft tissue reactions. It is also possible that a higher concentration of morphogenes might be triggered by a more radical surgical dissection and débridement in PJI cases resulting in greater surgical trauma and insult to the periarticular soft tissues.33. In the current study, the knee tissue samples were Grade I HO, even in the two septic revision cases. One potential explanation for these low-grade ossifications around the knee joint might be the lower soft tissue mass compared to the hip resulting in the release of a lower concentration of morphogenes which result in less ossification.Most tissue samples in this study were harvested from hip joints , whereas 6 samples (12%) were taken from knee joints. Heterotopic ossification around the knee joint has already been observed before and presents a known long-term complication in orthopaedic knee surgery35. Polyethylene wear being a SLIM-Type I synovitis generating material has not been described so far and might have an influence on implant survivorship as well as on soft tissue reactions and ectopic bone formations.We also encountered 6 samples (12%) with radiological suspicion of HO which turned out to be SLIM-Type 1 wear-induced synovitis (WIS). Five of these samples were taken from hip joints, whereas one sample was taken from a knee joint. Since not only the vast majority of total hip and knee arthroplasties, but also all our inlays/onlays used in this study were made from polyethylene, wear is known to generate particles resulting in osteolysis which is a relatively common cause of revision surgery in total hip arthroplasty17, which mainly relates to hip joints.Based on these results, we propose the first scoring system for Heterotopic Ossification according to intraoperatively taken tissue samples. This method might be a way to express histopathological findings in three simple letters and therethrough characterize the grade of ossification. Since this classification is applicable for all affected human joints suffering HO, it represents a further development of the Brooker classificationIn conclusion, HO predominantly appears as low-grade ossifications in total hip and knee arthroplasty. Additionally, WIS is a special type of synovial infection that was associated with polyethylene inlays/onlays in all of the used implants (hip and knee protheses) in this study. A potential impact of this WIS on ossification gensis has to be clarified by further research.27, which is a useful tool especially in septic revision cases. We know that sometime infections are not only limited to an implant infection including the periprosthetic membrane but also affect the adjacent, peri-implant bone which then leads to (chronic) osteomyelitis as it sometimes can be observed in long time low-grade infections38.The proposed scoring system might be helpful for grading HO according to intraoperative findings independent from the affected joint. Since it not only involves the grade of ossification but also captures necrotic tissue findings, this score is also applicable in more severe or longer ongoing infect situations. Lastly it also involves the HOES score for osteomyelitisLimitations of this study are a relatively small sample size with just a small number of high-grade ossifications and missing records for potential preoperatively taken NSAID. Furthermore, we did not perform a postoperative follow-up to see if patients suffered HO development again and if it might be associated with an intraoperative ossification grade in any kind.Further research is needed to investigate any potential correlation of HO activity and the grade of ossification. For activity measuring, scintigrams might help to graduate the metabolic activity of the chondral tissue. Lastly, clinical treatment suggestions should be worked out based on this activity level and HO grade.


ADAMTS9, AKR1B10, CXXC5, FST, FOXL1, GRPR, ITGA2, SOX17, STARD4, VGF) and four from LNCaP  were able to distinguish irradiated prostate cancer patients into early and late relapse groups. Moreover, in-depth in vitro validations for VGF (Neurosecretory protein VGF) showed that siRNA-mediated gene silencing increased the radiosensitivity of DU145 and LNCaP cells. Our computational approach enabled to predict novel radioresistance driver gene candidates. Additional preclinical and clinical studies are required to further validate the role of VGF and other candidate genes as potential biomarkers for the prediction of radiotherapy responses and as potential targets for radiosensitization of prostate cancer.Radiation therapy is an important and effective treatment option for prostate cancer, but high-risk patients are prone to relapse due to radioresistance of cancer cells. Molecular mechanisms that contribute to radioresistance are not fully understood. Novel computational strategies are needed to identify radioresistance driver genes from hundreds of gene copy number alterations. We developed a network-based approach based on lasso regression in combination with network propagation for the analysis of prostate cancer cell lines with acquired radioresistance to identify clinically relevant marker genes associated with radioresistance in prostate cancer patients. We analyzed established radioresistant cell lines of the prostate cancer cell lines DU145 and LNCaP and compared their gene copy number and expression profiles to their radiosensitive parental cells. We found that radioresistant DU145 showed much more gene copy number alterations than LNCaP and their gene expression profiles were highly cell line specific. We learned a genome-wide prostate cancer-specific gene regulatory network and quantified impacts of differentially expressed genes with directly underlying copy number alterations on known radioresistance marker genes. This revealed several potential driver candidates involved in the regulation of cancer-relevant processes. Importantly, we found that ten driver candidates from DU145 ( Prostate cancer cell lines represent an important model system to characterize molecular alterations that contribute to radioresistance, but irradiation can cause deletions and amplifications of DNA segments that affect hundreds of genes. This in combination with the small number of cell lines that are usually considered does not allow a straight-forward identification of driver genes by standard statistical methods. Therefore, we developed a network-based approach to analyze gene copy number and expression profiles of such cell lines enabling to identify potential driver genes associated with radioresistance of prostate cancer. We used lasso regression in combination with a significance test for lasso to learn a genome-wide prostate cancer-specific gene regulatory network. We used this network for network flow computations to determine impacts of gene copy number alterations on known radioresistance marker genes. Mapping to prostate cancer samples and additional filtering allowed us to identify 14 driver gene candidates that distinguished irradiated prostate cancer patients into early and late relapse groups. In-depth literature analysis and wet-lab validations suggest that our method can predict novel radioresistance driver genes. Additional preclinical and clinical studies are required to further validate these genes for the prediction of radiotherapy responses and as potential targets to radiosensitize prostate cancer. Radiation therapy and surgery with or without anti-androgen treatment are key therapies for prostate carcinoma. Depending on the stage of tumor and type of applied irradiation, up to 90% of prostate cancer patients can be permanently cured by radiotherapy –3. NeverStill, clinical, pathological and biological factors for the prediction of treatment outcomes are of great importance for the personalization of prostate cancer treatment. The current pre-treatment risk stratification system for prostate cancer is based on the analysis of prostate-specific antigen, clinical T-stage and Gleason scores to guide medical decision making . This coOver the last years, different large-scale studies were performed to obtain a better general characterization of prostate cancer at the molecular level. This has contributed to the identification of molecular subtypes, recurrent gene mutations and DNA copy number alterations, and the characterization of signaling and DNA repair pathways involved in the development of prostate cancer e.g. –26). Esp. Esp26])Irradiation of prostate cancer cells causes DNA double strand breaks and cells that survive this highly toxic damage can show complex genomic alterations such as large deletions or amplifications of chromosomal regions due to error-prone DNA repair . Many geVGF: Neurosecretory protein VGF) providing further evidence that our computational approach can contribute to the identification of genes involved in radioresistance.Here, we developed an approach for the network-based analysis of prostate cancer cell lines with acquired radioresistance to identify clinically relevant marker genes associated with radioresistance in prostate cancer patients . We consP = 7.16 ⋅ 10-56) and a non-significant overlap of 47 genes with increased copy numbers widespread across the genome.We considered radioresistant cell lines of the prostate cancer cell lines DU145 and LNCaP that were established in  and furtWe also compared the gene copy number alterations of radioresistant and radiosensitive DU145 and LNCaP to normal male reference DNA to better understand the observed differences between both cell lines . We founP < 7.46 ⋅ 10-8, Fisher’s exact test, BCL10, EPB41L4A, SPRED1, SERPINB5) and commonly overexpressed genes included e.g. SEMA4A involved in cell-cell signaling and migration, NROB1 associated with stem cell pluripotency, and genes involved in cytokine signaling  [We analyzed gene expression for DU145 and LNCaP to identify differentially expressed genes between established radioresistant DU145 and LNCaP and their radiosensitive parental cell line . We used, IL3RA) .We found similar patterns of differential expression among known cancer-relevant signaling pathways for both cell lines . FurtherPRDM1 and RNF217 had a reduced copy number and showed reduced expression in radioresistant compared to radiosensitive LNCaP.We analyzed which genes with copy number alterations showed altered expression. LNCaP showed more gene expression alterations than gene copy number alterations  and only 8.9%  of genes with copy number alterations showed altered expression. 66 of these 102 genes showed putative direct impacts of the underlying copy number alteration on the expression level , but only 7.3%  of genes with altered copy numbers also showed altered expression. 447 of these 1,144 genes showed expression changes in the same direction  and 3b. Generally, all genes with copy number alterations and consistent expression responses in the same direction represent putative direct driver candidates that could be involved in the manifestation of radioresistance.To determine which of the radioresistance driver candidates with altered expression and underlying copy number alteration putatively contribute to the manifestation of radioresistance, we computed direct and indirect impacts of these candidates on the expression of known radioresistance marker genes . To realq < 0.01). These driver candidates comprise overexpressed genes with increased copy number and underexpressed genes with decreased copy number. Potential driver candidates were distributed across the whole DU145 genome, whereas they were more focally distributed in LNCaP  and transcriptional regulators  [SOX17, a transcription factor important for embryonic development and cell fate determination, and the tumor suppressors SEPINB5 and PTRO were underexpressed with underlying reduced copy number [Considering the 162 driver candidates identified from DU145 , Fig 4a,1, LEF1) . Furthery number . GeneralCDH19, DCC, FERMT1, FYN, VNN2 except CLEC1A and KAL1) [ARHGAP18, DUSP10, PAK7, PDGFC, RNF217) [DCC and GPRC5A were underexpressed with underlying reduced copy number.Considering the 27 driver candidates identified from LNCaP , Fig 4b,nd KAL1) . Again g RNF217) . FurtherKAL1 located on chromosome X was found as common high impact gene in DU145 and LNCaP  and the four driver candidates from LNCaP and was clearly more pronounced for larger insignificant p-values .Further, we investigated if the disease status after initial treatment of irradiated patients had biased the observed separations into early and late relapse groups, but we did not find any significant difference with respect to the distribution of patients with complete and non-complete remission after initial treatment between both groups . As for irradiated patients . This is comparable to the overexpression of VGF in radioresistant LNCaP without a directly underlying VGF copy number alteration and supports that increased VGF expression could contribute to increased radioresistance.We found that VGF in parental DU145 and LNCaP cells grown under sphere-forming conditions  gave unspecific bands that were not consistent with the corresponding RT-qPCR data  in combination with further filtering revealed ten candidates from DU145  and four from LNCaP  that were able to distinguish irradiated prostate cancer patients from TCGA into early and late relapse groups. A detailed discussion of these candidate genes is given in Therefore, we developed a network-based method to jointly analyze the gene copy number and expression profiles of an individual cell line to distinguish potential drivers from passengers. The essential basis of this approach was the prostate cancer-specific gene regulatory network that we learned from gene expression and copy number data of 541 prostate cancer patients from TCGA. This network inference was very time and resource consuming requiring 670 hours on a high-performance compute server (Taurus ZIH TUD). Validations on data of 768 cancer cell lines from , 41 confVGF, a neuroendocrine factor, that was highly overexpressed in DU145 and LNCaP radioresistant prostate cancer cell lines and whose high expression was associated with shorter disease-free survival of irradiated prostate cancer patients. VGF was originally identified in a pheochromocytoma cell line in response to the addition of the nerve growth factor (NGF) [VGF is an important regulator of metabolism and endoplasmic reticulum (ER) stress in neurons and endocrine cells [in vitro models, mouse xenografts and analysis of patient outcomes showed that VGF expression is associated with resistance to EGFR inhibitors and further induces epithelial-mesenchymal transition (EMT) and tumor cell dissemination [VGF has been shown to be preferentially expressed in glioblastoma stem cells promoting glioblastoma stem cell survival and stemness and to further support survival of differentiated glioblastoma cells to promote tumor growth [in vitro validation experiments confirmed an upregulation of VGF expression in additionally analyzed independent radioresistant DU145 and LNCaP clones, showed that VGF is highly expressed under sphere forming conditions, and further demonstrated that VGF knockdowns lead to increased radiosensitivity. These results suggest that VGF is involved in radioresistance of prostate cancer. This is also supported by our findings for the prostate cancer cell line PC3.We experimentally validated the novel radioresistance marker gene candidate or (NGF) . VGF is ne cells –58, wherne cells , 60, butmination , 51. In r growth . Our prer growth . In accoVGF in response to siRNA-mediated gene silencing, we tried two available antibodies  and we additionally performed RT-qPCR analysis of VGF expression as control in parallel. Although we observed a pronounced knockdown of VGF by RT-qPCR, we did not observe the specific VGF band by Western blotting, which can be explained by the observation of substantial background signals. Therefore, we focused on PCR-based analysis of VGF expression in our validation studies.For Western blotting analysis of VGF in DU145 than in LNCaP may also have influenced the VGF knockdown efficiency observed for both cell lines. Nevertheless, our clonogenic assays clearly indicate that VGF could be involved in the regulation of radioresistance.We observed that VGF knockdowns were more efficient in DU145 than in LNCaP, but fewer LNCaP cells survived irradiation. The relation between knockdown efficiency and cell survival after irradiation is complex. Different factors can contribute to cell line specific radioresistance. We already know from our prior studies , 34 thatin vitro characterization of DU145 and LNCaP is limited to the identification of molecular alterations that are associated with intrinsic cellular radioresistance. Additional preclinical and clinical studies are necessary to further analyze the revealed marker genes in in vivo studies. Especially the tumor microenvironment and immune signatures of tumors can be altered by radiation therapies influencing tumor progression and therapy response [in vitro cancer models have been reported over the last years [in vitro which was not observed in corresponding PC3 xenografts [in vivo situation.Further, our response –12. Thusst years  and specst years . For exast years . Therefost years , 66. Anonografts . Still, VGF suggest that our network-based approach can predict potentially clinically relevant driver candidates involved in radioresistance of prostate cancer.In summary, our detailed literature analysis and results of radiobiological assays for the maker gene A detailed flow chart of our developed data analysis pipeline is shown in 2-ratio that compares the probe-specific DNA copy number in a radioresistant cell line relative to its radiosensitive counterpart . aCGH profiles were sorted according to chromosomal locations of probes and further segmented into chromosomal regions of constant copy number using DNAcopy [2-ratio gene copy number values were used to determine genes with increased or reduced copy number in radioresistant DU145 or LNCaP relative to their non-resistant counterpart using an absolute log2-ratio cutoff of 0.1 (Array-based comparative genomic hybridization (aCGH) was used to compare the genomes of radioresistant to radiosensitive cell lines for DU145 and LNCaP and to compare these genomes to normal reference DNA . Experiments were done on Agilent’s SurePrint G3 Human CGH Microarray Kit 2x400K  and performed and standardized as described in . Normali DNAcopy . Correspf of 0.1 . The cho2-ratio profile comparing radioresistant to radiosensitive cell lines to account for cell line specific expression characteristics. This training was done with standard settings and initial state-specific means of -1.25 (underexpressed), 0 (unchanged), and 1.25 (overexpressed). We used state-posterior decoding to assign each gene to its most likely underlying state  in radioresistant relative to radiosensitive cell lines  and performed as described in . Hybridill lines . Gene exWe learned a prostate cancer-specific gene regulatory network to predict potential impacts of gene copy number alterations in DU145 and LNCaP on known radioresistance marker genes. We downloaded aCGH profiles and gene expression data of 541 prostate cancer patients from TCGA and processed them as described in . In addia, b) how strong gene a acts on the expression of gene b by computing the impact that flows from gene a to gene b via all possible network paths in the prostate cancer-specific gene regulatory network connecting both genes under consideration of the predictive power of individual genes. More weight was given to genes with greater positive correlations than to genes with smaller positive correlations utilizing gene-specific correlation estimates obtained from cancer cell lines  and at the level of individual genes also significantly better than for random networks of same complexity derived by degree-preserving network permutations (paired t-test: P < 0.02). Corresponding estimated conservative false discovery rates were between 14% and 22% [To realize this, we first considered gene expression profiles of tumors before treatment to compute correlations between the expression of each potential radioresistance driver gene and the months until relapse  considering all 12 of 32 irradiated patients that had a relapse . Next, w and 22%  and more and 22%  , supporting that our selection of driver gene candidates based on the small cohort of irradiated patients was robust .In addition, we used the ExaLT algorithm  to compus robust . We alsoFurther, we used the determined optimal marker gene-specific expression cutoffs to analyze the 182 non-irradiated TCGA prostate cancer patients to determine those markers that were exclusively associated with relapse of irradiated patients but not with relapse of non-irradiated patients.2. DU145 and PC3 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma-Aldrich) and LNCaP cells in RPMI-1640 medium (Sigma-Aldrich) containing 10% fetal bovine serum  and 1 mM L-glutamine (Sigma-Aldrich). The analyzed radioresistant cell lines of DU145 and LNCaP were established in [Prostate cancer cell lines DU145, LNCaP and PC3 were purchased from the American Type Culture Collection  and cultured according to the manufacturers recommendations in a humidified 37°C incubator supplemented with 5% COished in  and furtished in . In moreished in ). Colonyished in ). CorresTo evaluate the self-renewal potential, cells were grown as non-adherent multicellular cell aggregates (spheres). Cells were plated at a density of 1,000 cells/2 mL/well in 6-well ultra-low attachment plates (Corning) in MEBM medium (Lonza) supplemented with 4 μg/mL insulin (Sigma-Aldrich), B27 (Invitrogen), 20 ng/mL EGF (Peprotech), and 20 ng/mL FGF (Peprotech). Media containing supplements were refreshed once a week and spheres with a size > 100 μm were assayed after 14 days using Axiovert 25 microscope (Zeiss) or were automatically scanned using the Celigo S Imaging Cell Cytometer (Brooks).VGF expression, cells were transfected with RNAiMAX (Life Technologies GmbH) according to the manufacturer’s protocol. The siRNA target sequences were obtained from the Life Technologies website and corresponding RNA duplexes were synthesized by Eurofins. The sequences were VGF siRNA 1: sense GGAAGAAGCAGCUGAAGCUdCdT; antisense AGCUUCAGCUGCUUCUUCCdTdC and VGF siRNA 2: sense GGAGGAGCUGGAGAAUUACdAdT; antisense GUAAUUCUCCAGCUCCUCCdTdG for targeted knockdowns of VGF. Scrambled siRNA 1: sense UGCGCUAGGCCUCGGUUGCdTdT; antisense GCAACCGAGGCCUAGCGCAdTdT, scrambled siRNA 2: sense: AGGUAGUGUAAUCGCCUUGdTdT; antisense CAAGGCGAUUACACUACCUdTdT, and scrambled siRNA 3: sense GCAGCUAUAUGAAUGUUGUdTdT; antisense ACAACAUUCAUAUAGCUGCdTdT were used as negative control. In addition, knockdown efficiencies of VGF siRNA 1 and 2 were analyzed by RT-qPCR in comparison to scrambled siRNAs considering three biological replicates for DU145 and PC3 and two for LNCaP. Seven technical replicates were done for each biological replicate.For knockdown of Cells were plated at a density of 500 cells/well in 6-well plates in complete medium and irradiated with doses of 2, 4 and 6 Gy of 200 kV X-rays  filtered with 0.5 mm Cu. The absorbed dose was measured using a Duplex dosimeter (PTW). After 10 days, the colonies were fixed with 10% formaldehyde (VWR) and stained with 0.05% crystal violet (Sigma-Aldrich). Colonies containing > 50 cells were counted using a stereo microscope (Zeiss). The plating efficiency (PE) was calculated as ratio between the number of colonies and the number of cells plated. The surviving fraction (SF) was calculated as ration between the PE of irradiated cells divided by PE of corresponding non-irradiated control cells. We also learned linear-quadratic (LQ) models to obtain a functional representation of the surviving fraction for each cell line using the R package ‘CFAssay’  with staS1 Text(PDF)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 Fig(PDF)Click here for additional data file.S7 Fig(PDF)Click here for additional data file.S8 Fig(PDF)Click here for additional data file.S9 Fig(PDF)Click here for additional data file.S10 Fig(PDF)Click here for additional data file.S11 Fig(PDF)Click here for additional data file.S1 Table(SEG)Click here for additional data file.S2 Table(XLS)Click here for additional data file.S3 Table(XLS)Click here for additional data file.S4 Table(XLS)Click here for additional data file.S5 Table(XLS)Click here for additional data file.S6 Table(XLS)Click here for additional data file.S7 Table(XLS)Click here for additional data file.S8 Table(TSV)Click here for additional data file.


Diamond is the hardest natural material, but its practical strength is low and its elastic deformability extremely limited. While recent experiments have demonstrated that diamond nanoneedles can sustain exceptionally large elastic tensile strains with high tensile strengths, the size- and orientation-dependence of these properties remains unknown. Here we report maximum achievable tensile strain and strength of diamond nanoneedles with various diameters, oriented in <100>, <110> and <111> -directions, using in situ transmission electron microscopy. We show that reversible elastic deformation depends both on nanoneedle diameter and orientation. <100> -oriented nanoneedles with a diameter of 60 nm exhibit highest elastic tensile strain (13.4%) and tensile strength (125 GPa). These values are comparable with the theoretical elasticity and Griffith strength limits of diamond, respectively. Our experimental data, together with first principles simulations, indicate that maximum achievable elastic strain and strength are primarily determined by surface conditions of the nanoneedles. While diamond is the strongest natural material, it fails to reach its theoretical elasticity limits and is brittle. Here, the authors show that thin <100>-orientated diamond nanoneedles can reach diamond’s theoretical strength and elasticity limits in tension. However, virtually no practical bulk materials survive under the ideal maximum stress because of the presence of internal defects and surface flaws. Based on experimental observations, Griffith3 proposed a more useful theoretical tensile strength of ~E/9, where E is the Young’s modulus of the solid. With further development6, the theoretical tensile strength is considered of the order of E/10. Two facts regarding defects guide the direction in designing ultra-strong materials toward reducing material dimension to nano- or even atomic-scales:8 (1) probability of finding a defect decreases with material volume, and (2) maximum size of defect is limited by the overall dimension of the material. By reducing material dimensions, internal defects and surface flaws are significantly reduced; hence material failure is primarily controlled by the intrinsic limits of atomic bonds, making it possible to achieve the theoretical strength of the material11.The ideal strength of a crystalline solid is defined as the maximum stress a perfect crystal lattice can withstand at 0 K12, carbon nanotubes (>100 GPa)13, and Graphene (100–130 GPa)11 in nanomechanical tests. Particularly, uniaxial tensile strength of graphene has been shown to reach the theoretical limits of E/9, the highest tensile strength experimentally achieved to date14. Because of the extremely high bulk modulus and hardness, diamond has historically been considered as the strongest bulk material15. However, it is still challenging to directly measure the tensile strength of diamond due to its poor deformability and relatively high brittleness17. Mechanical properties and fracture behavior of diamond are mostly tested by indentation19 or compression in the diamond-anvil cell20. Based on Hertzian indentation, the tensile strength of diamond is measured to be 20 GPa21, which is far below the ideal strength of ~225 GPa calculated by first principles23 and Griffith theoretical strength of ~122 GPa. The low tensile strength of bulk diamond is primarily attributed to inelastic relaxation induced by the movements of defects and the premature failure caused by the propagation of microcracks24.Near theoretical strengths have been achieved for silicon nanowires (20 GPa)25, corresponding to an estimated maximum tensile stress of ~98 GPa. The scarcity of internal defects coupled with the smoothness of the surface is considered the key to reaching this ultrahigh tensile strength. Although this work provides a strategy for elastic strain engineering, the lack of understanding of size, orientation and surface-defect dependence of elastic deformation prevents quantified specifications of diamond properties for electronic and optical applications. Here we tackle this challenge by measuring deformation of high-surface-quality diamond nanoneedles with various diameters and orientations.Notably, diamond nanoneedles with sub-micrometer diameters can be reversibly deformed with local tensile strains up to ~8.9% by in situ bending inside a scanning electron microscopeWe fabricate high-quality single-crystalline diamond nanoneedles using focused ion beam (FIB) milling coupled with argon plasma thinning. Either <100>, or <110>, or <111> directions are along the axes of the nanoneedles. State-of-the-art nanomechanical bending experiments are conducted in situ inside a transmission electron microscope (TEM). Experimental observations are complemented with detailed computational simulations by the finite element method (FEM) and first principles calculations to determine local strain and stress prior to failure and to understand atomistic mechanisms of fracture.26. These defects are mainly located in regions with diameters larger than 100 nm . The initial d-spacing of (200) lattice planes of undeformed nanoneedle is measured to be 1.78 Å  has been reached.We calculated stress-strain relations under uniaxial tensile for single-crystal diamond along [100], [110], and [111] by first principles calculations Fig. , to comp28, mechanical instability occurs at the point where any elastic modulus approaches zero. Such instability is inherently correlated with changes of atomic configurations at large strains. Along any of the three uniaxial tension directions, C–C bonds elongate monotonically with increasing strain  and fracture strength (125 GPa) of diamond nanoneedles allow great amounts of strain energy to be injected into diamond crystals without inelastic relaxation, thereby increasing the ability to fine-tune physical and chemical properties of diamondOur results indicate that achievable fracture tensile elastic strain and strength of diamond nanoneedles depend strongly on diameter, orientation and surface state. We show that by optimizing these properties, ultralarge elastic strain and ultrahigh strength can be achieved in diamond at nanoscales, allowing fine-tuning the physical and chemical properties. The same strategy may be applied to other brittle covalent solids for deep elastic strain engineering.We used natural type Ia diamond to fabricate nanoneedles by FIB milling with a current of 0.5 nA under a voltage of 30 kV. Residual amorphous carbon on as-FIB-milled crystalline surface was then removed by Argon plasma thinning by gradually decreasing voltages from 2.0 kV to 1.5, 1.0, and finally 0.8 kV.Characterization of the prepared diamond nanoneedles was carried out using an aberration-corrected FEI Themis Z scanning transmission electron microscope operating at 300 kV. In situ bending experiments were then carried out in a JEM 2100 microscope equipped with a XNano in situ TEM holder . To the first-order approximation by neglecting crystal anisotropy, the longitudinal compression stress on the needle is given by Euler’s critical stress σcr, according to Eq. Supplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6Peer Review File